CN108048531A - A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation - Google Patents

Abstract

The invention discloses a kind of super retardance fluorescence quantifying PCR methods of highly sensitive detection rare mutation.Present invention employs specific primers and probe technique, can realize the quick detection of blood sample point mutation.The advantage of the present invention includes：(1) LNA modifications are added on the basis of conventional ARMS primers or additionally introduce base mismatch at 3 ' last two three, ends, abrupt climatic change sensitivity is neither influenced, in turn ensures the specificity of mutant-specific primers detection single base mutation；(2) introduce with the modification of wild type complete complementary retardance probe, taking into account further reduces wild type background interference on the basis of saltant type amplification efficiency；(3) on the basis of high specific modification retardance probe is introduced, ARMS primers can detect the different bases mutant form in same site simultaneously, only realized so as to fulfill the multiple sites of same gene with several systems and annex detection；(4) high sensitivity；(5) detection speed is fast.

Description

A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation

Technical field

The present invention relates to clinical diagnosis biology fields, and in particular to a kind of highly sensitive detection rare mutation surpasses Block fluorescence quantifying PCR method.

Background technology

As people deepen continuously to tumour generating process, it is now recognized that cancer generation is related with gene mutation, cause Tumour cell uncontrolled proliferation, therefore can tumor screening be carried out according to gene mutation situation, then carry out tumor susceptibility gene Detection, if it find that it is specific abnormal, targetedly suggesting effect can be played.Chemotherapy is that current clinical treatment is pernicious swollen One of most important means of knurl, still, with its extensive utilization clinically, resistance problems become increasingly conspicuous, and tumour cell is normal Can often drug resistance be generated to chemotherapeutics and cause patient for treatment no longer sensitive, ultimately result in chemotherapy failure even palindromia, Wherein EGFR gene T790M mutation account for more than the 60% of clinical drug-resistant NSCLC patient, and K-ras gene appearances and colorectal cancer (CRC) treatment is closely related, and the mCRC patient for carrying Wild-Type K-ras Genes benefits bigger from the treatment of EGFR inhibitor, And the mCRC patient of K-ras gene mutations can not benefit from EGFR inhibitor Combination chemotherapy.

Traditional tumour detects sample acquisition, generally requires by performing the operation or puncturing original position, and wound, Wu Fajin are generated to patient The limitations such as Mobile state monitoring, and new " liquid biopsy " obtains free nucleic acid or cell from blood plasma for the body fluid sample of representative, And then applied to early diagnosis of tumor, the selection of targeted drug and dynamic curative effect monitoring etc., but due to tumour cell or nucleic acid Content in peripheral blood is at a fairly low, thus blood plasma Molecular Detection to the sensitivity requirement of detection technique compared with tissue higher.

There are many method of detection in Gene Mutation at present, including such as DNA direct Sequencings, pyrosequencing (Pyrosequencing), denaturing high-performance liquid chromatography (HDPLC), high-resolution solubility curve technology (HRM), limitation small pieces Segment length polymorphism analysis method (RFLP), ARMS-PCR etc..Wherein DNA direct Sequencings are the goldstandard of abrupt climatic change, but the party Method has the following disadvantages：The sensibility of detection is not high enough, if the content of mutator accounts for less than the 10% of genomic DNA total amount When, then it can't detect the presence of sudden change sample with direct sequencing；And operating process is complicated, detection time is longer, to operator Member's requirement is high；Non- stopped pipe operation is related to the operation after PCR amplification, therefore easily contaminated, and false negative rate is higher, causes result It is undesirable；The interpretation subjectivity of sequencing result is strong；It is limited once to test the sample size of detection, it at most can only be 8-24.Therefore Direct sequencing is difficult to generally carry out in clinic.ARMS-PCR methods can reach 1% sensitivity, for tumor tissues sample It disclosure satisfy that detection demand, but for sampling convenient blood sample, such as the circulating tumor cell in blood plasma or blood, swell Knurl DNA content is often below 1%, ARMS-PCR methods and is not enough to be detected blood, has limited to clinician to targeting medicine The judgement of object curative effect.In addition, the resistance mutation generated during clinical application, pervious method due to sensitivity not enough It can not be detected.Therefore, the gene of mutation is detected from these low content samples, it is necessary to look for a kind of sensitivity high, specific By force, simple and easy to do, the simple mutation detection methods of result judgement.

The content of the invention

The object of the present invention is to provide a kind of super retardance fluorescence quantifying PCR methods of highly sensitive detection rare mutation.

The present invention provides a kind of for detecting the primer combination of probe of specific site in nucleic acid, for following (a1) or (a2)：

(a1) by general sense primer, general reverse primer, logical inspection probe, specific amplified sense primer and retardance probe groups Into；

The general sense primer is general sense primer-I or general sense primer-II；The general reverse primer is General reverse primer-I or general reverse primer-II；The combination of the general sense primer-I and the general reverse primer-I For expanding target sequence；The target sequence is the segment in the nucleic acid and contains the specific site；Draw the general upstream The specific site is not contained in object-I and the general reverse primer-I；The general sense primer-II is by general upstream Primer-I has identical by the substitution of one or several nucleotide and/or missing and/or addition and with general sense primer-I The DNA molecular of function；The general reverse primer-II is the substitution by one or several nucleotide by general reverse primer-I And/or it lacks and/or adds and there is the DNA molecular of identical function with general reverse primer-I；

The logical inspection probe is reversely logical inspection probe or positive logical inspection probe；The reversely logical inspection probe is visited for reversely logical inspection Pin-I or reversely logical inspection probe-II；The reversely logical inspection probe-I with it is a certain beyond specific site described in the target sequence Section reverse complemental；The reversely logical inspection probe-II is by the substitution of one or several nucleotide by reversely logical inspection probe-I And/or it lacks and/or adds and examine DNA molecular of the probe-I with identical function with reversely logical；The forward direction leads to inspection probe The logical inspection probe-I of forward direction or positive logical inspection probe-II；The positive logical inspection probe-I and specific site described in the target sequence A certain section in addition is identical；The positive logical inspection probe-II is that the logical inspection probe-I of forward direction is passed through one or several nucleotide Substitution and/or missing and/or addition and the DNA molecular with positive logical inspection probe-I with identical function；The logical inspection probe Two ends be respectively adopted fluorophor and quenching group modification；

