WO2023077482A1

WO2023077482A1 - Combination of mnp markers of mycobacterium tuberculosis, primer pair combination, kit, and uses of combination, primer pair combination and kit

Info

Publication number: WO2023077482A1
Application number: PCT/CN2021/129160
Authority: WO
Inventors: 肖华锋; 高利芬; 陈利红; 李甜甜; 周俊飞; 彭海; 李论; 方治伟; 万人静
Original assignee: 江汉大学
Priority date: 2021-11-06
Filing date: 2021-11-06
Publication date: 2023-05-11

Abstract

Disclosed in the present invention are a combination of MNP markers of mycobacterium tuberculosis, a primer pair combination and kit for detecting the combination of the MNP markers, and the uses of the combination, the primer pair combination and the kit. The combination of the MNP markers comprises markers MNP-1 to MNP-17 on an AL123456 genome, and the specific nucleotide sequences are as shown in SEQ ID NO. 1-SEQ ID NO. 17. The primer pair combination has nucleotide sequences as shown in SEQ ID NO. 18-SEQ ID NO. 51. The combination of the MNP markers can specifically identify mycobacterium tuberculosis and finely distinguish different varieties; and the primers do not interfere with each other.

Description

A MNP marker combination, primer pair combination, kit and application of Mycobacterium tuberculosis

technical field

The embodiment of the present invention relates to the field of biotechnology, in particular to a combination of MNP markers of Mycobacterium tuberculosis, a combination of primer pairs, a kit and applications thereof.

Background technique

Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as Mycobacterium tuberculosis. Mycobacterium tuberculosis can invade susceptible organisms through respiratory tract, digestive tract or skin damage, causing tuberculosis in various tissues and organs. Mycobacterium tuberculosis can be inhaled through droplets or dust containing bacteria, so tuberculosis is more common. Tuberculosis is a worldwide disease and one of the most concerned public health problems. Although in recent years, with the production of vaccines and early vaccination of children, the incidence of tuberculosis has shown a downward trend, but its harm to human health is still very serious. Rapid and accurate detection and reporting of Mycobacterium tuberculosis is one of the important links in the prevention and control of tuberculosis, and is the basis for controlling the incidence and prevalence of tuberculosis.

Mycobacterium tuberculosis is a typical pathogenic microorganism, and it is also a pathogenic microorganism that is often studied in the laboratory. The genetic variation of experimental microorganisms will lead to genetically different strains with the same name in different laboratories or in the same laboratory in different periods, resulting in non-reproducible and non-comparable experimental results. A consensus has been reached on the validation of human Hella cells prior to publication. Therefore, the detection of pathogenic microorganisms must not only be able to detect Mycobacterium tuberculosis sensitively and accurately, but also be able to detect low-frequency mutations in the population, which is a challenge to the existing detection technology.

The classic detection methods of Mycobacterium tuberculosis, including isolation and culture, PCR technology, whole genome and metagenomic sequencing, etc., have one or more problems in terms of time length, operation complexity, detection throughput, accuracy and sensitivity of detection of mutations, cost, etc. limited. The targeted molecular marker detection technology that combines ultra-multiplex PCR amplification and high-throughput sequencing can enrich target microorganisms in samples with low microbial content, avoiding the isolation and culture steps of whole-genome pathogenic bacteria and the disadvantages caused by metagenomic sequencing. A large amount of data waste and background noise have the advantages of less sample requirements, accurate diagnostic results, saving data volume, and detecting low-frequency variations.

The molecular markers detected by existing targeted detection technologies mainly include SNP and SSR markers. SSR markers are recognized as the most polymorphic markers, but their number is small in microorganisms; SNP markers are huge in number, densely distributed, and are dimorphic markers, and the polymorphism of a single SNP marker is not enough to capture potential alleles in microbial populations genetic diversity. Therefore, the development of new molecular markers with high polymorphism and their detection technology has become an urgent technical problem to be solved.

