WO2016019900A1

WO2016019900A1 - Multielement gene composition and use therefor

Info

Publication number: WO2016019900A1
Application number: PCT/CN2015/086369
Authority: WO
Inventors: 王建铭; 吴开春; 韩晓亮; 聂勇战
Original assignee: 博尔诚（北京）科技有限公司; 博诚研究中心
Priority date: 2014-08-08
Filing date: 2015-08-07
Publication date: 2016-02-11
Also published as: CN104745681A; CN104745681B

Abstract

The present invention provides a use of a composition in preparing a test kit for detecting cell proliferation anomalies in a subject. The composition comprises a nucleic acid for detecting the degree of methylation in at least one target region of Septin9 and RNF180 genes as well as fragments thereof. The presence of cell proliferation anomalies is shown by synthesizing the Septin9 and RNF180 methylation detection results.

Description

Multi-gene composition and use thereof

The present application claims priority to the patent application filed on Aug. 8, 2014, entitled "Multiple-Generation Compositions and Uses", Application No. 2014103898628.

Technical field

The present invention relates to genetic testing, and more particularly to the use of a composition for the preparation of a kit for detecting abnormalities in cell proliferation in an individual.

Background technique

Diseases caused by abnormal cell proliferation, such as cancer, are a major public health problem. In the United States, the number of deaths due to cancer accounts for about 25% of the total death toll. Among them, gastric cancer is the fourth most commonly diagnosed cancer in the world, and it ranks second in all cancers and is regarded as one of the world's largest health nemesis. Especially in many Asian countries, including China, gastric cancer ranks first among all kinds of malignant tumors, and its morbidity and mortality are high.

Moreover, gastric cancer often does not have any special symptoms at an early stage, and most patients diagnosed with gastric cancer are already in advanced stage, which delays the timing of treatment. Moreover, so far, advanced gastric cancer is still an incurable disease. In general, the average five-year survival rate of gastric cancer patients is about 22%, while the five-year survival rate of patients with advanced gastric cancer is less than 5%. Although chemotherapy has become one of the indispensable methods for the treatment of gastric cancer, it is often accompanied by side effects of different severity, and the effect is not good. Therefore, there is an urgent need for a diagnostic method for cancers with high sensitivity and specificity, and in particular, a method for diagnosis and detection of gastric cancer with high sensitivity and specificity is urgently needed.

With the continuous development of biotechnology, methods for diagnosing diseases using genetic testing have received wide attention. Among them, DNA methylation is an important part of epigenetics, not only in maintaining normal cell function, but also plays an important role in cancer development, that is, the change of methylation status is an important factor causing cancer. The changes include a decrease in the overall methylation level of the genome and an abnormal increase in the degree of local methylation of the CpG island, resulting in instability of the genome and non-expression of the tumor suppressor gene. If the active allele in the tumor suppressor gene is inactivated, the probability of developing cancer increases. Therefore, methylation research provides a new basis for early prediction, classification, grading and prognosis of cancer, and is one of the current research hotspots.

Summary of the invention

Accordingly, the present invention provides the use of a composition for the preparation of a kit for detecting abnormalities in cell proliferation in an individual.

Accordingly, according to a first aspect of the present invention, there is provided a use of a composition for the preparation of a kit for detecting a cell proliferative abnormality in an individual, the composition comprising the same for detecting the Septin9 and RNF180 genes and A nucleic acid having a degree of methylation in at least one of the target regions in the fragment, to indicate the presence of a cell proliferative abnormality by synthesizing the methylation test results of Septin9 and RNF180.

Typically, the nucleic acid comprises at least 15 oligonucleotide long fragments of RNF180, wherein the oligonucleotide comprises at least one CpG dinucleotide sequence, and the presence of CpG methylation indicates the presence of the condition.

In certain preferred embodiments, the nucleotide long fragment of RNF180 comprises at least 15 nucleotides selected from SEQ ID No: 10 to SEQ ID No: 12 that are identical, complementary, or hybridized under moderately stringent or stringent conditions. Nucleotides and their complementary sequences.

Preferably, the nucleotide long fragment of RNF180 comprises a sequence selected from SEQ ID No: 1 to SEQ ID No: 9 and a complement thereof, which are identical, complementary or under moderately stringent or stringent conditions.

Typically, the composition further comprises an agent that converts the 5-position unmethylated cytosine base of the gene to uracil or other base that is detectably different from the cytosine in hybridization performance. Preferably, the reagent is bisulfite.

Moreover, according to certain preferred embodiments, the cell proliferative disorder is cancer. For example, the cell proliferative abnormality is gastric cancer.

Typically, the biological sample of the individual for detection is selected from the group consisting of a cell line, histological sections, tissue biopsy/paraffin-embedded tissue, body fluids, feces, colonic effluent, urine, plasma, serum, whole blood, isolated Blood cells, cells isolated from blood, or a combination thereof.

Preferably, the biological sample of the individual is plasma.

In this application, Septin9 and RNF180 are combined to detect gastric cancer. Septin9 can overcome some of the specific problems of RNF180 itself, such as the intersection of gastritis and gastric cancer, the possible influence of age on RNF180 content, and Septin9 can also increase gastric cancer detection. Positive rate. By combining the nucleic acid sequences used to detect the methylation of the Septin9 and RNF180 genes and their fragments, respectively, the sensitivity and specificity of cancer detection, especially the sensitivity and specificity of gastric cancer detection, were obtained. Improve, thus ensuring the correctness and reliability of the test results.

In particular, according to certain embodiments, some gastric cancers are negative for RNF180, while Septin9 is positive, and this portion of gastric cancer can be detected with Septin9, which can increase sensitivity by about 3%. The methylation assay of Septin9 can overcome and compensate for the lack of sensitivity in the methylation assay of RNF180 alone, and more accurately demonstrate the existence of cell proliferation abnormalities. According to other specific embodiments, the age and chronic gastritis can cause a non-specific increase in the content of methylation of RNF180 in peripheral blood, which has a great influence on the specificity and reliability of gastric cancer detection. The factors affecting the Septin9 gene affected by age and chronic gastritis are negligible, so Septin9 can be used to confirm RNF180 detection of gastric cancer. About 40% of gastric cancers are positive for both Septin9 and RNF180. The positive criteria for both are positive, and 90% specificity can be achieved. The methylation assay of Septin9 can overcome and compensate for the lack of specificity in the methylation assay of RNF180 alone, and more accurately demonstrate the existence of cell proliferation abnormalities.

Moreover, by using real-time PCR to analyze DNA in plasma samples, simultaneous dual-channel detection of two biomarkers, Septin9 and RNF180, can be conveniently performed, and can be based on the cycle threshold (Ct) value of real-time PCR. Quickly and conveniently determine if a sample is positive, providing a non-invasive method for detecting rapid cancer.

Finally, the present application also discloses at least one set of well-designed, optimized primers and primer combinations for detecting whether the RNF180 gene and its fragments are methylated, and a screening method thereof, to ensure that the detection effect is optimized. .

Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or identical to those described herein can be applied in an experimental or practical application, the materials and methods are described herein below. In the event of a conflict, the present specification, including the definitions thereof, is intended to be illustrative and not limiting.

Other features and advantages of the invention will be described in the detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features of the present invention will be further described by the following description in conjunction with the accompanying drawings. It is to be understood that the appended drawings are not intended to The figures are not necessarily to scale unless otherwise indicated, and like reference numerals indicate like parts.

Figure 1 shows that in the methylated DNA multiplex assay of Septin9 and RNF180, neither of the four sets of RNF180 primers and probes affected the determination of Septin9.

Figure 2 shows that the positive results of methylated DNA multiplex detection of gastric cancer by Septin9 and RNF180 complement each other.

Figure 3 shows the sensitivity and specificity of methylated DNA multivariate (RS19) of Septin9 and RNF180 for normal, gastric cancer.

Figure 4A shows a graph of methylation content of RNF180 versus age.

Figure 4B shows a schematic representation of the specificity and sensitivity of Septin9 and RNF180 binding to improve gastric cancer detection.

The best way to implement the invention

Unless otherwise indicated, the practice of the present application will employ conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and genetic techniques, all of which are within the skill of the art. Such techniques are described in detail in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al, 1989); Oligonucleotide Synthesis (MJ Gait, 1984 edition); Animal Cell Culture (RI Freshney, 1987 edition); Methods in Enzymology (American Academic Press, Inc.); Current Protocols in Molecular Biology (FMAusubel et al, 1987 edition, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al, 1994 edition). Primers, probes and kits for use in the present application can be prepared using standard techniques well known in the art.

The technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. Singleton et al, Dictionary of Microbiology and Molecular Biology, Second Edition, J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, Fourth Edition, John Wiley & Sons (New York, NY 1992) The general terminology is provided to those skilled in the art for the various terms used in this application.

definition

"Cancer" as used herein means and includes any malignancy, or malignant tumour, or any disease state with uncontrolled or inappropriate cell proliferation, and includes but is not limited to Any disease characterized by uncontrolled or inappropriate cell proliferation.

The "gastric cancer" and "stomach cancer" of the present application have the same meaning and mean cancer of the stomach or stomach cells. These cancers may be adenocarcinomas that occur in the inner lining of the stomach (mucosa) and may occur in the pyloric portion of the stomach, the corpus of the stomach, or the cardia (lower, body, and upper).

The "gastric cancer cell" of the present application means a cell having characteristics of gastric cancer, and includes pre-cancerous cells.

"Pre-cancerous" of the present application means a cell that is in an early stage of transformation into cancer cells or that tends to be converted into cancer cells. Such cells may exhibit one or more phenotypic traits with cancer cell characteristics.

As used herein, "biomarker" refers to a substance such as a gene, a variable associated with a disease, which can be used as an indicator or predictor of that disease. The presence or risk of a disease can be inferred from this parameter of the biomarker without the need to determine the disease itself.

The "nucleic acid", "nucleic acid sequence" and the like in the present application refer to a polynucleotide, which may be gDNA, cDNA or RNA, or may be single-stranded or double-stranded. The term also includes peptide nucleic acids (PNA), or any chemical DNA or RNA species. "cDNA" refers to DNA that is replicated from naturally occurring mRNA. "gDNA" refers to genomic DNA. Combinations of these materials (i.e., portions of gDNA and recombinant nucleic acids partially cDNA) can also be made.

As used herein, "operably linked" and "operably linked" refer to a functionally binding nucleic acid sequence.