The specific amplified sense primer is specific amplified sense primer-I or specific amplified sense primer-II or special expansion Increase sense primer-III；The specific amplified sense primer-I meets following three condition：With a certain section in the target sequence It is identical with nucleotide after mutation that identical, 3 ' terminal nucleotides correspond to the specific site, 3 ' terminal nucleotides；The specific amplified Sense primer-II is the substitution by one or several nucleotide in addition to the specific site by specific amplification primer-I And/or it lacks and/or adds and there is the DNA molecular of identical function with specific amplified sense primer-I；On the specific amplified Trip primer-III is that 1-5 nucleotide in specific amplified sense primer-I or specific amplified sense primer-II is carried out LNA to repair Adorn obtained DNA molecular；

The retardance probe is retardance probe-I or retardance probe-II or retardance probe-III；The retardance probe-I meets Two following conditions：, some nucleotide pair identical with a certain section in the target sequence answers the specific site and the core Thuja acid is identical with nucleotide before mutation；The retardance probe-II passes through for that will block probe-I in addition to the specific site The substitution of one or several nucleotide and/or missing and/or addition and the DNA molecular with retardance probe-I with identical function； The retardance probe-III is the DNA for modifying the 1-5 nucleotide progress LNA blocked in probe-I or retardance probe-II Molecule；3 ' ends of the retardance probe carry out blocking modification；

(a2) by general sense primer, general reverse primer, logical inspection probe, specific amplified anti-sense primer and retardance probe groups Into；

The general sense primer and general reverse primer are the general sense primer described in (a1)；

The logical inspection probe is the logical inspection probe described in (a1)；

The specific amplified anti-sense primer is specific amplified anti-sense primer-I or specific amplified anti-sense primer-II or special expansion Increase anti-sense primer-III；The specific amplified anti-sense primer-I meets following three condition：With a certain section in the target sequence It is complementary that reverse complemental, 3 ' terminal nucleotides correspond to the specific site, 3 ' terminal nucleotides and nucleotide after mutation；It is described special It is by one or several nucleotide in addition to the specific site by specific amplified anti-sense primer-I to expand anti-sense primer-II Substitution and/or missing and/or addition and the DNA molecular with specific amplified anti-sense primer-I with identical function；It is described special It is to carry out 1-5 nucleotide in specific amplified anti-sense primer-I or specific amplified anti-sense primer-II to expand sense primer-III The DNA molecular that LNA is modified；

The retardance probe is retardance probe-IV or retardance probe-V or retardance probe-VI；The retardance probe-IV is full Sufficient two following conditions：The specific site is answered with a certain section reverse complemental, some nucleotide pair in the target sequence And the nucleotide and nucleotide before mutation are complementary；The retardance probe-V passes through for that will block probe-IV except the specific site The substitution of one or several nucleotide in addition and/or missing and/or addition and there is identical function with retardance probe-IV DNA molecular；The retardance probe-VI is that the 1-5 nucleotide that will block probe-IV or block in probe-V carries out LNA modifications Obtained DNA molecular；3 ' ends of the retardance probe carry out blocking modification；

The specific site is the site that target point mutation occurs in nucleic acid；On the specific site, target point does not occur The Nucleotide designation of mutation is nucleotide before mutation, and the Nucleotide designation of target point mutation occurs as nucleotide after mutation.

It is described to block modification concretely amination, double deoxidation or phosphorylation modification.

The logical inspection probe may be selected 1-5 nucleotide and carry out LNA modifications.

MGB modifications can be used in the logical inspection probe.When being modified using MGB, described logical inspection probe one end uses fluorophor Modification, the other end uses and the quenching group of MGB couplings is modified or the MGB base group modifications of function are quenched using band.

The size of the target sequence is 100-350bp.

The size of the general sense primer and the general reverse primer is 13-25nt.

The size of the logical inspection probe is 12-30nt.

The size of the specific amplified sense primer or the specific amplified anti-sense primer is 12-25nt.

The size of the retardance probe is 12-30nt.

The specific amplified sense primer can introduce the base mismatch different from target sequence from 3 ' 2-3, ends.

The specific amplified anti-sense primer can introduce the base mismatch not complementary with target sequence from 3 ' 2-3, ends.

FAM fluorophors mark specifically can be used in described logical inspection probe one end, and the other end specifically can be used BHQ1 and base is quenched Group's mark.In an embodiment of the present invention, the FAM fluorophors mark is located at 5 ' ends of logical inspection probe, and the BHQ1 is quenched Group mark is located at 3 ' ends of logical inspection probe.

The retardance probe can specifically carry out amido modified.In an embodiment of the present invention, it is described amido modified positioned at resistance 3 ' ends of stagnant probe.

The TM values of the general sense primer and the general reverse primer are 56-66 DEG C.

The TM values of the logical inspection probe are 66-72 DEG C.

The TM values of the retardance probe are 66-72 DEG C.

When the primer combination of probe described in (a1) is used to detect specific site, detection method is as follows：(a) with determined nucleic acid For template, quantitative fluorescent PCR is carried out using the general sense primer, general reverse primer, logical inspection probe；(b) with core to be measured Acid is template, and fluorescent quantitation is carried out using the specific amplified sense primer, general reverse primer, logical inspection probe and retardance probe PCR；(c) if the quantitative fluorescent PCR of step (a) can obtain positive amplification and the quantitative fluorescent PCR of step (b) cannot obtain It is more than or equal to specific site described in 37, determined nucleic acid to positive amplification or ct values for nucleic acid before mutation, if step (a) is glimmering The quantitative fluorescent PCR that Fluorescent Quantitative PCR can obtain positive amplification and step (b) can obtain positive amplification and ct values are less than 37th, specific site described in determined nucleic acid is mutant nucleic acid.