The present invention develops a species-specific novel molecular marker-MNP marker. MNP markers refer to polymorphic markers caused by multiple nucleotides in an upper region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) rich allelic types, with 2 ⁿ allele types on a single MNP marker, higher than SSR and SNP markers; (2) strong species discrimination ability , only a small amount of MNP markers can be used to identify species and their taxonomic levels, and the identification of species is fine. The MNP labeling method based on super multiplex PCR combined with next-generation high-throughput sequencing technology to detect MNP markers has the following advantages: (1) The output is base sequence, without parallel experiments, and a standardized database can be built for sharing; (2) High Efficiency, using sample DNA barcodes, breaking through the limitation of the number of sequencing samples, and can type tens of thousands of MNP markers in hundreds of samples at one time; (3) High sensitivity, using multiplex PCR to detect multiple targets at a time, avoiding single Target amplification failures lead to high false negatives and low sensitivity; (4) high accuracy, using a second-generation high-throughput sequencer to sequence the amplified product hundreds of times.

In view of the above advantages and characteristics, MNP marker and its detection technology MNP marker method can realize the classification and traceability of multi-allelic genotypes in populations, and has application potential in the identification of pathogenic microorganisms, fingerprint database construction, and genetic variation detection. At present, in microorganisms, there is no report on MNP labeling, and there is a lack of corresponding technology. The development, screening and application of MNP markers have a good application basis in plants.

Therefore, there is an urgent need to develop MNP markers and detection primers for identifying Mycobacterium tuberculosis in pathogenic microorganisms. The combination of markers and primers developed in the present invention will be used to formulate national standards for pathogen detection (plan number 20201830-T-469), which will be released by the end of 2021.

Contents of the invention

The purpose of the present invention is to provide a MNP marker combination of Mycobacterium tuberculosis, a primer pair combination, a kit and its application, which can identify and detect mutations of Mycobacterium tuberculosis, and have multi-target, high-throughput, high-sensitivity and precision The typing effect.

In the first aspect of the present invention, a MNP marker combination of Mycobacterium tuberculosis is provided, which refers to the MNP marker combination that is screened on the Mycobacterium tuberculosis genome to distinguish it from other species and have multiple nucleosides within the species. Genomic regions with acid polymorphisms, including 17 markers from MNP-1 to MNP-17 on the Mycobacterium tuberculosis reference sequence Genebank number AL123456.

In the above technical scheme, the nucleotide sequence marked by MNP-1-MNP-17 is specifically shown in NO.1-SEQ ID NO.17.

It is further explained in Table 1 of the specification, wherein the starting and ending positions of the marked MNP markers are determined based on the sequence of AL123456.

In a second aspect of the present invention, there is provided a combination of multiple PCR primer pairs for detecting the MNP marker combination, the multiple PCR primer pair combination includes 17 pairs of primers, and the specific primer pair combination nucleotide sequence is as SEQ As shown in ID NO.18-SEQ ID NO.51, wherein ID NO.18-SEQ ID NO.34 is the upper primer, and ID NO.35-SEQ ID NO.51 is the lower primer.

In the above technical scheme, each MNP-labeled primer includes an upper primer and a lower primer, as shown in Table 1 of the specification.

In a third aspect of the present invention, a detection kit for detecting the MNP marker combination of Mycobacterium tuberculosis is provided, the kit includes the primer pair combination; further, the kit also includes Multiplex PCR Master Mix. As well as the application of the marker combination, primer pair combination and kit in the detection of Mycobacterium tuberculosis for non-diagnostic purposes, and in the preparation of detection products for Mycobacterium tuberculosis.

In the fourth aspect of the present invention, there is provided the MNP marker combination of Mycobacterium tuberculosis or the combination of multiple PCR primers or the detection kit in the identification of Mycobacterium tuberculosis and the construction of DNA fingerprint database , Application in genetic variation detection.