As used herein, "stringent hybridization conditions" and "highly stringent" refer to the conditions under which a probe hybridizes to its target sequence, typically in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and different in different environments. Longer sequences hybridize specifically at higher temperatures. For detailed guidance on nucleic acid hybridization, reference can be made to Tijssen, Biochemistry and Molecular Biology Techniques - Nucleic Acid Probe Hybridization, "Review of Hybridization Principles and Nucleic Acid Assay Strategies". Generally, stringent conditions are about 5-10 ° C below the melting point (Tm) of a particular nucleic acid at a defined ionic strength pH. At the temperature of Tm (at defined ionic strength, pH and nucleic acid concentration), 50% of the probes complementary to the target hybridize to the target sequence in a balanced manner. Stringent conditions can also be achieved by adding destabilizing agents. For selective or specific hybridization, the positive signal is twice, preferably 10 times, the background hybridization. Exemplary stringent hybridization conditions are as follows: hybridization at 42 degrees Celsius in a solution of 50% formamide, 5x SSC and 1% SDS, or at 65 degrees C in a solution of 5x SSC and 1% SDS, then at 0.2xSSC Wash with 65% Celsius in a solution of 0.1% SDS.

Also, nucleic acids that hybridize under stringent conditions are substantially similar if the polypeptides encoded by the nucleic acids are substantially similar. In this case, the nucleic acid is typically hybridized under moderately stringent hybridization conditions. By way of example, "medium stringent hybridization conditions" include hybridization at 37 degrees C in a solution of 40% formamide, 1 M sodium chloride and 1% SDS, and a 45 degree Celsius wash in a 1 x SSC solution. It will be apparent to one of ordinary skill in the art that guidance in obtaining the conditions for achieving the same stringency can be obtained in the prior art. For PCR, a temperature of around 36 degrees Celsius is typically suitable for low stringency amplification, while based on the length of the primer, the annealing temperature ranges from 32 degrees Celsius to 48 degrees Celsius. For highly stringent PCR amplifications, typically at 62 degrees Celsius, and based on the length and specificity of the primers, the annealing temperature for highly stringent hybridization ranges from 50 degrees Celsius to 65 degrees Celsius. Typical conditions for highly stringent and low-rigidity cycling include: 30 seconds to 2 minutes in a sustained denaturation phase at 90-95 degrees Celsius, 30 seconds to 2 minutes in a continuous annealing phase, and a continuous expansion phase at about 72 degrees Celsius. 1 to 2 minutes. Tools and guidance for low and high stringency amplification reactions can be obtained in the prior art.

As used herein, "oligonucleotide" refers to a molecule composed of two or more nucleotides, preferably a molecule composed of three or more nucleotides, the exact size of which may depend on a number of factors. This in turn is determined by the ultimate function and use of the oligonucleotide. In certain embodiments, an oligonucleotide can comprise a length of from 10 nucleotides to 100 nucleotides. In certain embodiments, the oligonucleotides can comprise a length of from 10 nucleotides to 30 nucleotides, or can have a length of 20 and 25 nucleotides. In some specific embodiments, oligonucleotides shorter than these lengths are also suitable.

As used herein, "primer" means when placed under conditions that induce the synthesis of a primer extension product that is complementary to a nucleic acid strand, ie, in the presence of a nucleotide and an elicitor such as DNA or RNA polymerase, and at a suitable temperature and At pH, an oligonucleotide that can serve as a starting point for synthesis, whether it is naturally occurring or synthetically produced in a purified restriction digest. The primers may be single-stranded or double-stranded and must be sufficiently long to initiate synthesis of the desired extension product in the presence of an elicitor. The exact length of the primer depends on a number of factors, including temperature, source of the primer, and method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least or more than about 10, or 15, or 20, or 25 or more nucleotides, depending on the complexity of the target sequence, but may contain more Less nucleotides or more nucleotides. Factors involved in determining the appropriate length of the primer are well known to those skilled in the art.

A "primer pair" as used herein refers to a primer pair that hybridizes to the opposite strand of a target DNA molecule or to a target DNA region flanked by a nucleotide sequence to be amplified.

A "primer site" as used herein refers to a region of a target DNA or other nucleic acid to which a primer hybridizes.

A "probe" as used herein, when referring to a nucleic acid sequence, is used in its ordinary meaning to mean a selected nucleic acid sequence that hybridizes to a target sequence under specified conditions and that can be used to detect the presence of the target sequence. Those skilled in the art will appreciate that in some cases, probes can also be used as primers, and primers can be used as probes.

As used herein, "DNA methylation" refers to the addition of a methyl group to position 5 of cytosine (C), which is usually, but not necessarily, the case of a CpG (behind cytosine) dinucleotide. As used herein, "increased degree of methylation" or "significant degree of methylation" refers to the presence of at least one methylated C nucleotide in a DNA sequence, wherein a normal control sample (eg, from a non-cancer cell or tissue sample) Corresponding C in the extracted DNA sample or DNA sample treated for methylation of DNA residues) is unmethylated, in certain embodiments, at least 2, 3, 4, 5, 6, 7, 8 9, 10, or more C may be methylated, wherein C at these positions in the control DNA sample is unmethylated.

In embodiments, a variety of different methods can be used to detect DNA methylation changes. Methods for detecting DNA methylation include, for example, methylation-sensitive restriction endonuclease (MSRE) assays, methylation-specific or methylation-sensitive assays using southern or polymerase chain reaction (PCR) assays. PCR (MS-PCR), methylation-sensitive single nucleotide primer extension (Ms-SnuPE), high-resolution melting (HRM) analysis, bisulfite sequencing, pyrosequencing, methylation-specific single-strand Conformation analysis (MS-SSCA), combined bisulfite restriction analysis (COBRA), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE), methylation specific melting curve analysis (MS-MCA), A Base-specific denaturing high performance liquid chromatography (MS-DHPLC), methylation specific microarray (MSO). These assays can be PCR assays, quantitative assays using fluorescent labels, or southern blot assays.

"Methylation assay" as used herein refers to any assay that determines the methylation status of one or more CpG dinucleotide sequences within a DNA sequence.

A "biological sample" or "sample" of the present application includes tissue sections such as biopsy and autopsy samples, and frozen sections obtained for histological purposes, or a processed form of any of these samples. Biological samples include blood and blood components or products (eg, serum, plasma, platelets, red blood cells, etc.), sputum or saliva, lymphoid and lingual tissues, cultured cells such as primary cultures, explants, and transformed cells, Feces, urine, stomach biopsy, etc. Biological samples are typically obtained from eukaryotes, which may be mammals, may be primates, and may be human subjects.

"Biopsy" of the present application refers to the process of taking a tissue sample for diagnostic or prognostic evaluation, and also refers to the tissue sample itself. Any biopsy technique known in the art can be used in the diagnostic and prognostic methods of the present invention. The biopsy technique used depends on the type of tissue to be assessed (eg tongue, colon, forefront) Factors such as gland, kidney, bladder, lymph nodes, liver, bone marrow, blood cells, stomach tissue, etc.). Representative biopsy techniques include, but are not limited to, excisional biopsy, excisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy, and may include colonoscopy. A variety of biopsy techniques are well known to those skilled in the art, and they require a small number of experiments to select and use from among these techniques.

An "isolated" nucleic acid molecule of the present application refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are normally associated with the isolated nucleic acid molecule. Thus, an "isolated" nucleic acid molecule includes, but is not limited to, a nucleic acid molecule that does not have a sequence that naturally flanks one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (eg, by cDNA or genomic DNA fragments produced by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are typically introduced into a vector (eg, a cloning vector or expression vector) to facilitate manipulation or production of the fusion nucleic acid molecule. Furthermore, an isolated nucleic acid molecule can include an engineered nucleic acid molecule, such as a recombinant or synthetic nucleic acid molecule. Nucleic acid molecules present in hundreds to millions of other nucleic acid molecules in a nucleic acid library (eg, a cDNA or genomic library) or a portion of a gel (eg, agarose or polyacrylamide) containing restriction-digested genomic DNA It is not considered to be an isolated nucleic acid.

A "cell" of the present application may be isolated, may be contained within a population of cells, may be in culture, or may be included in a living individual, and may be a mammalian cell, and may be a human cell. Likewise, "tissue" can include any number of cells and can be included in or can be isolated from a living individual.

As used herein, "purified" or "substantially purified", when used to refer to a nucleic acid, refers to nucleic acids isolated from their natural environment such that they comprise at least about 75 of all nucleic acids or organic chemicals in a given sample. %, 80%, 85%, 90% or 95%. Herein, nucleic acid purity can be assessed by agarose gel and EtBr staining.

"Detection" as used herein refers to any process of observing a change in a marker or marker in a biological sample, such as a change in the methylation status of a marker or the expression level of a nucleic acid or protein sequence, whether or not a marker is actually detected or The marker changes. In other words, the behavior of detecting a marker or marker change of a sample is "detection" even if the marker is determined to be absent or below the sensitivity level. Detection can be quantitative, semi-quantitative or non-quantitative and can be based on comparison to one or more control samples. It will be appreciated that detecting colon cancer as disclosed herein includes detecting pre-cancerous cells that begin to develop into gastric cancer cells or are about to develop into gastric cancer cells, or have an increased propensity to develop into gastric cancer cells. Detecting gastric cancer can also include detecting a probable probability of death or a possible prognosis of the disease condition.

"Homology", "identity" and "similarity" as used herein, refer to sequence similarity between two nucleic acid molecules. The "homology", "identity" or "similarity" can be determined by comparing the positions in each sequence, which can be aligned for comparison purposes. When the equivalent positions in the compared sequences are occupied by the same base, the molecules are identical at that position; when the equivalent sites are occupied by residues of the same or similar amino acids (eg, similar in spatial or charged nature) The molecule may be said to be homologous (similar) at this position. Expression of homology/similarity or percent identity refers to a function of the number of identical or similar amino acids at the positions shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than 25% identity with a sequence of the invention. The loss of residues (amino acids or nucleic acids) or the presence of redundant residues also reduces identity and homology/similarity when comparing two sequences. In a specific embodiment, for two or more sequences or subsequences, the assay is performed using a BLAST or BLAST 2.0 sequence comparison algorithm having default parameters as described below or by, for example, the National Center for Biotechnology (National Center for Biotechnology) Information (NCBI) is determined by manual comparison and visual inspection provided online. When comparing and aligning the maximum correspondence on the comparison window or designated area, if their sequence is about 60 in the specified area, the identity is about 60. %, or about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more High, can be considered to be substantially or significantly homologous, similar or identical. This definition also relates to or can be used to test the complement of a sequence. Thus, to the extent permitted by the background herein, for example, if the nucleotide sequence can be predicted to be naturally occurring in the DNA duplex, or can occur naturally in the form of one or both of the complementary strands, A nucleotide sequence that is complementary to a sequence or variant thereof is itself considered "similar" to a target sequence, and when referring to a "similar" nucleic acid sequence, includes a single-stranded sequence, its complementary sequence, a double-stranded strand complex A sequence capable of encoding the same or similar polypeptide product, and any permissible variant of any of the above. The case where the similarity must be limited to the analysis of a single nucleic acid strand sequence may include, for example, detection and quantification of expression of a particular RNA sequence or coding sequence in a cell. This definition also includes sequences with deletions and/or additions, as well as sequences with substitutions. In embodiments, the identity or similarity may be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, 24, 25 or more. Over the region of the polynucleotide, or at a length of more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 Or on a region of more than about 100 nucleotides.