When the primed probe described in (a2) is used to detect specific site, detection method is as follows：(a) using determined nucleic acid as mould Plate carries out quantitative fluorescent PCR using the general sense primer, general reverse primer, logical inspection probe；(b) using determined nucleic acid as Template carries out quantitative fluorescent PCR using the specific amplified anti-sense primer, general sense primer, logical inspection probe and retardance probe； (c) if the quantitative fluorescent PCR of step (a) can obtain positive amplification and the quantitative fluorescent PCR of step (b) cannot obtain sun Property amplification or ct values be more than or equal to 37, specific site described in determined nucleic acid for nucleic acid before being mutated, if the fluorescence of step (a) is determined The quantitative fluorescent PCR that amount PCR can obtain positive amplification and step (b) positive amplification can be obtained and ct values less than 37, treat It is mutant nucleic acid to survey specific site described in nucleic acid.

When nucleic acid corresponds to various mutations nucleic acid before there is a mutation on the specific site, can make The detection of various mutations is realized with a specific amplification primer.

The present invention also protects a kind of method for detecting the specific site in nucleic acid, is method A or method B.

The method A includes the following steps：(a) using determined nucleic acid as template, using the primer combination of probe described in (a1) In general sense primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(b) using determined nucleic acid as template, adopt With specific amplified sense primer, general reverse primer, logical inspection probe and the retardance probe in the primer combination of probe described in (a1) Carry out quantitative fluorescent PCR；(c) if the quantitative fluorescent PCR of step (a) can obtain positive amplification and the fluorescence of step (b) Quantitative PCR cannot obtain positive amplification or ct values are more than or equal to specific site described in 37, determined nucleic acid for nucleic acid before mutation, such as The quantitative fluorescent PCR of fruit step (a) can obtain positive amplification and the quantitative fluorescent PCR of step (b) can obtain positive expansion It is mutant nucleic acid that increasing and ct values, which are less than specific site described in 37, determined nucleic acid,.

The method B expands following steps：(a) using determined nucleic acid as template, using the primer combination of probe described in (a2) In general sense primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(b) using determined nucleic acid as template, adopt With specific amplified anti-sense primer, general sense primer, logical inspection probe and the retardance probe in the primer combination of probe described in (a2) Carry out quantitative fluorescent PCR；(c) if the quantitative fluorescent PCR of step (a) can obtain positive amplification and the fluorescence of step (b) Quantitative PCR cannot obtain positive amplification or ct values are more than or equal to specific site described in 37, determined nucleic acid for nucleic acid before mutation, such as The quantitative fluorescent PCR of fruit step (a) can obtain positive amplification and the quantitative fluorescent PCR of step (b) can obtain positive expansion It is mutant nucleic acid that increasing and ct values, which are less than specific site described in 37, determined nucleic acid,.

In the method A or method B, the specific site is the site that target point mutation occurs in nucleic acid；It is described special On site, the Nucleotide designation of target point mutation does not occur as nucleotide before mutation, the Nucleotide designation of target point mutation occurs For nucleotide after mutation.

In the method A, the PCR reaction systems of the step (a) are concretely：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM, general 0.2 μM of sense primer- 0.5 μM, 0.2 μM -0.5 μM of general reverse primer leads to 0.2 μM -0.5 μM of probe of inspection, 50 × ROX (Invitrogen, article No.： 12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.

In the method A, the PCR response procedures of the step (a) are concretely：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C 30s, 72 DEG C of 30s, 40 Xun Huans.

In the method A, the PCR reaction systems of the step (b) are concretely：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM, 0.2 μM of general reverse primer- 0.5 μM, 0.2 μM -0.5 μM of specific amplified sense primer leads to and examines 0.2 μM -0.5 μM of probe, 0.1 μM -0.5 μM of probe of retardance, and 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.

In the method A, the PCR response procedures of the step (b) are concretely：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C 30s, 72 DEG C of 30s, 40 Xun Huans.Fluorescence signal is detected in reaction process.

In the method B, the PCR reaction systems of the step (a) are concretely：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM, general 0.2 μM of sense primer- 0.5 μM, 0.2 μM -0.5 μM of general reverse primer leads to 0.2 μM -0.5 μM of probe of inspection, 50 × ROX (Invitrogen, article No.： 12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.

In the method B, the PCR response procedures of the step (a) are concretely：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C 30s, 72 DEG C of 30s, 40 Xun Huans.

In the method B, the PCR reaction systems of the step (b) are concretely：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM, general 0.2 μM of sense primer- 0.5 μM, 0.2 μM -0.5 μM of specific amplified anti-sense primer leads to and examines 0.2 μM -0.5 μM of probe, and 0.1 μM of probe of retardance probe retardance - 0.5 μM, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.

In the method B, the PCR response procedures of the step (b) are concretely：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C 30s, 72 DEG C of 30s, 40 Xun Huans.Fluorescence signal is detected in reaction process.

The present invention also protects a kind of primer combination of probe for being used to detect and whether there is EGFR gene T790M mutation in nucleic acid (primer combination of probe first), by general sense primer, general reverse primer, logical inspection probe, specific amplified anti-sense primer and retardance Probe forms；The general sense primer is the single strand dna shown in the sequence 1 of sequence table；The general reverse primer is Single strand dna shown in the sequence 2 of sequence table；The specific amplified anti-sense primer is single-stranded shown in the sequence 3 of sequence table DNA molecular, wherein carrying out LNA modifications from 5 ' the 5th nucleotide in end, the 6th nucleotide and the 7th nucleotide；The logical inspection is visited Pin is the single strand dna shown in the sequence 4 of sequence table, and 5 ' ends are marked using FAM fluorophors, and base is quenched using BHQ1 in 3 ' ends Group's mark；The retardance probe is the single strand dna shown in the sequence 5 of sequence table, wherein holding the 5th nucleotide, 6 from 5 ' Position nucleotide and the 7th nucleotide carry out LNA modifications, and 3 ' ends carry out amido modified.