In the above-mentioned application, at first be to obtain the bacterial total DNA of the sample to be tested; Utilize the kit of the present invention to carry out the first round of multiplex PCR amplification to the total DNA and the blank control, and the number of cycles is not higher than 25; After the amplified product is purified, add sample tags and next-generation sequencing adapters based on the second round of PCR amplification; quantify the second-round amplified product after purification; detect multiple strains by combining the second-round amplified product, etc. High-throughput sequencing is carried out after the amounts are mixed; the sequencing results are compared to the reference sequence of Mycobacterium tuberculosis, and the number of detected sequences and genotype data in the total DNA are obtained. According to the number of Mycobacterium tuberculosis sequencing sequences and the number of detected MNP markers obtained in the total DNA and the blank control, data quality control and data analysis are performed on the sequencing data of the total DNA to obtain the number of detected MNP markers , the number of sequencing sequences covering each of the MNP markers and the genotype data of the MNP markers.

When used for the identification of Mycobacterium tuberculosis, according to the number of sequencing sequences of Mycobacterium tuberculosis detected in the sample to be tested and the blank control and the number of detected MNP sites, after quality control, it is determined whether the sample to be tested contains Nucleic acid of Mycobacterium tuberculosis. Wherein, the quality control scheme and determination method is to use the DNA of Mycobacterium tuberculosis with known copy number as the detection sample, evaluate the sensitivity, accuracy and specificity of the kit to detect Mycobacterium tuberculosis, formulate the The quality control scheme and determination method when the kit detects Mycobacterium tuberculosis.

When used for the detection of genetic variation in Mycobacterium tuberculosis, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation between strains includes using the above kit and method to obtain the genotype data of each of the 17 MNP markers of the strains to be compared. Through genotype comparison, analyze whether there are differences in the main genotypes of the strains to be compared on the 17 MNP markers. If there is a variation in at least one main genotype of the MNP marker in the strain to be compared, it is determined that there is a genetic variation between the two. As an alternative, single-plex PCR can also be used to amplify the 17 markers of the strains to be compared, and then perform Sanger sequencing on the amplified products. After obtaining the sequence, compare the genotypes of each MNP marker of the strains to be compared. right. If there are MNP markers with inconsistent main genotypes, there is variation among the strains to be compared. When detecting the genetic variation within the strain, it is judged by statistical model whether the MNP markers in the strain to be tested detect sub-genotypes other than the main genotype. If the test strain has a subgenotype in at least one MNP marker, it is determined that there is genetic variation within the test strain.

When used to construct the DNA fingerprint database of Mycobacterium tuberculosis, the genotype data of the MNP marker of the Mycobacterium tuberculosis identified from the sample is entered into the database file to constitute the DNA fingerprint database of Mycobacterium tuberculosis; When different samples, by comparing with the DNA fingerprint database of Mycobacterium tuberculosis, identify whether the Mycobacterium tuberculosis in the sample and the bacterial strain in the database have the main genotype in the MNP marker (with more than one MNP marker in one MNP marker). The genotype supported by 50% of the sequenced fragments) difference, Mycobacterium tuberculosis with main genotype difference in at least one MNP marker is a new variant type, which is included in the DNA fingerprint database.

When used for Mycobacterium tuberculosis typing, it is to identify the Mycobacterium tuberculosis in the sample to be tested, and obtain the genotype of each MNP site; collect the genome sequence of Mycobacterium tuberculosis published on the Internet and the constructed The Mycobacterium tuberculosis DNA fingerprint database constitutes the Mycobacterium tuberculosis reference sequence library; compare the genotype of Mycobacterium tuberculosis in the sample to be tested with the reference sequence library of Mycobacterium tuberculosis, and screen for genetically consistent or most For close strains, the type of Mycobacterium tuberculosis in the sample to be tested is obtained. According to the comparison result with the reference sequence library, it is identified whether the Mycobacterium tuberculosis in the sample is an existing type or a new variant, so as to realize fine typing of Mycobacterium tuberculosis.