"Amplification" as used herein refers to the process of obtaining multiple copies of a particular locus of a nucleic acid, such as genomic DNA or cDNA. Amplification can be achieved using any of a variety of known means including, but not limited to, polymerase chain reaction (PCR), transcription-based amplification, and strand displacement amplification (SDA).

"Standard amplification conditions" as used herein refers to the essential components and cycling conditions of an amplification reaction mixture, including template nucleic acid denaturation, oligonucleotide primer and template nucleic acid annealing, and primer extension by a polymerase to produce Multiple cycles of amplification products.

"Polymerase chain reaction" or "PCR" as used herein refers to a technique in which denaturation, annealing with primers, and elongation with DNA polymerase are used to amplify the copy number of the target DNA sequence to about ¹⁰⁶ times. Or more. The polymerase chain reaction process for amplifying nucleic acids can be found in U.S. Patent Nos. 4,683,195 and 4,683,202.

The "fluorescence-based real-time PCR" of the present application means a method of adding a fluorescent group to a PCR reaction system, monitoring the entire PCR process in real time using fluorescence signal accumulation, and finally performing quantitative analysis of an unknown template by a standard curve. In this PCR technique, there is a very important concept, the cycle threshold, also known as the Ct value. C stands for Cycle, and t stands for threshold (threshold, critical value). The meaning of Ct value is the number of cycles experienced by the fluorescent signal in each reaction tube when it reaches a set threshold. For example, the fluorescence threshold is set as follows: the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the default (default) setting of the fluorescence threshold is the standard deviation of the fluorescent signal of 3-15 cycles. 10 times.

The "cut off value of real-time PCR" of the present application means a critical Ct value which is positive for the determination of a certain biomarker. According to some specific real-time modes of the present application, "Cut Off value is based on a certain amount of sample data, based on statistical processing," which may be based on sensitivity or specificity required. Requirements vary. In the overview, this critical Ct value will be further exemplified.

The "sensitivity" of the present application means the proportion of cancer detected from a certain cancer sample, and the calculation formula is: sensitivity = (detected cancer / all cancers), and "specificity" means that normal human samples are detected normally. The ratio is calculated as specificity = (undetected negative / total negative).

A "marker" or "detectable moiety" of the present application is a component detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (eg, enzymes commonly used in ELISA), biotin, digoxin or haptens, and proteins that can be prepared to be detectable, for example, by radioactivity The label is incorporated into the peptide or used to detect antibodies that specifically react with the peptide.

Nucleic acid molecules can be detected using a variety of different methods. Nucleic acid detection methods include, for example, PCR and nucleic acid hybridization (for example, Southern blot, Northern blot, or in situ hybridization). Specifically, an oligonucleotide (for example, an oligonucleotide primer) capable of amplifying a target nucleic acid can be used in a PCR reaction. A PCR method generally includes the steps of obtaining a sample, isolating nucleic acid from the sample (eg, DNA, RNA, or both) and contacting the nucleic acid with one or more oligonucleotide primers that specifically hybridize to the template nucleic acid under conditions that enable amplification of the template nucleic acid. An amplification product is produced in the presence of a template nucleic acid. Conditions for nucleic acid amplification and amplification product detection are known to those skilled in the art. A number of improvements have been developed to basic PCR techniques including, but not limited to, anchored PCR, RACE PCR, RT-PCR, and ligase chain reaction (LCR). The primer pairs in the amplification reaction must anneal to the opposite strand of the template nucleic acid and should be kept at a suitable distance from one another such that the polymerase can efficiently polymerize across the region and allow the amplification product to be readily detected, for example, using electrophoresis. For example, oligonucleotide primers can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) to help design primers with similar melting temperatures. Typically, oligonucleotide primers are 10-30 or 40 or 50 nucleotides in length (eg, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 in length) , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49 or 50 nucleotides), but the oligonucleotide primers can be longer or shorter as long as appropriate amplification conditions are used.

Detection of amplification products or hybridization complexes is typically accomplished using detectable labels. The term "label", when referring to a nucleic acid, is intended to include direct labeling of a nucleic acid by coupling (ie, physically linking) a detectable substance to a nucleic acid, and by reacting with another reagent that directly labels the detectable substance. Indirect labeling of nucleic acids. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and antibiotics Biotin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; Examples of materials include luminol; examples of biological luminescent materials include luciferase, luciferin, and aequorin. Examples of indirect labeling include end labeling of the nucleic acid with biotin such that the nucleic acid can be detected with fluorescently labeled streptavidin.

Overview

The present application provides a method for detecting cancer by using the Septin9 and RNF180 genes in combination, and corresponding compositions, kits, and nucleic acid sequences to diagnose and detect cancer in a non-invasive manner, with high efficiency and sensitivity. It can be found by specific embodiments that the combined use of Septin9 and RNF180 in cancer, especially in gastric cancer, greatly increases the sensitivity or specificity of the assay.

The following are specific embodiments of the compositions, kits, nucleic acid sequences, and detection methods of the present application. It will be appreciated that a variety of other embodiments may be implemented in view of the general description provided above.

In a first set of embodiments, a composition for the diagnosis or detection of a cell proliferative disorder in a biological sample is disclosed, the composition comprising for detecting a methyl group in at least one target region of the Septin9 and RNF180 genes and fragments thereof Degree of nucleic acid. Specifically, the composition includes not only a nucleic acid sequence for detecting the degree of methylation in at least one target region of the Septin9 gene and a fragment thereof, but also a methyl group for detecting at least one target region of the RNF180 gene and a fragment thereof. The degree of nucleic acid sequence.

The human Septin 9 gene (also known as MLL septin-like fusion protein, MLLseptin-like fusion protein MSF-A, Slpa, Eseptin, Msf, septin-like protein ovary/septin (Ov/Brseptin) and Septin D1) is located on chromosome 17q25. Within the group AC068594.15.1.168501, it is a member of the Septin gene family.

For example, SEQ ID No: 13 provides the sequence of the CpG-rich promoter region of Septin9.

The sequence of SEQ ID No: 13 is as follows:

CGTTACCCGAGTTGTAAAGGGCGGCTCCCTGTGTCTGCCCCGCTGCACCGATACACCGAGCTGCGCACGGTGCCCAGCGCAGGGAGAACAAATGATCATCTGTCCAACGCGCCCATTTACAGGTGAGGAAACTAAGGCTCCAACTCAATCGACGCACTCTGCCCTTTTGATTACCAGAAAAGTAGCAGGACAGGTGTCCTGTCCCGCCCTACCCCGGCCCACTAAGCCGGCACCCCGGCTCCGACCCCCGGCTGTGCCCGGCGCCGCCGCGGTGCCCGGCGCCGCCGCCTCGCCCGGCGGGGCCGCCCGGAGCGCCCGCACCTCCGCCCGCTTCCACCTGGCCGGGCCCGCCCCGCCCGGACTCGGGACTGGGAAGTGCGGCGACTCCCGGAACCAGCCATTGGCGCCAGCGCGGGGAGCTGGGGGTGCAGAGCTGCGGGCGCGGCGGGCCACGCAGGCGGCCCCCACCCCCGGCCTGGCCTGGTCTGGTCTGGTCTGCGCTGCCGCGCGGGGGCGCCCCCTCCCAGGCCCGGCGCCCGCCAGCCCCGCTCCGCCAGGTGCAGCGCAGCGCAGGGGTGGGCGGGGGTGGGGCTCGGCGCGCACGTTCACGGGGCGGGGAGGGGGCGGGTCAGGGGCGGGACCACAGCCGGCTGGGCCGGGGTTCTATGCGCATCTCCGGGGAGGGGCGGGGCGGGGGCGGGGCCGGGGCGGGGCCCGGTCGGTGCACTCCAGACGGCGGGCCGCCCCCTCTTCCCGCCTTCCTACTACCGGCCCAGGATTAGCGCCCTGGGAGCGCGCGCC CCGCTGCCTCGCCGCCACACTTTCCTGGGAGCGGCGGCCACGGAGGCACCATGAAGAAGTCTTACTCAGGTGGGCTTCGCGCCCGGGGTGGGGAGGGGTCGGTGTCCCGGGACCAGCGCTGCTCACCTGAGTGCCTGCGGCCGGGAGTGGCGAGGCGCCCCCGGAGCTGAGCGAGTCCCCGCGGCGGGCACACTGCAGGTCGAGTTCCTCCCAGGACAGGGCCGCTGTCGGGCCGCTTTCGACCTGAGCCGACCGTCCCCTGCGCTGTCTCCAGCCCTTGCTCGAGTGTCGGAGGGGCTGCCCTGGGGGACGCTCCCTCTTCCTCGCCCCTTGCACCCTCGCAGGAATCGCTGACTTTCCAGGTCGGCCGGGTGCTTTGGGTCCCTGTGCGTCTGTGTGGGTGAATGGGGTCGGGGCTAGGTGGAGGGGTGTCCTTGGGTTCAGCCTCTAGGGCTGGTGGTCCAGGCCGCAGCATCCTTTCTTCGGATTCTCTTCGGTTTCTCCTCTACTTAGTGGGGCACGGGACGGCCTCCAGATGGGACCGTCCAGCAGCGCCCAAACTTGGCGACTCGGGTTCACGTTTTGCGCTCAGGACGCCGCCCGC

Members of the Septin gene family are involved in a variety of cellular functions ranging from vesicle trafficking to cytokinesis. The disruption of Septin 9 results in incomplete cell division, and Septin 9 and other proteins have been shown to be fusion partners of the proto-oncogene MLL, suggesting a role in tumorigenesis.

A nucleic acid sequence for detecting the degree of methylation in at least one of the target regions of the Septin9 gene and fragments thereof comprises: hybridizing to or complementary to or under moderately stringent or stringent conditions, at least hybridizing to a contiguous sequence selected from the group consisting of SEQ ID NO: 9 base long fragment.