The present invention also protects a kind of primer combination of probe for being used to detect in nucleic acid with the presence or absence of K-ras gene mutations (to draw Object probe combinations second), by general sense primer, general reverse primer, logical inspection probe, specific amplified sense primer and retardance probe Composition；

The general sense primer is as shown in the sequence 8 of sequence table；

The general reverse primer is as shown in the sequence 9 of sequence table；

The specific amplified sense primer is following (b1) and/or (b2) and/or (b3)：

(b1) single strand dna shown in the sequence 10 of sequence table；

(b2) single strand dna shown in the sequence 11 of sequence table；

(b3) single strand dna shown in the sequence 12 of sequence table；

The logical inspection probe is as shown in the sequence 13 of sequence table, wherein from 5 ' the 7th nucleotide in end, the 8th nucleotide, the 11 nucleotide, the 12nd nucleotide and the 14th nucleotide carry out LNA modifications；The sequence 14 of the retardance probe such as sequence table Shown, wherein carrying out LNA modifications from 5 ' the 7th nucleotide in end, the 9th nucleotide and the 10th nucleotide, 3 ' ends carry out amino Modification.

The present invention also protects a kind of detect with the presence or absence of the method for EGFR gene T790M mutation in nucleic acid, including walking as follows Suddenly：(1) using determined nucleic acid as template, using the general sense primer, general reverse primer, logical described in primer combination of probe first It examines probe and carries out quantitative fluorescent PCR；(2) using determined nucleic acid as template, using the specific amplified described in primer combination of probe first Anti-sense primer, general sense primer, logical inspection probe and retardance probe carry out quantitative fluorescent PCR；(3) if the fluorescence of step (1) The quantitative fluorescent PCR that quantitative PCR can obtain positive amplification and step (2) cannot obtain positive amplification or ct values are more than or equal to 37th, there is no EGFR gene T790M in determined nucleic acid to be mutated, if the quantitative fluorescent PCR of step (1) can obtain positive amplification And the quantitative fluorescent PCR of step (2) can obtain positive amplification and ct values are less than there are EGFR genes in 37, determined nucleic acid T790M is mutated.

The present invention also protection detection nucleic acid in the presence or absence of 2 exon of K-ras genes mutation method, be method first or Method second or method third.

The method first includes the following steps：(1) using determined nucleic acid as template, using in the primer combination of probe second General sense primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, using institute The specific amplified sense primer (b1) in primer combination of probe second, general reverse primer, logical inspection probe and retardance probe is stated to carry out Quantitative fluorescent PCR；(3) if the quantitative fluorescent PCR of step (1) can obtain positive amplification and the fluorescent quantitation of step (2) PCR cannot obtain positive amplification or ct values to dash forward more than or equal to there is no 2 exon G12A of K-ras genes in 37, determined nucleic acid Become and be mutated there is no 2 exon G12V of K-ras genes and 2 exon G12D of K-ras genes is not present and be mutated, if step Suddenly the quantitative fluorescent PCR of (1) can obtain positive amplification and step (2) quantitative fluorescent PCR can obtain positive amplification and Ct values to be dashed forward less than there are 2 exon G12A of K-ras genes mutation or 2 exon G12V of K-ras genes in 37, determined nucleic acid Become or 2 exon G12D of K-ras genes is mutated；

Method second includes the following steps：(1) using determined nucleic acid as template, using general in the primer combination of probe second Sense primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, drawn using described Specific amplified sense primer (b2), general reverse primer, logical inspection probe and retardance probe in object probe combinations second carry out fluorescence Quantitative PCR；(3) if the quantitative fluorescent PCR of step (1) can obtain the quantitative fluorescent PCR of positive amplification and step (2) not It can obtain positive amplification or ct values and be more than or equal in 37, determined nucleic acid to be mutated there is no 2 exon G12S of K-ras genes and not There are 2 exon G12C of K-ras genes mutation and there is no 2 exon G12R of K-ras genes mutation, if step (1) Quantitative fluorescent PCR can obtain the quantitative fluorescent PCR of positive amplification and step (2) and can obtain positive amplification and ct values are small There are 2 exon G12S of K-ras genes mutation or 2 exon G12C of K-ras genes mutation or K- in 37, determined nucleic acid 2 exon G12R of ras genes is mutated；

Method third includes the following steps：(1) using determined nucleic acid as template, using general in the primer combination of probe second Sense primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, drawn using described Specific amplified sense primer (b3), general reverse primer, logical inspection probe and retardance probe in object probe combinations second carry out fluorescence Quantitative PCR；(3) if the quantitative fluorescent PCR of step (1) can obtain the quantitative fluorescent PCR of positive amplification and step (2) not It can obtain positive amplification or ct values to be mutated more than or equal to there is no 2 exon G13D of K-ras genes in 37, determined nucleic acid, such as The quantitative fluorescent PCR of fruit step (1) can obtain positive amplification and the quantitative fluorescent PCR of step (2) can obtain positive expansion Increase and ct values are less than there are 2 exon G13D of K-ras genes mutation in 37, determined nucleic acid.