Compared with the prior art, the present invention has the following advantages:

The invention provides an MNP marker combination of Mycobacterium tuberculosis, a primer pair combination, a kit and applications thereof. The provided 17 MNP markers of Mycobacterium tuberculosis and their primer combinations can be used for multiplex PCR amplification, combined with the next-generation sequencing platform to sequence the amplified products, meeting the high-throughput, high-efficiency, The requirements for high accuracy, high sensitivity and culture-free detection meet the requirements for the construction of a standard and shareable fingerprint database for Mycobacterium tuberculosis; meet the requirements for accurate detection of genetic variation between strains of Mycobacterium tuberculosis and the identification of pure Mycobacterium tuberculosis Synthetic and heterozygous needs.

The present invention is the first in the field of Mycobacterium tuberculosis, and there are no relevant literature reports; MNP markers are mainly developed based on reference sequences, and large-scale identification of Mycobacterium tuberculosis from other species can be mined based on the resequencing data of the reported representative races of Mycobacterium tuberculosis , MNP markers with polymorphic and conserved sequences on both sides of Mycobacterium tuberculosis species; MNP marker detection primers suitable for multiplex PCR amplification can be designed through the conserved sequences on both sides of the MNP marker; then according to the test results of the standard, A set of primer combinations and detection methods with the largest polymorphism and high specificity and the best MNP marker compatibility can be screened out, and used for the detection of Mycobacterium tuberculosis, the construction of DNA fingerprints, and the genetic variation within and between strains In detection and other related applications, it provides technical support for scientific research, scientific monitoring and prevention of Mycobacterium tuberculosis.

Description of drawings

Figure 1 is a schematic diagram of MNP marker polymorphism;

Fig. 2 is the flow chart of screening and primer design of Mycobacterium tuberculosis MNP marker;

Fig. 3 is the detection flowchart of MNP mark;

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

It should be noted that, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field of the present invention. Terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the examples of the present invention can be purchased from the market or prepared by existing methods.

Example 1 The screening of Mycobacterium tuberculosis MNP marker combination and the design of multiple PCR amplification primers

S1, Screening of Mycobacterium tuberculosis MNP marker combinations

Based on the genome sequences of 501 different isolates of Mycobacterium tuberculosis published online, 17 MNP markers were obtained through sequence alignment. For species that do not have genomic data on the Internet, the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected markers, the genome sequences of at least 10 isolates are generally used as references.

The 17 MNP markers screened are shown in Table 1:

The MNP markers described in Table 1 and the starting positions of the detection primers on the reference genome

The step S1 specifically includes:

selecting one of the representative races of the Mycobacterium tuberculosis as a reference genome, and comparing the genome sequence with the reference genome to obtain the single nucleotide polymorphism marker of the Mycobacterium tuberculosis;

On the reference genome, use 100-300bp as the window, and perform window translation with 1bp as the step size, and screen to obtain multiple candidate MNP marker regions, wherein the candidate MNP marker regions contain ≥ 2 of the single nucleotides Variation markers, and the single nucleotide polymorphism markers do not exist on the 30bp sequences at both ends. In the region of the candidate polynucleotide polymorphism markers, the regions with a discrimination degree DP≥0.2 are selected as MNP markers; wherein , DP=d/t, t is the comparison logarithm of all races in the region of the candidate polynucleotide polymorphism marker, and d is at least Sample logarithm of the difference between two SNPs.

As an optional implementation, other step lengths can also be used when screening on the reference genome with a window of 100-300 bp. In this embodiment, the step size is 1 bp, which is conducive to comprehensive screening.

S2, the design of multiple PCR amplification primers

The multiple PCR amplification primers labeled with MNP are designed by primer design software. The primer design follows that the primers do not interfere with each other. All primers can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be used in one amplification reaction. normal expansion.

In this embodiment, the primers used to identify the MNP marker are shown in Table 1 and SEQ ID NO.18-SEQ ID NO.51.

S3. Evaluation of detection efficiency of primer combinations

The method for detecting the MNP markers is to use a PCR master mix suitable for multiplex PCR, amplify all MNP markers at one time through multiplex PCR, sequence the amplified products through second-generation high-throughput sequencing, and analyze the sequencing data , evaluating the compatibility of the primer combination based on the detected markers.