According to a specific embodiment, the nucleic acid sequence for detecting the degree of methylation in at least one target region of the RNF180 gene and its fragment may comprise: hybridizing to or complementary to or under moderately stringent or stringent conditions, as follows The promoter region of RNF180 is shown to be at least 15 nucleotides of SEQ ID No: 10 and its complement, and SEQ ID No: 10 corresponds to Genbank accession number: NMi001113561chr5163497153-63497758. The underlined "transcription start site" is indicated.

According to certain preferred embodiments, primers and probes can be designed based on the sequence of SEQ ID No: 10. Sequences suitable for use as primers and probes for PCR amplification, may comprise any suitable length, for example may comprise at least 15 nucleotides, or may comprise at least 20, 25, 30, 35, 40, 45 or More than 50 nucleotides. In these embodiments, the nucleic acid sequence can have a similarity to the sequence of SEQ ID NO: 10 or its complement, about 95%, 96%, 97%, 98% or 99%.

SEQ ID No: 10 (-234 bp to +372 bp at the start of transcription)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGC TT TCCGCGGGG TTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCCGGGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAGGCT

Preferably, the nucleic acid sequence for detecting the degree of methylation in at least one of the target regions of the RNF180 gene and fragments thereof comprises: equating to or complementary to or hybridizing under moderately stringent or stringent conditions to SEQ ID No: At least 15 nucleotides of 11 and its complement.

SEQ ID No: 11 (-167 bp to +135 bp of the transcription start site)

CGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGC TTTCCGCGGGG TTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCC

Further preferably, the nucleic acid sequence for detecting the degree of methylation in at least one of the target regions of the RNF180 gene and its fragments may comprise: hybridizing to or complementary to or under moderately stringent or stringent conditions, SEQ ID No as shown below : at least 15 nucleotides of 12 and its complement.

SEQ ID No: 12 (-43 bp to +135 bp of the transcription start site)

GTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGC TTTC CGCGGGG TTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCC GGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCC

Therefore, TaqMan probes and primers can be designed to detect DNA methylation of the RNF180 gene promoter region (-234 bp to +372 bp at the transcription start site), and the preferred promoter region to be detected is -167 bp to the transcription start site. +135 bp, more preferably the promoter region detected is -43 bp to +135 bp at the start of transcription, and the size of the amplicon used for detection ranges from 66 bp to 130 bp.

For example, in certain embodiments, the target region of SEQ ID No: 12 (-43 bp to +135 bp at the start of transcription) is designed to detect methylation in at least one target region of the RNF180 gene and its fragments. The extent of primers and probes. Therefore, depending on the application, many sets of probes and primer combinations can be designed, and the performance of each set of probes and primer combinations may differ. Therefore, in order to screen for efficient primers and probes, the present application utilizes artificial methylation templates and unmethylated templates, as well as cancer (eg, gastric cancer) and normal DNA as templates for the following sets of probes and probes. Primer combinations were screened:

1. Design primers and probes for the promoter region of the RNF180 gene.

2. Design artificial methylated DNA and unmethylated DNA.

3. Using artificial methylated DNA and non-methylated DNA to select primers and probes, artificial methylated DNA is amplified, and artificial unmethylated does not show amplification.

4. Screen primers and probes using DNA extracted from normal leukocyte DNA.

5. If there is little amplification or amplification in normal human leukocyte DNA, primers and probes are screened using DNA extracted from human gastric cancer tissue.

6. Screening primers and probes for DNA extracted from normal plasma at the clinical study stage.

7. If there is no amplification or amplification in normal plasma during the clinical study phase, primers and probes are screened using DNA extracted from gastric cancer patients.

By the above screening, the following sequences SEQ ID NO: 1-9 were constructed as primers and probes:

Primer: SEQ ID No 1 (180F7) 5'-GTTCGAGGTCGCGGGGTC-3'

Probe: SEQ ID No 2 (180P7) 5'-CAL Fluor

Red-AACGCTCGAACTATACCTACAACCCC-BHQ2-3'

Primer: SEQ ID No 3 (180R7) 5'-ACAAAAACCAAACCCCGCG-3'

Primer: SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

Probe: SEQ ID No 5 (180P2) 5'-CAL Fluor

Red-CCGACGACGACGATACCG-BHQ2-3'

Primer: SEQ ID No 6 (180R2) 5'-ACAACCAAACTCTAAAAACTCG-3'

Probe: SEQ ID No 7 (180P14) 5'-CAL Fluor

Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3'

Primer: SEQ ID No 8 (180R135) 5'-AAAACCTCCAACTTCACACCC-3'

Primer: SEQ ID No 9 (180R14) 5'-CGCCAACAACCAAACTCTAA-3'

Further, the Applicant has designed at least one primer and probe combination based on the above-mentioned primers and probes screened, wherein the following four combinations are preferred:

A, primer and probe combination 1 (amplifier 66 bp, transcription start site -43 bp to +23 bp)

SEQ ID No 1 (180F7) 5'-GTTCGAGGTCGCGGGGTC-3'

SEQ ID No 2 (180P7) 5'-CAL Fluor

Red-AACGCTCGAACTATACCTACAACCCC-BHQ2-3' SEQ ID No 3

(180R7) 5'-ACAAAAACCAAACCCCGCG-3'

B, primer and probe combination 2 (amplifier 86bp, +5bp to +91bp of transcription start site)

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 5 (180P2) 5'-CAL Fluor

Red-CCGACGACGACGATACCG-BHQ2-3'

SEQ ID No 6(180R2) 5'-ACAACCAAACTCTAAAAACTCG-3'

C, primer and probe combination 3 (amplicon 130 bp, +5 bp to +135 bp of transcription start site)

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 7 (180P14) 5'-CAL Fluor

Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3'

SEQ ID No 8 (180R135)

5'-AAAACCTCCAACTTCACACCC-3’

D, primer and probe combination 4 (amplifier 91 bp, +5 bp to +96 bp of transcription start site)

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 7 (180P14) 5'-CAL Fluor

Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3'

SEQ ID No 9 (180R14) 5'-CGCCAACAACCAAACTCTAA -3'

The binding sites of the above primers and probe combinations to the above gene sequences are further clarified below (wherein the same shape underline indicates the corresponding portion).

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCG

The binding site is indicated:

SEQ ID No 1 (180F7) 5'- GTTCGAGGTCGCGGGGTC -3'

AGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCAC GTCCGAGGCCGCGGGGTC

SEQ ID No 2 (180P7) 5'-CAL Fluor Red-

ATACCTACAACCCC -BHQ2-3'

ACAGCTCGAGCGCT TTC

CGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAG

5'-

-3' SEQ ID No 3 (180R7)

CCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCCGGGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAGGCT

The AGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGC binding site is indicated:

BHQ2-3' SEQ ID No 5 (180P2)

SEQ ID No 4 (180F24) 5'-

-3'5'-CAL Fluor Red- CCGACGACGACGATACCG - ACAGCTCGAGCGCTTTCC

CGCTTCCCGTCTCGCCGAAC CGGCATCGCCGCCGCCGG AG

CCGCAG

TGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCCG

5'-

-3' SEQ ID No 6 (180R2)

GGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTGGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAGGCT

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTGGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGC binding site:

SEQ ID No 4 (180F24) 5'- GCGGGGTTTGGTTTTTGT -3' SEQ ID No 7 (180P14) 5'-

ACAGCTCGAGCGCTTTCC GCGGGGTTTGGCTCCTGT CGCTTCCCGTCTCGCCGAACCGGCATCGCCGC

-BHQ2-3'

TCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACG

G

SEQ ID No 8 (180R135) 5'-

-3'

D, primer and probe combination 4 (amplifier 91, +5 bp to +96 bp of transcription start site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGC The binding site is indicated:

SEQ ID No 4 (180F24) 5'- GCGGGGTTTGGTTTTTGT -3' SEQ ID No 7 (180P14) 5'-

ACAGCTCGAGCGCTTTCC GCGGGGTTTGGCTCCTGT CGCTTCCCGTCTCGCCGAACCGGCATCGCCGC

-BHQ2-3'

GCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCCG

5'-

-3' SEQ ID No 9 (180R14)

In certain embodiments, the composition further comprises a reagent that converts a 5-position unmethylated cytosine base of a gene into uracil or other base that is detectably different from cytosine in hybridization performance. . For example, the reagent can be bisulfite.

In certain embodiments, the cell proliferative disorder is cancer. For example, the cell proliferative abnormality is gastric cancer. First, the multivariate gene composition for joint assay of the present application can be applied to various types of cancer including liver cancer, gastric cancer, colon cancer, and blood cancer, but it is particularly effective in diagnosis and detection of gastric cancer. For example, in an exemplary embodiment, the above-described primer and probe combination 2 can be used as a primer and probe for gastric cancer detection, so that 100% sensitivity can be obtained when distinguishing gastric cancer from a normal person.

Further, for use in the above described or suggested applications of the present application, in a second set of embodiments, a kit for detecting cell proliferative abnormalities in an individual, comprising for detecting the Septin9 and RNF180 genes and A nucleic acid having a degree of methylation in at least one of the target regions of the fragment, indicating the presence of a cell proliferative abnormality by combining the results of methylation detection of Septin9 and RNF180.

The kit includes a composition for diagnosing or detecting a cell proliferative disorder in a biological sample disclosed in the first set of embodiments. The description of the composition is similar to the first set of embodiments and will not be repeated here.

Such kits can include a carrier that is divided for sealingly housing one or more containers, such as vials, vials, etc., each container including a separate component to be used in the method. For example, one of the containers may include a probe that is or may be detectably labeled.

Typically, the kit of the present application will include a container for holding a patient's biological sample and or instructions for using and interpreting the results of the kit. In particular, the kit of the present application includes materials required from a commercial and user perspective. Includes instructions for holding the patient's biological sample in a container, buffer, diluent, filter, needle, syringe, and insert package. Labels can be used on the container to illustrate the components for a particular therapeutic or non-therapeutic application, and can also be stated to be used in vivo or in vitro, such as those described above.

The kits of the present application have a variety of embodiments. A typical embodiment is a kit comprising a container, a label on the container, and components within the container; wherein the component comprises for detecting at least one of the Septin9 and RNF180 genes and fragments thereof The degree of methylation of the nucleic acid in the region, the label on the container indicates that the component can be used to assess the degree of DNA methylation of the sample, and how to use the kit. The kit may further comprise a set of instructions and materials for preparing a tissue sample and applying the composition of the present application to the sample. The kit may include an agent for converting a 5-methyl unmethylated cytosine base of a gene into uracil or other base which is detectably different from cytosine in hybridization properties, such as bisulfite.