Present invention employs specific primers and probe technique, can realize the quick inspection of blood sample point mutation It surveys.The advantage of the present invention includes：(1) primed probe design on, added on the basis of conventional ARMS primers LNA modification or Base mismatch is additionally introduced at 3 ' two, ends-three, end, neither influence abrupt climatic change sensitivity is compared with tradition ARMS, and ensure The specificity of mutant-specific primers detection single base mutations；(2) in primed probe design, introduce completely mutual with wild type The modification retardance probe of benefit, taking into account further reduces wild type background interference on the basis of saltant type amplification efficiency；(3) introducing On the basis of high specific modification retardance probe, ARMS primers can detect the different bases mutant form in same site simultaneously Formula, sensitivity do not interfere with each other influence, are only realized so as to fulfill the multiple sites of same gene with several systems and annex detection； (4) high sensitivity, up to 0.01%, the circulating tumor cell being more suitable in blood detects detection sensitivity；(5) detection speed Degree is fast, and detection process only needs can be completed for 120 minutes, takes the half for only needing digital pcr.

Description of the drawings

Fig. 1 is super occlusion detection system primed probe design principle figure.

Fig. 2 is to carry out EGFR wild types using the EGFR gene T790M Fluorescence PCR control tests system of universal primer Plasmid and T790M mutant plasmids PCR amplification graphs.

Fig. 3 is to carry out EGFR wild types using the EGFR gene T790M Fluorescence PCR abrupt climatic changes system of special primer Plasmid and T790M mutant plasmids PCR amplification graphs.

Fig. 4 is to surpass retardance inspection using the EGFR gene T790M Fluorescence PCRs of special primer combination lock nucleic acid retardance probe Survey system carries out EGFR wild plasmids and T790M mutant plasmids PCR amplification graphs.

Fig. 5 surpasses occlusion detection system sensitivity curve figure for EGFR gene T790M Fluorescence PCRs.

Fig. 6 be using universal primer control test system carry out K-ras wild plasmids and G12A, G12V, G12D, G12S, G12C, G12R, G13D mutant plasmids PCR amplification graph.

Fig. 7 be using the abrupt climatic change system of G12AVD special primers carry out K-ras wild plasmids and G12A, G12V, G12D, G12S, G12C, G12R, G13D mutant plasmids PCR amplification graph.

Fig. 8 be using the abrupt climatic change system of G12SCR special primers carry out K-ras wild plasmids and G12A, G12V, G12D, G12S, G12C, G12R, G13D mutant plasmids PCR amplification graph.

Fig. 9 be using the abrupt climatic change system of G13D special primers carry out K-ras wild plasmids and G12A, G12V, G12D, G12S, G12C, G12R, G13D mutant plasmids PCR amplification graph.

Figure 10 is the super occlusion detection system progress K-ras open countries for blocking probe using G12AVD special primer combinations lock nucleic acid Raw type plasmid and G12A, G12V, G12D mutant plasmids PCR amplification graph.

Figure 11 is the super occlusion detection system progress K-ras open countries for blocking probe using G12SCR special primer combinations lock nucleic acid Raw type plasmid and G12R, G12S, G12R mutant plasmids PCR amplification graph.

Figure 12 is wild using the super occlusion detection system progress K-ras of G13D special primer combinations lock nucleic acid retardance probe Type plasmid and G13D mutant plasmids PCR amplification graphs.

Figure 13 is to surpass retardance system using G12AVD to detect G12A mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 14 is to surpass retardance system using G12AVD to detect G12V mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 15 is to surpass retardance system using G12AVD to detect G12D mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 16 is to surpass retardance system using G12SCR to detect G12S mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 17 is to surpass retardance system using G12SCR to detect G12C mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 18 is to surpass retardance system using G12SCR to detect G12R mutant plasmids gradient dilutions sensitivity technique amplification song Line chart.

Figure 19 is to surpass retardance system using G13D to detect G13D mutant plasmids gradient dilution sensitivity technique amplification curves Figure.

Specific embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Average.

The design of embodiment 1, super retardance fluorescence quantification PCR primer probe

For detect the primer combination of probe in gene rare mutation site by upstream universal primer F, downstream universal primer R, Logical inspection type probe P, specific amplification primer AP and lock nucleic acid retardance probe BL compositions.

The upstream universal primer F, downstream universal primer R and logical inspection type probe P sequences derive from the open country of detection gene Raw type and saltant type homologous region；The upstream universal primer F and downstream universal primer R length are 13-25nt, and TM values are 56-66 ℃；The logical inspection type probe P length is 12-30nt, and TM values are 66-72 DEG C；The logical inspection type probe P is modified using LNA or MGB Modification；LNA modification quantity is 1-5,4 kinds of base monomers of ATCG can LNA modifications, 5 ' and 3 ' ends mark glimmering respectively Light group and quenching group；The MGB modifies the quenching group of 5 ' end mark fluorophors, 3 ' end marks and MGB couplings Or band is quenched the MGB groups of function (MGB is a minor groove binders in itself, and itself can be coupled one when synthesis A quencher, it is whole to be connected to 3 ' end of probe again, quenching group can not also be coupled, quenching group is individually first connected to probe 3 ' ends, then meet MGB).

The specific amplification primer AP can be that sense primer (identical with genic mutation type sequence to be detected) or downstream are drawn Object (and genic mutation type sequence to be detected is complementary)；The length of the specific amplification primer AP is 12-25nt, and length is shorter, special The opposite sex is stronger.3 ' the terminal bases of the specific amplification primer AP are designed as site where mutation；The specific amplification primer AP LNA can be contained to modify, the modification quantity containing LNA is 1-5,4 kinds of base monomers of ATCG can LNA modifications, the position of LNA modifications It rests against and is held among nearly primer with 5 '；The specific amplification primer AP can be in additionally 1 mispairing of increase of 2-3 reciprocal of 3 ' ends Base, to increase specificity；When the mutation of different bases occurs for same site, specific amplification primer AP can be designed as and together The different mutagenesis templates specific binding of different bases occurs for one site.