Use the purchased Mycobacterium tuberculosis DNA standard with known copy number (article number: BDS-BW-047, Guangzhou Bangdesheng Biotechnology Co., Ltd.) to prepare a mock sample of Mycobacterium tuberculosis, that is, tuberculosis with known copy number The mycobacterium standard was added to human genomic DNA (2ng/reaction) to prepare a template with 1000 copies/reaction, and the primer combination was used to perform repeated detection by the MNP marker detection method. Based on the principles of high species discrimination and species-specific MNP marker screening and high-efficiency amplification and stable amplification primer screening principles, 17 MNP markers and their detection primer pair combinations provided by the present invention were finally screened out.

Performance evaluation and threshold formulation of MNP marker combination and kit for identifying Mycobacterium tuberculosis described in Example 2

In this example, the DNA of Mycobacterium tuberculosis was added into Into human genomic DNA, prepare 1 copy/reaction, 10 copies/reaction, and 100 copies/reaction of M. tuberculosis mock samples. At the same time, an equal volume of sterile water was set as a blank control. In this embodiment, there are 4 samples in total, and 3 repeated libraries are constructed for each sample every day, and the detection is performed continuously for 4 days, that is, 12 sets of sequencing data are obtained for each sample, as shown in Table 2. According to the number of sequencing fragments and the number of markers of Mycobacterium tuberculosis MNP markers detected in the blank control and Mycobacterium tuberculosis mock samples in 12 repeated experiments, the reproducibility, accuracy and sensitivity of the detection method were evaluated, and the quality Thresholds for controlling system contamination and detection of target pathogens.

The detection process of MNP markers is shown in Figure 3.

Sensitivity and stability analysis of MNP marker combinations and kits for identifying Mycobacterium tuberculosis described in Table 2

As shown in Table 2, in the 12 sets of data of 1 copy/reaction, 1-2 MNP sites can be detected, and in the blank control part can also detect 1 site; in the 12 sets of 10 copies/reaction In the group data, at least 7 MNP markers can be stably detected, which is much higher than the number of MNP sites detected in the blank control, indicating that the kit can stably and sensitively detect tuberculosis as low as 10 copies/reaction mycobacteria.

2. Evaluation of the reproducibility and accuracy of MNP markers and kits for detecting Mycobacterium tuberculosis

Based on whether the genotypes of the co-detected markers were reproducible in two replicates, the reproducibility and accuracy of the MNP marker detection method for detecting Mycobacterium tuberculosis were evaluated. Specifically, 12 sets of data of 100 copies of samples were compared in pairs. The results are shown in Table 3. The number of MNP markers with differences in main genotypes is 0; It is the principle of accuracy, the accuracy rate a=1-(1-r)/2=0.5+0.5r, r represents the recurrence rate, that is, the ratio of the number of reproducible markers of the main genotype to the number of common markers. In the reproducibility test of this project, the logarithm of the difference of the main genotype of the MNP marker between different libraries and different database construction batches of each sample is 0, the reproducibility rate r=100%, and the accuracy rate a=100%.

Table 3 Evaluation of reproducibility and accuracy of MNP marker detection methods for Mycobacterium tuberculosis

Based on this, the kit can accurately and sensitively detect Mycobacterium tuberculosis as low as 10 copies/reaction.

3. Threshold determination of Mycobacterium tuberculosis detected by MNP marker detection kit

As shown in Table 3, the sequence aligned to Mycobacterium tuberculosis can be detected in 1 copy/reaction sample, covering at least 1 MNP marker. In some blank controls, the sequence of Mycobacterium tuberculosis was also detected. Due to the extreme sensitivity of the MNP marker detection method, data contamination during the detection process can easily lead to false positives. Therefore, the following quality control plan was formulated in this example.