In a third set of embodiments, a method of detecting a cell proliferative disorder in an individual, comprising: determining methylation in at least one target region of a Septin9 and RNF180 gene and a fragment thereof in a biological sample isolated from the individual To the extent that the presence of cell proliferative abnormalities is indicated by the results of methylation detection by combining Septin9 and RNF180.

Typically, the method according to the present application further comprises the step of using an agent to convert the 5-unmethylated cytosine base of the gene into uracil or other base which is detectably different from the cytosine in terms of hybridization performance.

The bisulfite modification of DNA is a known tool for assessing the methylation status of CpG. 5-methylcytosine is the most common covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of transcription, genetic imprinting, and tumorigenesis. Therefore, confirmation of 5-methylcytosine as a genetic information component has considerable significance. However, 5-methylcytosine cannot be identified by sequencing because 5-methylcytosine has the same base pairing behavior as cytosine. In addition, epigenetic information carried by 5-methylcytosine is completely lost, for example, during PCR amplification.

The most commonly used method for the analysis of the presence of 5-methylcytosine in DNA is based on the specific reaction of bisulfite with cytosine, whereby after subsequent alkaline hydrolysis, cytosine is converted to a corresponding thymine in the pairing behavior. Uracil. But it is important that 5-methylcytosine remains unmodified under these conditions. As a result, the original DNA is transformed in such a manner that the methylcytosine originally unable to distinguish it from the cytosine in its hybridization behavior can now be conventionally known as the only remaining cytosine. Sub-biological techniques detect, for example, by amplification and hybridization. All of these techniques are based on different base pairing characteristics and can now be fully utilized.

Thus, typically, the present application provides a combination of bisulfite technology in combination with one or more methylation assays for determining the methylation status of CpG dinucleotide sequences within the Septin9 and RNF180 gene sequences. Genomic CpG dinucleotides can be methylated or unmethylated (either as up-and down-methylated, respectively). However, the method of the invention is suitable for the analysis of heterogeneous biological samples, such as low concentrations of tumor cells in blood or feces. Thus, when analyzing the methylation status of CpG positions in such samples, one skilled in the art can use quantitative assays to determine the level of methylation (eg, percentage, number of parts, ratio, ratio, or degree) at a particular CpG position. Instead of methylation status. Accordingly, the term methylation status or methylation status should also be taken to mean a value that reflects the degree of methylation at the CpG position. Unless explicitly stated, the terms "hypermethylation" or "upper methylation" shall be taken to mean that the level of methylation exceeds a particular threshold, where the threshold may be an average or medium representative of a given population. The value of the value of the methylation level, or preferably the optimized critical level. "Critical" may also be referred to herein as "threshold". In the context of the present invention, the terms "methylated", "super" for all CpG positions within or associated with a gene or genomic sequence selected from the following sequences (eg, in a promoter or regulatory region) "Methylated" or "upper methylated" shall be taken to include methylation at a methylation level above zero (0)% (or its equivalent) of the critical value, said sequence being Septin9 and RNF180 as described above. gene sequence.

In certain embodiments, the methods of the present application specifically comprise contacting the reagent-treated Septin9 and RNF180 genes or fragments thereof with an amplification enzyme and a primer such that the processed gene or fragment is amplified to produce The amplification product is either not amplified; the amplification product is detected with a probe; and the degree of methylation of at least one CpG dinucleotide of the genetic DNA sequence of Septin9 and RNF180 is determined based on the presence or absence of the amplification.

And, typically, the contacting or amplifying comprises applying at least one method selected from the group consisting of using a thermostable DNA polymerase as the amplification enzyme; using a polymerase lacking 5'-3' exonuclease activity; Polymerase chain reaction (PCR) is used; an amplification product nucleic acid molecule with a detectable label is produced.

That is, the degree of methylation is preferably determined by PCR, such as "fluorescence-based real-time PCR technology" (Eads et al, Cancer Res. 59: 2302-2306, 1999), Ms-SNuPETM (methylation sensitive) Single nucleotide primer extension) reaction (Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997), methylation specific PCR ("MSP"; Herman et al, Proc. Natl. Acad. Sci. USA 93: 9821-9826, 1996; U.S. Patent 5,786,146) and methylated CpG island amplification ("MCA"; Toyota et al, Cancer Res. 59: 2307-12, 1999) and the like are used to determine the degree of methylation of at least one CpG dinucleotide of the genetic DNA sequence of Septin9 and RNF180.

Among them, the "fluorescence-based real-time PCR" assay is a high-throughput quantitative methylation assay using fluorescence-based real-time PCR (TaqMan_) technology that does not require further manipulation after the PCR step (Eads et al., Cancer Res. 59) :2302-2306, 1999). Briefly, the "fluorescence-based real-time PCR" method begins with a mixed sample of genomic DNA according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil) in sulfurous acid The sodium hydrogen reaction is converted to a mixing pool of methylation-dependent sequence differences. Fluorescence-based PCR is then performed in a "biased" reaction (using PCR primers that overlap known CpG dinucleotides). Sequence differences can be made at the level of the amplification process as well as at the level of the fluorescence detection process.

The "Fluorescence-Based Real-Time PCR" assay can be used as a quantitative test for methylation patterns in genomic DNA samples where sequence discrimination occurs at the probe hybridization level. In this quantitative approach, the PCR reaction provides methylation-specific amplification in the presence of fluorescent probes that overlap specific putative methylation sites. A non-offset control for the amount of input DNA is provided by the reaction in which neither the primer nor the probe covers any CpG dinucleotide. The "fluorescence-based real-time PCR" method can be used with any suitable probe, such as "TaqMan_", "Lightcycler_", and the like.

The TaqMan_ probe is double labeled for fluorescent "reporter" and "quench" molecules and is designed to be specific to a relatively high GC content region such that it is about 10 higher than the forward or reverse primer in the PCR cycle. The temperature of °C is melted. This allows the TaqMan_ probe to remain sufficiently hybridized during the PCR annealing/extension step. When Taq polymerase synthesizes a new strand in PCR, it eventually encounters an annealed TaqMan_probe. The 5' to 3' endonuclease activity of Taq polymerase will then be displaced by digestion of the TaqMan_probe, thereby releasing the fluorescent reporter molecule for quantitative detection of its now unrefined signal using a real-time fluorescence detection system.

Typical reagents for "fluorescence-based real-time PCR" analysis (eg, which can be found in kits based on "fluorescence-based real-time PCR") can include, but are not limited to, for specific genes (or bisulfite) PCR primers for processed DNA sequences or CpG islands; TaqMan_ or Lightcycler_ probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

And, in particular, in a preferred embodiment, the method comprises the steps of:

In the first step, a tissue sample to be analyzed is obtained. The source can be any suitable source, such as cell lines, histological sections, biopsy tissue, paraffin embedded tissue, body fluids, feces, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, isolated from blood. Cells and their There is a possible combination. Preferably, the source of DNA is feces or body fluid selected from the group consisting of colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood.

Genomic DNA is then isolated from the sample. Separation can be by any standard means in the prior art, including the use of commercially available kits. Briefly, when the DNA of interest is encapsulated in a cell membrane, the biological sample must be disrupted and cleaved by enzymatic, chemical or mechanical means. Proteins and other contaminants are subsequently removed, for example by digestion of protein kinase K. The genomic DNA is then recovered from the solution. This can be accomplished by a variety of methods including salting out, organic extraction or binding DNA to a solid support. The choice of method is influenced by a number of factors, including time, cost, and amount of DNA required.

When the sample DNA is not encapsulated in a cell membrane (e.g., circulating DNA from a blood sample), standard methods of isolating and/or purifying DNA in the prior art can be used. These methods include the use of protein degradation agents such as chaotropic salts such as guanidine hydrochloride or urea; or detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide. Other methods include, but are not limited to, ethanol precipitation or propanol precipitation, vacuum concentration by centrifugation, and the like. Those skilled in the art can also utilize devices such as filters such as ultrafiltration, silicon surfaces or membranes, magnetic particles, polystyrene particles, polystyrene surfaces, positively charged surfaces, and positively charged membranes, charged membranes, charged Surface, charged conversion film, charged conversion surface.

Once the nucleic acid is extracted, the genomic double-stranded DNA is used for analysis.

In a second step of the method, the genomic DNA sample is treated such that a cytosine base that is unmethylated at the 5' position is converted to uracil, thymine, or not used for cytosine in hybridization behavior. Another base. This should be understood as "pretreatment" or "processing" as described herein.

This is preferably achieved by treatment with a bisulfite reagent. The term "bisulfite reagent" refers to an agent comprising bisulfite, disulfite, acid sulfite or a combination thereof, as disclosed herein, which can be used to distinguish between methylation and unmethylation. CpG dinucleotide sequence. Such treatments are known in the art (for example, PCT/EP2004/011715, which is incorporated herein in its entirety by reference). Preferably, the bisulfite treatment is carried out in the presence of a denaturing solvent such as, but not limited to, a normal alkyl diol, especially diethylene glycol dimethyl ether (DME), or in dioxane or The reaction is carried out in the presence of a dioxane derivative. In a preferred embodiment, the denaturation solvent is used at a concentration of from 1% to 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of a scavenger such as, but not limited to, a chroman derivative such as 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid or Trihydroxybenzoic acid and its derivatives, such as gallic acid (see: PCT/EP2004/011715, which is incorporated herein by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature of from 30 ° C to 70 ° C, wherein the temperature is increased to a short time during the reaction to More than 85 ° C (see: PCT/EP2004/011715, which is incorporated herein by reference in its entirety). The bisulfite treated DNA is preferably purified prior to quantification. This can be done by any method known in the art, such as, but not limited to, ultrafiltration, preferably by a Microcon(TM) column (manufactured by Millipore(TM)).

In a third step of the method, fragments of the treated DNA are amplified using the primer oligonucleotides of the invention and the amplification enzyme. Amplification of several DNA fragments can be performed simultaneously in the same reaction vessel. Typically, the amplification reaction is carried out using polymerase chain reaction (PCR). Preferably, the amplification product is from 100 to 2,000 base pairs in length.

For the detection of methylation of the Septin9 gene and its fragments, primers and probes for Septin9 are used, for example:

Primer: SEQ ID No 14 GTAGTAGTTAGTTTAGTATTTATTTT

Primer: SEQ ID No 15 CCCACCAACCATCATAT

Probe: SEQ ID No 16 GAACCCCGCGATCAACGCG

For the detection of methylation of the RNF180 gene and its fragments, primers and probes screened for RNF180 by the above screening methods were utilized. For example, in a preferred embodiment, any combination of the above-described primer and probe

combinations

1, 2, 3, 4 can be utilized.