The lock nucleic acid retardance probe BL sequences include cls gene wild type to be checked and saltant type difference site, and and treat Detection gene wild-type sequence exactly matches (identical or reverse complemental)；3 ' the ends of described lock nucleic acid retardance probe BL can be with The method of modifying used is amination, double deoxidation, phosphorylation etc.；The lock nucleic acid blocks probe BL length for 12-30nt, TM values For 66-72 DEG C；The lock nucleic acid retardance probe BL is combined same template strand with forward primer；The lock nucleic acid retardance probe BL's TM values are worth 5-10 DEG C high than forward primer TM；The lock nucleic acid retardance probe BL can contain LNA and modify, and quantity is modified containing LNA For 1-5,4 kinds of base monomers of ATCG can LNA modifications；The difference site base of the lock nucleic acid retardance probe BL is repaiied containing LNA Decorations.

The target sequence optimization length of the primer combination of probe is 100-350bp.

The design principle figure of the primer combination of probe is as shown in Figure 1.

The foundation of embodiment 2, super retardance fluorescent quantitative PCR detection method

1st, the total serum IgE of sample to be tested is extracted, and reverse transcription is cDNA.

2nd, using the cDNA that step 1 obtains as template, control PCR reactions and super retardance PCR reactions are carried out；

Compare PCR reaction systems (by taking 20 μ L systems as an example)：10 × PCR Buffer (Vazyme, article No.：P122-d2)2μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM, general 0.2 μM -0.5 μM of sense primer F, under general 0.2 μM -0.5 μM of primer R is swum, leads to 0.2 μM -0.5 μM of inspection type probe P, 50 × ROX (Invitrogen, article No.：12223- 012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.

Compare PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.

Fluorescence signal is detected in reaction process.

When specific amplification primer is anti-sense primer, surpass retardance PCR reaction systems (by taking 20 μ L systems as an example)：10× PCRBuffer (Vazyme, article No.：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM General 0.2 μM -0.5 μM of sense primer F, 0.2 μM -0.5 μM of specific amplification primer AP lead to P0.2 μM -0.5 μM of inspection type probe, 0.1 μM -0.5 μM of lock nucleic acid retardance probe BL, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, Aqua sterilisa complements to 20 μ L.

When specific amplification primer is sense primer, surpass retardance PCR reaction systems (by taking 20 μ L systems as an example)：10× PCRBuffer (Vazyme, article No.：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl₂1.5mM 0.2 μM -0.5 μM of general reverse primer R, AP0.2 μM -0.5 μM of specific amplification primer lead to P0.2 μM -0.5 μM of inspection type probe, lock 0.1 μM -0.5 μM of nucleic acid retardance probe BL, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, go out Bacterium water complements to 20 μ L.

Super retardance PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.

Fluorescence signal is detected in reaction process.

3rd, judged that determination methods are as follows according to the reaction result of step 2：

Control PCR reaction system amplification curves have apparent S curve, surpass retardance PCR reaction systems without apparent S curve or ct More than or equal to 37, which is judged as wild type；Control PCR reaction system amplification curves have apparent S curve, surpass retardance PCR reaction systems also have apparent S curve and ct values are less than 37, which is judged as saltant type.

The super retardance quantitative fluorescent PCR of embodiment 3, EGFR gene T790M rare mutations

First, detection method

1st, according to the method for embodiment 1 (EGFR gene wild-type target sequence such as sequence table is mutated for EGFR gene T790M Sequence 6 shown in；EGFR gene T790M saltant types target sequence is as shown in the sequence 7 of sequence table) design primed probe, following institute Show：

General sense primer EGFR-T790M-F：5 '-CCTCCAGGAAGCCTACGTGATGG-3 ' (sequence 1)；

General reverse primer EGFR-T790M-R：5 '-CAGTTGAGCAGGTACTGGGAG-3 ' (sequence 2)；

Specific amplified anti-sense primer EGFR-T790M-AP：5 '-AGGG+C+A+TGAGCTGCA-3 ' (sequence 3)；Wherein "+" It is modified for LNA, "+" represents base modification below；

Logical inspection type probe EGFR-T790M-P：5 '-TGAGCTGCACGGTGGAGGTGA-3 ' (sequence 4)；Its middle probe 5 ' It holds and is marked for FAM, 3 ' ends mark for BHQ1；

Block probe EGFR-T790M-BL:5 '-AGGG+C+A+TGAGCTGCG-3 ' (sequence 5)；Wherein "+" is repaiied for LNA Decorations, "+" represent base modification below；The end of probe 3 ' is amido modified.

2nd, the double chain DNA molecule shown in artificial synthesized sequence 6 (EGFR gene wild-type target sequence) and double shown in sequence 7 Ssdna molecule (EGFR genetic mutation type target sequence).

3rd, by the EcoRV restriction enzyme sites of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 6, wild plasmid is obtained Wild plasmid is diluted to 1 × 10 by (sequence verification) using 1 × TE buffer solutions⁷The concentration of copy/mL obtains wild Type standard items.By the EcoRV restriction enzyme sites of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 7, mutant plasmids are obtained Mutant plasmids are used concentration as 1 × 10 by (sequence verification)⁷The wild type standard items dilution of copy/mL, is mutated Type plasmid concentration is 1 × 10³Copy/mL, 1 × 10⁴Copy/mL, 1 × 10⁵Copy/mL, 1 × 10⁶Copy/mL, 1 × 10⁷Copy/ The saltant type standard items of mL, it can be understood as saltant type standard items account for wild type standard items background content be 0.01% respectively, 0.1%th, 1%, 10%, 100%.

4th, it is 1 × 10 that the wild type standard items of step 3 preparation and concentration, which are respectively adopted,⁷The saltant type standard items of copy/mL As template, control PCR reactions and super retardance PCR reactions are carried out according to the method in embodiment 2, while carries out one group of mutation again PCR reacts.

It is mutated PCR reaction systems：10 × PCR Buffer (Vazyme, article No.：P122-d2) 2 μ L, dNTP (Vazyme, goods Number：P031-01) 0.2mM, MgCl₂1.5mM, general 0.2 μM -0.5 μM of sense primer F, AP0.2 μM of specific amplification primer - 0.5 μM, lead to 0.2 μM -0.5 μM of inspection type probe P, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ of template L, aqua sterilisa complement to 20 μ L.