The quality control plan is as follows:

1) The amount of sequencing data is greater than 5 million bases. The calculation is based on the fact that the number of MNP markers detected in each sample is 17, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 5 million bases, most samples can be guaranteed to cover each marker in one experiment. The number of sequencing fragments reaches 1000 times, ensuring accurate analysis of the base sequence of each MNP marker.

2) Determine whether the pollution is acceptable according to the signal index S of Mycobacterium tuberculosis in the test sample and the noise index P of Mycobacterium tuberculosis in the blank control, wherein:

Blank control noise index P=nc/Nc, wherein nc and Nc respectively represent the number of sequencing fragments of Mycobacterium tuberculosis in the blank control.

The signal index of the test sample S=nt/Nt, wherein nt and Nt respectively represent the number of sequencing fragments of Mycobacterium tuberculosis in the test sample.

3) Calculate the detection rate of MNP markers in the test sample, which refers to the ratio of the number of detected markers to the total number of designed markers.

As shown in Table 4, the mean value of the noise index of Mycobacterium tuberculosis in the blank control is 0.1%, while the mean value of the signal index in the sample of 1 copy is 0.3%, the signal of the sample of 1 copy and the blank control The average value of the noise ratio is 5.8. Therefore, the present invention stipulates that when the signal-to-noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable.

As shown in Table 4, the average value of the signal-to-noise ratio of 10 copies of the sample and the blank control is 52.2, and in the 12 sets of data of 10 copies/reaction, at least 7 MNP markers can be stably detected, accounting for the total markers 41.2%. Therefore, in the case of ensuring accuracy, this standard stipulates that the signal-to-noise ratio threshold of Mycobacterium tuberculosis is 30, that is, when the signal-to-noise ratio of Mycobacterium tuberculosis in the sample is greater than 30, and the marker detection rate is greater than or equal to 40%. When , it is determined that the nucleotide of Mycobacterium tuberculosis has been detected in the sample.

Table 4 The signal-to-noise ratio of Mycobacterium tuberculosis in each of the 12 detections of 4 samples

Therefore, the kit provided by the present invention can sensitively detect 10 copies/reaction Mycobacterium tuberculosis.

Table 33. Specificity evaluation of MNP marker detection kit for detecting Mycobacterium tuberculosis

Artificial Mycobacterium tuberculosis and Acinetobacter spp., Adenovirus, Bacillus anthracis, Bordetella holbachii, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Epstein-Barr virus, Haemophilus influenzae, varicella zoster Viruses, cytomegalovirus, herpes simplex virus, human bocavirus, Klebsiella pneumoniae, Legionella, Moraxella catarrhalis, Pseudomonas aeruginosa, Rickettsia, Staphylococcus aureus, pneumonia Streptococcus and Streptococcus pyogenes DNA are mixed together in equimolar amounts to prepare a mixed template, and sterile water is used as a blank control, and the method provided by the invention is used to detect the mixed template. Three repeated experiments were carried out, and 17 MNP sites of Mycobacterium tuberculosis could be specifically detected in the three repeated experiments, and the signal-to-noise ratios were 743.5, 812.4 and 752.6, respectively. After analysis according to the quality control scheme and the determination threshold, it was determined that the nucleic acid of Mycobacterium tuberculosis was detected, indicating that the MNP marker and the kit have high specificity in detecting target microorganisms in complex templates.

Example 3, Genetic variation detection between the same named Mycobacterium tuberculosis strains

In experimental research, the variation of strains with the same name will lead to incomparable and irreproducible experimental results. A total of 10 attenuated strains of Mycobacterium tuberculosis BCG provided by Huazhong Agricultural University, Wuhan Institute of Virology and Shihezi University were detected using the kit and MNP marker combination detection method, and the samples were named S1-S10 in turn, each The average sequencing coverage of the samples reached 2103 times, and all 17 MNP markers could be detected on average for each strain (Table 5). The fingerprints of 10 strains were compared in pairs, and the result was wrong! Reference source not found. As shown, the MNP fingerprints of the 3 copies of BCG stored in laboratories A and C are consistent, and among the 4 copies of laboratory B, 1 copy (S-5) and the 9 copies of BCG tested together with the same batch are consistent. There were major genotype differences for some markers (Table 6). Such genotype differences may lead to irreproducible experimental results obtained using this sample, affecting the communication and sharing of experimental results. It is recommended to note its fingerprint for the experimental results generated using the S-5 strain.