Fragments obtained by amplification can carry markers that can be detected directly or indirectly. Preferably, the label is in the form of a fluorescent label, a radionuclide or an attachable molecular fragment.

In the fourth step of the method, the amplification product obtained in the third step of the method is analyzed to determine the methylation status of the CpG dinucleotide prior to treatment.

In the fourth step, the detection of the amplified product is carried out by detecting the probe in real time. In the present invention, real-time PCR detection can be performed using standard commercial operations on various commercial real-time PCR instrumentation devices according to the prior art. Real-time PCR detection was performed on a Life Technologies instrument (7500 Fast) according to some embodiments. The PCR reaction mixture was buffered from bisulfite-transformed DNA template 25-40 ng and 300-600 nM primers, 150-300 nM probe, 1 UTaq polymerase, 50-400 uM of each dNTP, 1 to 10 mM MgCl2 and 2X PCR buffered to the final 2ul to 100ul volume. Continue at 85 to 99 ° C for 3-60 minutes to amplify the sample with pre-cycle, followed by an annealing of 35-55 cycles of 1 to 30 seconds at 50 to 72 ° C and an annealing of 5 to 90 at 45 to 80 ° C. Seconds, denatured at 85 to 99 ° C for 5 to 90 seconds.

The gene fragment was detected by probe specific to the RNF180 and Septin9 promoter regions containing 5-methylcytosine by observing amplification only on the methylated RNF180 gene and the Septin9 gene fragment. Moreover, in certain embodiments, β-actin is used as an internal reference for PCR, a β-actin amplicon is created by using a primer complementary to the β-actin sequence, and β-muscle is detected with a specific probe. Actin amplicon. Each sample was subjected to at least one real-time PCR, and in some embodiments, two real-time PCR assays were performed.

In the fifth step of the method, the methylation status of at least one CpG dinucleotide of the gene DNA sequence of the Septin9 and RNF180, respectively, is determined by the cycle threshold Ct of the polymerase chain reaction, and then further The method comprises the following steps: A) comparing the PCR Ct value corresponding to Septin9 of the sample to be measured and the preset cut value of Septin 9 (ie, the critical Ct value), thereby determining whether the analysis result based on the Septin9 gene is positive; B) comparing the Detecting a PCR Ct value corresponding to the sample RNF 180 and a preset cut value of the RNF 180, thereby determining whether the analysis result based on the RNF180 gene is positive; c) synthesizing the results of the steps A) and B) to determine the sample Whether the final analysis result is positive.

According to a specific embodiment of the present application, gastric cancer and normal critical Ct values relative to Septin9 and RNF180 are determined based on the average Ct values of Septin9 and RNF180 of a certain number of gastric cancer samples and normal samples. In certain preferred embodiments, the kit for measuring the methylation status of at least one CpG dinucleotide of the gene DNA sequence of Septin9 is purchased from Epigenomics (Germany), so Septin 9 critical Ct can be determined according to the Epigenomics specification. The value is 45Ct. The critical Ct value of RNF180 is determined based on the average Ct value of RNF180 of a certain number of gastric cancer samples and normal samples. Moreover, the critical Ct value of RNF180 is also related to the actual required sensitivity. The higher the sensitivity requirement, the larger the selected critical Ct value. In some embodiments, the RNF 180 threshold is about 45 Ct.

Moreover, the present application also allows for the use of different methodologies to analyze Ct values. For example, using ΔCt or dCT, actin Ct as an internal control for PCR, and Septin 9 Ct minus actin Ct gave dCT values for Septin9. Similarly, RNF180 Ct subtracts actin Ct to obtain the dCT value of RNF180. Accordingly, if ΔCt or dCT is employed as the detection standard, then in the fifth step of the method, the methylation status of at least one CpG dinucleotide of the gene DNA sequence of the Septin9 and RNF180, respectively, is represented by polymerization. The cycle threshold Ct value of the enzyme chain reaction is determined, and then further comprising the steps of: A) comparing the PCR ΔCt value corresponding to Septin9 of the sample to be measured with a preset Δcut value of Septin 9 (ie, a critical Ct value), thereby determining based on Septin9 Whether the analysis result of the gene is positive; B) comparing the PCR ΔCt value corresponding to the RNF180 of the sample to be measured with the preset Δcut value of the RNF 180, thereby determining the analysis knot based on the RNF180 gene Whether the result is positive; c) synthesize the results of the steps A) and B) to determine whether the most comprehensive analysis result of the sample is positive.

In summary, the present application utilizes the above-described compositions, nucleic acid sequences, kits and uses thereof, and the above detection methods, by jointly utilizing nucleic acid sequences for detecting methylation of the Septin9 and RNF180 genes and fragments thereof, respectively. The sensitivity and specificity of cancer detection, especially the sensitivity and specificity of gastric cancer detection, are improved, thereby ensuring the correctness and reliability of the detection results.

Specific embodiments will be described in detail below.

Example

Example 1: Primer and probe screening

Because according to certain embodiments, since the kit for measuring the methylation status of at least one CpG dinucleotide of the gene DNA sequence of Septin9 is purchased from Epigenomics (Germany), it can be directly according to the instructions of the kit. Primers and probes for the genetic test of Septin9 were obtained. For the RNF180 gene, many sets of probes and primer combinations can be designed, and the performance of each set of probes and primer combinations may differ. Therefore, the probes and primers were screened in the following examples.

In this example, primers and probes for RNF180 were first screened using an artificial methylation template and an unmethylated template. This embodiment includes the following steps:

First, various primers and probes of RNF180 are designed so long as they hybridize to, complement to, or hybridize to at least 15 nucleotides selected from SEQ ID No: 10 to SEQ ID No: 12 under moderately stringent or stringent conditions. And its complementary sequence.

PCR amplification was then performed using artificially methylated oligonucleotide sequences and artificial unmethylated oligonucleotide sequences as templates, using different probes and primer combinations. Among them, the PCR amplification conditions employed in this experimental example were: real-time PCR was performed on a Life Technologies instrument (7500 Fast). The PCR reaction mixture consisted of bisulfite converted DNA template 35 ng and 450 nM primers, 225 nM probe, 1 UTaq polymerase, 200 um of each dNTP, 4.5 mM MgCl 2 and 2X PCR buffer to a final volume of 30 ul. The sample was amplified by pre-circulation at 94 ° C for 20 minutes, followed by 45 cycles of annealing at 62 ° C for 5 seconds, annealing at 55.5 ° C for 35 seconds, and denaturation at 93 ° C for 30 seconds.

Finally, the following four sets of suitable primers and probes were screened by PCR test results:

Primer and probe combination 1 (66 bp amplicon, -43 bp to +23 bp at the start of transcription)

SEQ ID No 1 (180F7) 5'-GTTCGAGGTCGCGGGGTC-3'

SEQ ID No 2 (180P7) 5'-CAL Fluor

Red-AACGCTCGAACTATACCTACAACCCC-BHQ2-3' SEQ ID No 3

(180R7) 5'-ACAAAAACCAAACCCCGCG-3'

Primer and probe combination 2 (amplifier 86bp, +5bp to +91bp of transcription start site)

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 5 (180P2) 5'-CAL Fluor

Red-CCGACGACGACGATACCG-BHQ2-3'

SEQ ID No 6(180R2) 5'-ACAACCAAACTCTAAAAACTCG-3'

Primer and probe combination 3 (amplicon 130 bp, +5 bp to +135 bp at the start of transcription)

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 7 (180P14) 5'-CAL Fluor

Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3'

SEQ ID No 8 (180R135)

5'-AAAACCTCCAACTTCACACCC-3’ Primer

SEQ ID No 4 (180F24) 5'-GCGGGGTTTGGTTTTTGT-3'

SEQ ID No 7 (180P14) 5'-CAL Fluor

Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3'

SEQ ID No 9(180R14) 5'-CGCCAACAACCAAACTCTAA -3'

Conclusion: The artificial methylated oligonucleotide template was amplified, and the artificial unmethylated oligonucleotide template was not amplified, indicating that the primer and probe design were correct. Four sets of primers and probes can distinguish between methylated and unmethylated templates, and can be used as primers and probes in the RNF180 assay. Although the effects of the different probes and primer combinations are different, the above four sets of probes are suitable for use as primers and probes in the RNF180 assay.

Next, primers and probes for RNF180 were further screened using cancer and normal DNA as templates.

Samples of 4 cases of gastric cancer (S1-S4), 2 cases of colorectal cancer (C1-C2), 2 cases of lung cancer (L1-L2), 1 case of blood cancer (Jurkat) and 1 case of normal (WBC) were obtained. Genomic DNA was extracted from 4 cases of gastric cancer, 2 cases of intestinal cancer, 2 cases of lung cancer, 1 case of blood cancer and 1 normal person. Jurkat cell genomic DNA was used as a positive control and normal DNA was used as a negative control. All cancer samples were obtained from Borcheng. The normal human sample was obtained from BioReclamation IVT. The extraction of the DNA can be carried out by any standard means in the prior art. Specifically, in the present example, all human sample DNA was extracted by using the EPi proColon Plasma Quick Kit of Epigenomics.

The genomic DNA sample is then pretreated such that the cytosine base unmethylated at the 5' position is converted to uracil, thymine or another base that is not used for cytosine in hybridization behavior. In this example, the pretreatment is achieved by treatment with a bisulfite reagent. Modification of bisulfite DNA was carried out by using the EPi proColon Plasma Quick Kit.

Next, the pre-treated 4 cases of gastric cancer, 2 cases of intestinal cancer, 2 cases of lung cancer, 1 case of blood cancer, and 1 normal human genomic DNA sample were added to the four sets of RNF180 primers and probe sets, and 4 sets of RNF 180 were performed. The RNF180 PCR assay and the Septin9 PCR assay were tested in multiple assays by PCR and by adding primers and probes from Septin9. Septin9 PCR reagent was purchased from Epigenomics. Real-time PCR was performed on bisulfite converted DNA.

Among them, the PCR amplification conditions employed in this experimental example were: real-time PCR was performed on a Life Technologies instrument (7500 Fast). The PCR reaction mixture consisted of bisulfite converted DNA template 35 ng and 450 nM primers, 225 nM probe, 1 UTaq polymerase, 200 um of each dNTP, 4.5 mM MgCl 2 and 2X PCR buffer to a final volume of 30 ul. The sample was amplified by pre-circulation at 94 ° C for 20 minutes, followed by 45 cycles of annealing at 62 ° C for 5 seconds, annealing at 55.5 ° C for 35 seconds, and denaturation at 93 ° C for 30 seconds.