It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.In reaction process Detect FAM fluorescence signals.

Compareing PCR reaction detections, the results are shown in Figure 2.Being mutated PCR reaction detections, the results are shown in Figure 3.Super retardance PCR is anti- Answer testing result as shown in Figure 4.The result shows that in PCR reaction results are compareed, the Ct values of wild type and saltant type are each about 22.In PCR reaction results are mutated, the Ct values of saltant type are identical with the Ct values in control PCR reactions, are also 22；And because Specific amplification primer is used in reaction system, the Ct values of wild type are delayed, and illustrate that specific amplification primer introduces LNA lock nucleic acids After modification, primer length shortens, and specificity significantly increases.In super retardance PCR reaction results, the Ct values of saltant type are with compareing Ct values in PCR reactions are identical, are also 22；And because adding retardance probe in system, wild type does not obtain amplification curve, Illustrate that lock nucleic acid retardance probe adds in, further enhance reaction system specificity, be suitble to detect under high background wild type environment Rare mutation gene.

2nd, sensitivity

The saltant type standard items of the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted as mould Plate carries out super retardance PCR reactions, detection sensitivity according to the method in embodiment 2.

The results are shown in Figure 5.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 21.95；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.23；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 28.61；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 32.42；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.88；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

Embodiment 4, the 12nd codon mutation of 2 exon of K-ras genes, the super retardance fluorescence of the 13rd codon mutation are determined Measure PCR

First, method

1st, dash forward according to the method for embodiment 1 for the 12nd codon mutation of 2 exon of K-ras genes, the 13rd codon (2 exon wild-type target sequence of K-ras genes is as shown in the sequence 15 of sequence table for change；2 exon G12A of K-ras genes Saltant type target sequence is as shown in the sequence 16 of sequence table；2 exon G12V saltant type target sequences of K-ras genes are such as sequence table Shown in sequence 17；2 exon G12D saltant types target sequence of K-ras genes is as shown in the sequence 18 of sequence table；K-ras genes 2 Exon G12S saltant types target sequence is as shown in the sequence 19 of sequence table；2 exon G12C saltant type target sequences of K-ras genes Row are as shown in the sequence 20 of sequence table；21 institute of sequence of 2 exon G12R saltant types target sequence such as sequence table of K-ras genes Show；2 exon G13D saltant types target sequence of K-ras genes is as shown in the sequence 22 of sequence table) design primed probe, following institute Show：

General sense primer K-ras-F：5 '-GCCTGCTGAAAATGACTGAA-3 ' (sequence 8)；

General reverse primer K-ras-R：5 '-CCTGACATACTCCCAAGGAAAG-3 ' (sequence 9)；

For the specific amplified sense primer K-ras-G12AVD-AP in tri- sites of G12A, G12V, G12D：

5 '-AACTTGTGGTAGTTGGAGCTGC-3 ' (sequence 10)；

For the specific amplified sense primer K-ras-G12SCR-AP in tri- sites of G12S, G12C, G12R：

5 '-ATAAACTTGTGGTAGTTGGAGCTC-3 ' (sequence 11)；

For the specific amplified sense primer K-ras-G13D-AP in G13D sites：

5 '-GTGGTAGTTGGAGCTGGAGA-3 ' (sequence 12)；

Logical inspection type probe K-ras-P：5 '-ACTTGA+A+ACC+C+AA+GGTAC-3 ' (sequence 13)；Wherein "+" is LNA Modification, "+" represent base modification below；The end of probe 5 ' marks for FAM, and 3 ' ends mark for BHQ1；

Block probe K-ras-BL:5 '-TGGAGC+TG+G+TGGC-3 ' (sequence 14)；Wherein "+" is modified for LNA, "+" Represent base modification below；The end of probe 3 ' is amido modified.

2nd, the double chain DNA molecule shown in artificial synthesized sequence 15 (K-ras genes wild-type target sequence), shown in sequence 16 Double chain DNA molecule (K-ras genes G12V shown in double chain DNA molecule (K-ras gene G12A saltant types target sequence), sequence 17 Saltant type target sequence), the double chain DNA molecule (K-ras gene G12D saltant types target sequence) shown in sequence 18, shown in sequence 19 Double chain DNA molecule (K-ras genes G12C shown in double chain DNA molecule (K-ras gene G12S saltant types target sequence), sequence 20 Saltant type target sequence), shown in the double chain DNA molecule (K-ras gene G12R saltant types target sequence) shown in sequence 21 and sequence 22 Double chain DNA molecule (K-ras gene G13D saltant types target sequence).

3rd, by the EcoRV restriction enzyme sites of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 15, wild type matter is obtained Grain (sequence verification).By the EcoRV restriction enzyme sites of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 16, obtain G12A mutant plasmids (sequence verification).By the EcoRV enzymes of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 17 Enzyme site obtains G12V mutant plasmids (sequence verification).Double chain DNA molecule shown in sequence 18 is inserted into PUC57 matter The EcoRV restriction enzyme sites of grain, obtain G12D mutant plasmids (sequence verification).By the double chain DNA molecule shown in sequence 19 The EcoRV restriction enzyme sites of PUC57 plasmids are inserted into, obtain G12S mutant plasmids (sequence verification).It will be shown in sequence 20 Double chain DNA molecule is inserted into the EcoRV restriction enzyme sites of PUC57 plasmids, obtains G12C mutant plasmids (sequence verification).By sequence The EcoRV restriction enzyme sites of double chain DNA molecule insertion PUC57 plasmids shown in row 21, obtain G12R mutant plasmids and (have been sequenced Verification).By the EcoRV restriction enzyme sites of the double chain DNA molecule insertion PUC57 plasmids shown in sequence 22, G13D saltant type matter is obtained Grain (sequence verification).