Table 5 Detection and analysis of the same named strains in different laboratories

Table 6 Detection and analysis of the same named strains in different laboratories

Example 4, Detection of genetic variation within Mycobacterium tuberculosis strains

As a group organism, some individuals within the Mycobacterium tuberculosis group mutate, making the group no longer homozygous and forming a heterogeneous heterozygous group, which affects especially the stability and consistency of the microbial phenotype used in the test. Such variants appear as alleles outside of the marker's major genotype when the population is tested for molecular markers. When the variant individual has not yet accumulated, it only accounts for a very small part of the population, showing a low frequency allele type. Low-frequency allele types are often mixed with technical errors, making them difficult to distinguish with existing techniques. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that multiple errors are less likely to occur simultaneously than one error. The essence of the detection of genetic variation within Mycobacterium tuberculosis strains is to detect whether there is a minor allele other than the main genotype at the MNP site of the population, and to determine the authenticity of the detected minor allele.

The authenticity evaluation of the secondary allelic type in this embodiment is carried out as follows: first, the allelic type with strand preference (the ratio of the number of sequencing sequences covered on the DNA double strand) is excluded according to the following rules: the strand preference is greater than 10 times, or more than 5 times different from the strand preference of the main allele type.

The authenticity of genotypes without strand preference was determined based on the number and ratio of sequenced sequences in Table 7. Table 7 lists the calculations based on the BINOM.INV function under the probability guarantee of α=99.9999%, when e _max (n=1) and e _max (n≥2) are 1.03% and 0.0994%, respectively, in each marker The critical value of the sequence number of the allele type, only when the sequence number of the secondary allele type exceeds the critical value, it is determined as the real secondary allele type. When there are multiple candidate minor alleles, the P value of each candidate allele type is multiple-corrected, and the candidate alleles with FDR<0.5% are judged to be true minor allele types.

The parameters e _max (n=1) and e _max (n≥2) involved in Table 7 refer to the highest ratio of the number of sequencing sequences carrying the wrong alleles of n SNPs to the total number of sequencing sequences of the marker. This embodiment summarizes the error rates of all minor allele types of MNP markers detected in 10 homozygous strains of Mycobacterium tuberculosis, and _emax (n=1) and _emax (n≥2) are 1.03% respectively and 0.0994%.

Table 7 Partial sequencing depth to determine the critical value of the suballelic genotype

According to the above parameters, the nucleotides of S-5 are mixed into S according to the following 8 ratios: In -4 nucleotides, artificial heterozygous samples were prepared, and each sample was detected 3 times, and a total of 24 sequencing data were obtained. Through accurate comparison with the genotypes of MNP markers of S-4 and S-5, the genotype of S-5 was detected in 24 artificial heterozygous samples, and the concentration of S-5 in the artificial heterozygous samples accounted for The ratio was as low as 1/1000, illustrating the applicability of the developed MNP marker assay for the identification of M. tuberculosis to detect individuals with a low proportion of genetic variation within the strain population.

The construction of embodiment 5 Mycobacterium tuberculosis DNA fingerprint database

The DNA of all strains or samples used to construct the Mycobacterium tuberculosis DNA fingerprint database was extracted by conventional CTAB method and commercial kits, and the quality of DNA was detected by agarose gel and ultraviolet spectrophotometer. If the ratio of the absorbance value of the extracted DNA at 260nm to 230nm is greater than 2.0, the ratio of the absorbance value at 260nm to 280nm is between 1.6 and 1.8, the main band of DNA electrophoresis is obvious, and there is no obvious degradation and RNA residue, it means that the genomic DNA has reached Relevant quality requirements, follow-up experiments can be carried out.