Finally, the Ct values of real-time PCR of RNF180 gene in 4 cases of gastric cancer, 2 cases of colorectal cancer, 2 cases of lung cancer, 1 case of blood cancer and 1 normal person were measured, and 4 cases of gastric cancer and 2 cases of intestinal cancer were detected. The Ct values of real-time PCR of the Setptin9 gene in genomic DNA samples of 2 lung cancers, 1 blood cancer and 1 normal human (as shown in Figure 1). Figure 1 shows that in the methylation DNA multiplex assay of Septin9 and RNF180, the RNF180 four sets of primers and probes do not affect the determination of Septin9, and the abscissa SC_set1-4 represents the primers and probe sets 1-4, and the column strips of each group From left to right, it represents S1, S2, S3, S4, C1, C2, L1, L2, Jurkat and WBC, and the ordinate is the Ct value of Septin9, where S1-S4 represents 4 cases of gastric cancer, and C1-C2 represents 2 cases of colon. For cancer, L1-L2 represents 2 cases of lung cancer, Jurkat means 1 case of blood cancer (positive control), and WBC means 1 normal person (negative control).

It can be seen from the Ct value of real-time PCR of the RNF180 gene that RNF180 is highly amplified in cancer DNA and low in normal DNA. The specificity of the RNF180 primer and probe distinguishes between cancer DNA and normal DNA. RNF180 has different primer and probe combinations and the effect is different. The DNA of blood cancer Jurkat cells has a very high amplification of RNF180.

As can be seen from the Ct value of the real-time PCR of the Setptin9 gene, as shown in Figure 1, the RNF180 primers and probes had no effect on the detection of Septin9 in the multivariate assay. Primin9 primers and probes distinguish between cancer DNA and normal DNA, independent of RNF180. The different primer and probe combinations of RNF180 did not affect the determination of Septin9. The DNA of blood cancer Jurkat cells has a very high amplification of RNF180, but no amplification of Septin9. RNF180 and Septin9 have commonality in methylation in different cancers, but they also have personality.

According to the above examples, the four sets of probes and primers of the RNF180 designed by the present application are capable of distinguishing between cancer DNA and normal DNA. In the following examples, in vitro tests and clinical trials were further carried out using the probes and primers described above.

Example 2: In vitro assay of RNF180 and Septin9 on cancer and healthy human leukocyte DNA.

Samples of 4 cases of gastric cancer (S1-S4), 2 cases of intestinal cancer (C1-C2), 2 cases of lung cancer (L1-L2), 1 case of blood cancer (Jurkat) and 7 cases of normal (WBC1-7) were obtained. Genomic DNA was extracted from 4 cases of gastric cancer, 2 cases of intestinal cancer, 2 cases of lung cancer, 1 case of blood cancer and 7 cases of normal people. All cancer samples were obtained from Borcheng. The normal human sample was obtained from BioReclamation IVT. The extraction of the DNA can be carried out by any standard means in the prior art. Specifically, in the present example, all human sample DNA was extracted by using the EPi proColon Plasma Quick Kit of Epigenomics.

Then, the above-mentioned group 2 RNF180 primers and probes were added to the pre-treated 4 cases of gastric cancer, 2 cases of intestinal cancer, 2 cases of lung cancer, 1 case of blood cancer and 7 normal human genomic DNA samples for RNF 180 PCR test. , and the primers and probes of Septin9 were added to quantitatively detect the RNF180 PCR assay and the Septin9 PCR assay. Septin9 PCR reagent was purchased from Epigenomics. Real-time PCR was performed on bisulfite converted DNA.

Among them, the PCR amplification conditions adopted in this experimental example are: in Life Technologies Instruments Real-time PCR was performed on (7500 Fast). The PCR reaction mixture consisted of bisulfite converted DNA template 35 ng and 450 nM primers, 225 nM probe, 1 UTaq polymerase, 200 um of each dNTP, 4.5 mM MgCl 2 and 2X PCR buffer to a final volume of 30 ul. The sample was amplified by pre-circulation at 94 ° C for 20 minutes, followed by 45 cycles of annealing at 62 ° C for 5 seconds, annealing at 55.5 ° C for 35 seconds, and denaturation at 93 ° C for 30 seconds.

Finally, the Ct values of real-time PCR of RNF180 gene in 4 cases of gastric cancer, 2 cases of intestinal cancer, 2 cases of lung cancer, 1 case of blood cancer and 7 normal persons were measured, and 4 cases of gastric cancer and 2 cases of intestinal cancer were detected. The Ct values of the real-time PCR of the Setptin9 gene in the genomic DNA samples of 2 lung cancers, 1 blood cancer, and 7 normal humans are shown in Table 1-2 below.

Table 1: DNA samples of 4 cases of gastric cancer (S1-S4), 2 cases of colorectal cancer (C1-C2), 2 cases of lung cancer (L1-L2), and 1 case of blood cancer (Jurkat) for real-time PCR of RNF180 gene and Setptin9 gene Ct value

Table 2: Ct values of real-time PCR of RNF180 gene and Setptin9 gene in 7 normal human (WBC) DNA samples

Both Septin9 and RNF180 have a Ct value of 45 as the critical value (Cut), more than 45 normal, and less than 45 is cancer positive. "/" indicates that the Ct value exceeds 45.

The methylation DNA of Septin9 and RNF180 was used to detect cancer tissue DNA and healthy human leukocyte DNA. The results were as follows: four cases of gastric cancer were positive, and the sensitivity of RNF180 and Septin9 detection was 100%; both cases were positive for colon cancer, RNF180 and The sensitivity of Septin9 detection is 100%; one case of lung cancer RNF180 is positive, one case of lung cancer is RNF180 negative, the sensitivity of RNF180 detection is 50%, the sensitivity of Septin9 detection is 100%; one case of blood cancer is positive, the sensitivity of RNF180 detection is 100%, Septin9 detection The sensitivity was 0%; seven normal humans were negative for RNF180, specificity was 100%, and the specificity of Septin9 was 86%.

Example 3: Septin9 and RNF180 methylation DNA multivariate detection of plasma in patients with gastric cancer Step clinical outcome

Samples of 10 gastric cancer patients and 11 healthy individuals were obtained. Genomic DNA was extracted from 10 gastric cancer patients and 11 healthy individuals. All cancer samples were obtained from Borcheng. The normal human sample was obtained from BioReclamation IVT. The extraction of the DNA can be carried out by any standard means in the prior art. Specifically, in the present example, all of the sample DNA was extracted by using the EPi proColon Plasma Quick Kit of Epigenomics.

Then, the genomic DNA samples of the above-mentioned pretreated 10 gastric cancer patients and 11 healthy humans were added with the primers and probes of the second group RNF180, and the RNF 180 PCR test was carried out, and the primers and probes of Septin 9 were added, and the primers and probes were added. The RNF180 PCR assay and the Septin9 PCR assay were tested. Septin9 PCR reagent was purchased from Epigenomics. Real-time PCR was performed on bisulfite converted DNA.

Finally, the Ct values of real-time PCR of RNF180 gene in genomic DNA samples of 10 gastric cancer patients and 11 healthy individuals were measured, and the Ct of real-time PCR of Setptin9 gene in genomic DNA samples of 10 gastric cancer patients and 11 healthy humans. Values are shown in Table 3-4 below.

Table 3: Methylation DNA multivariate detection of Septin9 and RNF180 in 10 patients with gastric cancer

癌症样本Cancer sample	Ct RNF180Ct RNF180	Ct Septin9Ct Septin9	关联Association		诊断diagnosis
癌症样本Cancer sample	Ct RNF180Ct RNF180	Ct Septin9Ct Septin9	关联Association		诊断diagnosis	6/亚/男/636/Asia/Men/63	//	38.1938.19	互补Complementary	阳性Positive
35/亚/男/5835/Asia/Men/58	37.6237.62	//	互补 Complementary		阳性Positive	6/亚/男/636/Asia/Men/63	//	38.1938.19	互补Complementary	阳性Positive
35/亚/男/5835/Asia/Men/58	37.6237.62	//	互补 Complementary		阳性Positive	36/亚/女/5336/Asia/Female/53	34.4634.46	38.3438.34		阳性Positive
39/亚/女/5839/Asia/Female/58	31.8431.84	34.5134.51		阳性Positive		36/亚/女/5336/Asia/Female/53	34.4634.46	38.3438.34		阳性Positive
39/亚/女/5839/Asia/Female/58	31.8431.84	34.5134.51		阳性Positive	41/亚/男/5341/Asia/Men/53	33.2833.28	38.6738.67		阳性Positive
42/亚/女/5842/Asia/Female/58	36.9636.96	//	互补 Complementary		41/亚/男/5341/Asia/Men/53	33.2833.28	38.6738.67		阳性Positive	阳性Positive
42/亚/女/5842/Asia/Female/58	36.9636.96	//	互补 Complementary		43/亚/女/5643/Asia/Female/56	32.9032.90	36.0336.03		阳性Positive	阳性Positive

59/亚/男/5759/Asia/Men/57	34.5634.56	38.0038.00		阳性Positive
59/亚/男/5759/Asia/Men/57	34.5634.56	38.0038.00		阳性Positive	61/亚/男/6961/Asia/Men/69	34.7134.71	//	互补 Complementary		阳性Positive
68/亚/女/4968/Asian/Female/49	35.1535.15	No CtNo Ct	互补Complementary	阳性Positive	61/亚/男/6961/Asia/Men/69	34.7134.71	//	互补 Complementary		阳性Positive
68/亚/女/4968/Asian/Female/49	35.1535.15	No CtNo Ct	互补Complementary	阳性Positive	阳性率Positive rate	90％90%	70％70%		阳性Positive

Table 4: Methylation DNA multivariate detection of Septin9 and RNF180 in 6 healthy individuals

健康人血浆Healthy human plasma	Ct RNF180Ct RNF180	Ct Septin9Ct Septin9
健康人血浆Healthy human plasma	Ct RNF180Ct RNF180	Ct Septin9Ct Septin9	H1/亚/男/30H1/Asia/Men/30	//	//
H2/亚/女/27H2/Asia/Female/27	36.4536.45	//	H1/亚/男/30H1/Asia/Men/30	//	//
H2/亚/女/27H2/Asia/Female/27	36.4536.45	//	H3/亚/男/24H3/Asia/Men/24	//	//
H4/亚/女/45H4/Asia/Female/45	//	//	H3/亚/男/24H3/Asia/Men/24	//	//
H4/亚/女/45H4/Asia/Female/45	//	//	H5/亚/男/35H5/Asia/Men/35	//	//
H6/亚/男女/32H6/Asian/Men and Women/32	//	//	H5/亚/男/35H5/Asia/Men/35	//	//
H6/亚/男女/32H6/Asian/Men and Women/32	//	//	特异性Specificity	83％83%	100％100%

Table 5: Methylation DNA multivariate detection of Septin9 and RNF180 5 healthy people

Both RC1 and RC2 are references. The Ct values of PCR for RNF180 and Septin9 are all critical at 45, more than 45 normal, and less than 45 are cancer positive. "/" indicates that the Ct value exceeds 45.

In the methylation DNA multiplex assay of Septin9 and RNF180, Ct values below 45 were positive for gastric cancer, and combined with the positive results of methylated DNA detection of Septin9 and RNF180, and the positive results were complementary. The diagnosis results of 10 patients with gastric cancer were positive. The sensitivity of gastric cancer reaches 100%. Of the 11 healthy human negatives, 10 were negative and the specificity was 91%.

The Ct values of PCR of RNF180 and Septin9 of 10 gastric cancer patients in Table 3 are represented in the form of a histogram, as shown in FIG. The abscissa of Fig. 2 is 10 cases of gastric cancer, and the ordinate is the Ct value of Septin9 and RNF180.

The positive result of methylation DNA detection by Septin9 and RNF180 is that the positive results of methylation DNA detection of Septin9 and RNF180 are complementary, and the sensitivity of gastric cancer reaches 100%. The positive results of Septin9 and RNF180 are complementary in the diagnosis of gastric cancer.

Example 4: Detection of methylated DNA polymorphism (RS19) of Septin9 and RNF180 is normal, The sensitivity and specificity of gastric cancer.

Samples of 15 normal subjects and 74 gastric cancer patients were obtained. Genomic DNA in each sample was extracted. The extraction of the DNA can be carried out by any standard means in the prior art. Specifically, in the present example, all of the sample DNA was extracted by using the EPi proColon Plasma Quick Kit of Epigenomics.

Then, the primers and probes of the second group RNF180 were added to the genomic DNA samples of the pretreated 15 normal humans and 74 gastric cancer patients, and the RNF 180 PCR test was carried out, and the primers of Septin9 and β-actin were added. And probes, multivariate detection of RNF180 PCR test, Septin9 PCR assay and beta-actin PCR assay. Septin9 PCR reagent and beta-actin PCR reagent were purchased from Epigenomics. Real-time PCR was performed on bisulfite converted DNA.

Finally, the Ct values of real-time PCR of the Setptin9 gene were measured in genomic DNA samples from 15 normal subjects and 74 gastric cancer patients. The Ct values of real-time PCR of β-actin in genomic DNA samples of 15 normal subjects and 74 gastric cancer patients were measured. The Ct values of real-time PCR of RNF180 gene were detected in genomic DNA samples of 15 normal subjects and 74 gastric cancer patients. Actin Ct was used as an internal control. RNF180 Ct minus β-actin Ct gave the dCT value of RNF180, as shown in Figure 3.

Figure 3 also shows the sensitivity and specificity of the detection of gastric cancer with the ROC curve and the critical values of 10 and 15, respectively. When the cut-off value is 10, the sensitivity of detecting gastric cancer is 74%, and the specificity is 87%. When the cut-off value is 15, the sensitivity of detecting gastric cancer is 82%, and the specificity is 73%. Thus the dCut value, or Cut value, can vary depending on the sensitivity and specificity chosen. Considering the actual demand for sensitivity and specificity, it is preferable to have a dCut value of 10.

Example 5: Combination of Septin9 and RNF180 improves the specificity and sensitivity of gastric cancer detection

Samples were obtained from 15 normal subjects, 37 gastritis, and 74 gastric cancer patients. Genomic DNA in each sample was extracted. The extraction of the DNA can be carried out by any standard means in the prior art. Specifically, in the present example, all of the sample DNA was extracted by using the EPi proColon Plasma Quick Kit of Epigenomics.

Then, the above-mentioned pretreated genomic DNA sample is added with the primers and probes of the above second group RNF180, and the RNF 180 PCR test is performed, and Septin9 and β-actin are added. Primers and probes were used to detect RNF180 PCR assay, Septin9 PCR assay and β-actin PCR assay. Septin9 PCR reagent and beta-actin PCR reagent were purchased from Epigenomics. Real-time PCR was performed on bisulfite converted DNA.

Finally, the Ct values of real-time PCR of the Setptin9 gene in genomic DNA samples from 15 normal subjects, 37 gastritis and 74 gastric cancer patients were measured. The Ct value of the above DNA sample for real-time PCR of β-actin was measured. And the Ct value of the above-mentioned DNA sample for real-time PCR of the RNF180 gene was measured. Actin Ct was used as an internal control. The RCT180 Ct value was subtracted from β-actin Ct to obtain the dCT value of RNF180, and the Setptin9 Ct value was subtracted from the β-actin Ct value to obtain the dCT value of RNF180.

With the increase of age, the content of methylated RNF180 gene in peripheral blood increased, with age as the abscissa and dCT of RNF180 as the ordinate to obtain Figure 4A. Because older elders have more or less a certain degree of gastritis, it is difficult to distinguish whether it is an age factor or a gastritis factor that causes an increase in the methylation of RNF180 in peripheral blood. This has a great impact on the specificity and reliability of gastric cancer detection. As shown in Fig. 4A, in the senile stage, chronic gastritis (diamond) and gastric cancer (square) partially cross, affecting the detection of gastric cancer.

The dCT value of Setptin9 is plotted on the abscissa and the dCT value of RNF180 is plotted on the ordinate. As shown in FIG. 4B, when the critical value of dCT of Septin9 is selected to be 14, and the critical value of dCT of RNF180 is 10, the point in the left box represents the detection result of Septin9, and the detection result of RNF180 is also positive. The box on the right indicates that Septin9 is negative, and RNF180 is positive. For this example, the selection of Septin9 and RNF180 was positive at the same time, which improved the specificity of some gastric cancers. About 39% (29/74) of gastric cancers reached 90% coincidence rate (29/32), which effectively eliminated gastritis and The negative impact of age on RNF180. Therefore, combining the two biomarkers of Septin9 and RNF180 can reduce the negative impact of RNF180 non-specific high (age, gastritis) on the diagnosis of gastric cancer, improve the specificity of gastric cancer detection, and increase the sensitivity of gastric cancer detection.

Also, in terms of sensitivity, two cases of gastric cancer RNF180 were negative, but Septin9 was positive, and Septin9 was increased by 2.7% (2/74). Therefore, combining the two biomarkers of Septin9 and RNF180 can improve the specificity and sensitivity of gastric cancer detection.

In summary, with the increase of age, the content of methylated RNF180 gene in peripheral blood increased, but because older people have more or less a certain degree of gastritis, it is difficult to distinguish the age factor. It is also a factor of gastritis leading to an increase in the content of methylated RNF180 gene in peripheral blood. This has a great impact on the specificity and reliability of gastric cancer detection. The factors affecting the Septin9 gene affected by age and chronic gastritis are negligible, so Septin9 can be used to confirm RNF180 detection of gastric cancer.

In summary, in the present application, the sensitivity and specificity of cancer detection, especially the sensitivity and specificity of gastric cancer detection, are obtained by jointly utilizing nucleic acid sequences for detecting methylation of Septin9 and RNF180 genes and fragments thereof, respectively. The improvement is made to ensure the correctness and reliability of the test results. In particular, according to certain embodiments, some gastric cancers are negative for RNF180, while Septin9 is positive, and this portion of gastric cancer can be detected with Septin9, which can increase sensitivity by about 3%. According to other specific embodiments, the age and chronic gastritis can cause a non-specific increase in the content of methylation of RNF180 in peripheral blood, which has a great influence on the specificity and reliability of gastric cancer detection. The factors affecting the Septin9 gene affected by age and chronic gastritis are negligible, so Septin9 can be used to confirm RNF180 detection of gastric cancer. About 40% of gastric cancers are positive for both Septin9 and RNF180. The positive criteria for both are positive, and 90% specificity can be achieved.

All published literature and patent applications mentioned in the specification are indicative of the level of those skilled in the art. All publications and patent applications are hereby incorporated by reference in their entirety in their entirety in the extent of the disclosure of the disclosure of the disclosure of each of the disclosures of The mere mention of these published documents and patent applications is not to be construed as an adult. They are prior art documents with respect to the present application.

While the various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and not for purposes of limitation. The scope and spirit of the invention is to be determined only by the appended claims.

The terms and phrases used herein, and variations thereof, are to be interpreted as open and not restrictive. In some instances, the appearance of extensible vocabulary and phrases such as "one or more", "at least", "but not limited to", or other similar terms is not to be understood as an example in the absence of such an extended term. Intention or need to indicate a narrowing.

Claims

Use of a composition for the preparation of a kit for detecting a cell proliferative disorder in an individual, wherein the composition comprises a nucleic acid for detecting the degree of methylation in at least one target region of the Septin9 and RNF180 genes and fragments thereof The presence of cell proliferative abnormalities was demonstrated by the results of methylation detection by combining Septin9 and RNF180.
The use according to claim 1, wherein said nucleic acid comprises at least 15 oligonucleotide long fragments of RNF180, wherein said oligonucleotide comprises at least one CpG dinucleotide sequence, and the presence of CpG methylation Indicates the presence of the condition.
The use according to claim 2, wherein the nucleotide long fragment of RNF180 comprises at least 15 complementary to, complementary to, or under moderately stringent or stringent conditions to at least 15 selected from SEQ ID No: 10 to SEQ ID No: 12. Nucleotides and their complementary sequences.
The use according to claim 3, wherein the nucleotide long fragment of RNF180 comprises a sequence that is identical to, complementary to, or hybridized under medium or tight conditions to a sequence selected from the group consisting of SEQ ID No: 1 to SEQ ID No: Its complementary sequence.
The use according to claim 1, wherein the composition further comprises converting a 5-methyl unmethylated cytosine base of the gene into uracil or other base which is detectably different from cytosine in hybridization performance. Reagents.
The use according to claim 5 wherein the reagent is bisulfite.
The use according to any one of claims 1 to 6, wherein the cell proliferative abnormality is cancer.
The use according to claim 7, wherein the cell proliferative abnormality is gastric cancer.
The use according to claim 1, wherein the biological sample of the individual for detection is selected from the group consisting of a cell line, a histological section, a tissue biopsy/paraffin-embedded tissue, body fluid, feces, colonic effluent, urine, plasma, Serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof.
The use according to claim 9, wherein the biological sample of the individual is plasma.