Using the kit, the MNP fingerprints of the S1-S10BCG strains were obtained. Compare the fingerprints of 10 strains pairwise, and enter the fingerprints of strains with at least one MNP fingerprint difference into the database file to form the MNP fingerprint database of Mycobacterium tuberculosis; The MNP fingerprints are compared with the established MNP fingerprint database, and the MNP fingerprints of the strains with different main genotypes are entered into the constructed MNP fingerprint database, and the constructed fingerprint database is continuously updated and enriched. In addition, the constructed MNP fingerprint database is based on the gene sequences of the detected strains, so it is compatible with all high-throughput sequencing data and has the characteristics of being completely co-constructed and shared.

Embodiment 6, application in fine typing of Mycobacterium tuberculosis

First construct the reference sequence library of Mycobacterium tuberculosis, which consists of the published genome sequence of Mycobacterium tuberculosis and the constructed DNA fingerprint database of Mycobacterium tuberculosis; use the primer combination and MNP marker site described in Example 2 The detection method is to obtain the MNP fingerprint of Mycobacterium tuberculosis in each sample to be tested; compare the DNA fingerprint of each strain with the constructed reference sequence library, and screen to obtain the strain with the closest genetic distance to the sequence library; If the genotype is 100% identical to the existing strain, it is an existing variant, and if there is a main genotype difference in at least one MNP site, it is a new variant, and the fine typing of Mycobacterium tuberculosis can be achieved.

Such as Table 4 error! Reference source not found. In the genotype analysis results of the 10 strains shown, S-5 and the other 9 strains were different in the main genotypes of 5 MNP markers, and the 10 strains were divided into 2 types. Therefore, the method can achieve the single-base level of resolution of combined mycobacterium genotypes, and can realize fine typing of Mycobacterium tuberculosis in samples.

Finally, it should also be noted that the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, but also Other elements not expressly listed, or inherent to the process, method, article, or apparatus are also included.

Having described preferred embodiments of embodiments of the present invention, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, the appended claims are intended to be construed to cover the preferred embodiment and all changes and modifications which fall within the scope of the embodiments of the present invention.

Apparently, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. In this way, if the modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and equivalent technologies thereof, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

A MNP marker combination of Mycobacterium tuberculosis, characterized in that the MNP marker combination includes 17 markers, and the specific nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.17.
A multiplex PCR primer pair combination for detecting the Mycobacterium tuberculosis MNP marker combination described in claim 1, characterized in that the multiplex PCR primer pair combination includes 17 pairs of primers, and the specific primer pair combination nucleotide sequence is as follows: Shown in SEQ ID NO.18-SEQ ID NO.51.
A detection kit for detecting the MNP marker combination of Mycobacterium tuberculosis according to claim 1, characterized in that the detection kit comprises the primer pair combination according to claim 2.
The detection kit according to claim 3, characterized in that, the detection kit also includes a multiplex PCR master mix.
Application of the Mycobacterium tuberculosis MNP marker combination described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 in the detection of Mycobacterium tuberculosis for non-diagnostic purposes .
The MNP marker combination of mycobacterium tuberculosis described in claim 1 or the primer pair combination described in claim 2 or the detection kit described in any one of claims 3-4 is used in the detection product of preparing mycobacterium tuberculosis Applications.
The MNP marker combination of Mycobacterium tuberculosis described in claim 1 or the primer pair combination described in Claim 2 or the detection kit described in any one of Claims 3-4 are used in the detection of Mycobacterium tuberculosis strains inside and between bacterial strains Applications in Genetic Variation.
Application of the MNP marker combination of Mycobacterium tuberculosis described in claim 1 or the primer pair combination described in Claim 2 or the detection kit described in any one of claims 3-4 in the construction of Mycobacterium tuberculosis database.
The application of the MNP marker combination of Mycobacterium tuberculosis described in claim 1 or the combination of primers described in Claim 2 or the detection kit described in any one of claims 3-4 in the detection of Mycobacterium tuberculosis fine typing .