Wild plasmid is diluted to 1 × 10 using 1 × TE buffer solutions⁷The concentration of copy/mL obtains wild type standard Product.By G12A mutant plasmids, G12V mutant plasmids, G12D mutant plasmids, G12C mutant plasmids, G12S saltant type matter Concentration is respectively adopted as 1 × 10 in grain G12R mutant plasmids and G13D mutant plasmids⁷The wild type standard items of copy/mL are dilute It releases, obtains mutant plasmids concentration as 1 × 10³Copy/mL, 1 × 10⁴Copy/mL, 1 × 10⁵Copy/mL, 1 × 10⁶Copy/ mL、1×10⁷The saltant type standard items of copy/mL, it can be understood as saltant type standard items account for wild type standard items background content point It is not 0.01%, 0.1%, 1%, 10%, 100%.

4th, the wild type standard items and concentration obtained respectively using step 3 is 1 × 10⁷Each saltant type standard items of copy/mL For template, control PCR reactions are carried out according to the method in embodiment 2.The results are shown in Figure 6.

5th, the wild type standard items and concentration obtained respectively using step 3 is 1 × 10⁷Each saltant type standard items of copy/mL For template, mutation PCR reactions are carried out.

Detect the mutation PCR reaction systems in tri- sites of G12A, G12V, G12D：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl2 1.5mM, general reverse primer K-ras- R0.2 μM -0.5 μM, 0.2 μM -0.5 μM of specific amplified sense primer K-ras-G12AVD-AP, lead to inspection type probe K-ras-P 0.2 μM -0.5 μM, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L. It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.FAM is detected in reaction process Fluorescence signal.The results are shown in Figure 7.

Detect the mutation PCR reaction systems in tri- sites of G12S, G12C, G12R：10 × PCR Buffer (Vazyme, goods Number：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl2 1.5mM, general reverse primer K-ras- R0.2 μM -0.5 μM, 0.2 μM -0.5 μM of specific amplified sense primer K-ras-G12SCR-AP, lead to inspection type probe K-ras-P 0.2 μM -0.5 μM, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L. It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.FAM is detected in reaction process Fluorescence signal.The results are shown in Figure 8.

Detect the mutation PCR reaction systems in G13D sites：10 × PCR Buffer (Vazyme, article No.：P122-d2) 2 μ L, DNTP (Vazyme, article No.：P031-01 0.2 μM -0.5 μM of) 0.2mM, MgCl2 1.5mM, general reverse primer K-ras-F, it is special Different 0.2 μM -0.5 μM of amplification sense primer K-ras-G13D-AP leads to 0.2 μM -0.5 μM of inspection type probe K-ras-P, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.FAM fluorescence signals are detected in reaction process.As a result such as Fig. 9 It is shown.

6th, the wild type standard items and concentration obtained respectively using step 3 is 1 × 10⁷The G12A saltant types of copy/mL, G12V Saltant type and G12 modifications standard items are template, carry out super retardance PCR reactions.The results are shown in Figure 10.

Detect the super retardance PCR reaction systems in tri- sites of G12A, G12V, G12D：10 × PCR Buffer (Vazyme, Article No.：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl2 1.5mM, general reverse primer K- Ras-R0.2 μM -0.5 μM, 0.2 μM -0.5 μM of specific amplified sense primer K-ras-G12AVD-AP, lead to inspection type probe K-ras- 0.2 μM -0.5 μM of P blocks K-ras-BL0.1 μM -0.5 μM of probe, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C 30s, 40 Xun Huans.FAM fluorescence signals are detected in reaction process.

7th, the wild type standard items and concentration obtained respectively using step 3 is 1 × 10⁷The G12C saltant types of copy/mL, G12S Saltant type and G12R saltant types standard items are template, carry out super retardance PCR reactions.As a result as shown in figure 11.

Detect the super retardance PCR reaction systems in tri- sites of G12S, G12C, G12R：10 × PCR Buffer (Vazyme, Article No.：P122-d2) 2 μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl2 1.5mM, general reverse primer K- Ras-R0.2 μM -0.5 μM, 0.2 μM -0.5 μM of specific amplified sense primer K-ras-G12SCR-AP, lead to inspection type probe K-ras- 0.2 μM -0.5 μM of P blocks K-ras-BL0.1 μM -0.5 μM of probe, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, aqua sterilisa complement to 20 μ L.It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C 30s, 40 Xun Huans.FAM fluorescence signals are detected in reaction process.

8th, the wild type standard items and concentration obtained respectively using step 3 is 1 × 10⁷The G13D saltant type standards of copy/mL Product are template, carry out super retardance PCR reactions.As a result as shown in figure 12.

Detect the super retardance PCR reaction systems in G13D sites：10 × PCR Buffer (Vazyme, article No.：P122-d2)2μ L, dNTP (Vazyme, article No.：P031-01) 0.2mM, MgCl2 1.5mM, K-ras-R0.2 μM -0.5 μM of general reverse primer, 0.2 μM -0.5 μM of specific amplified sense primer K-ras-G13D-AP leads to 0.2 μM -0.5 μM of inspection type probe K-ras-P, retardance K-ras-BL0.1 μM -0.5 μM of probe, 50 × ROX (Invitrogen, article No.：12223-012) 0.4 μ L, 2 μ L of template, sterilizing Water complements to 20 μ L.It is mutated PCR response procedures：95 DEG C 3 minutes；95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.Reaction FAM fluorescence signals are detected in the process.

Above-mentioned experimental result is as follows：

In PCR reaction systems are compareed, the ct values of wild type and each saltant type are 24.00 ± 0.10.

In the mutation PCR reaction systems in tri- sites of detection G12A, G12V, G12D, the Ct values of G12A, G12V, G12D 24.00 ± 0.10 are may each be about, G12S, G12C, G12R, G13D plasmid Ct values are about 34.00 ± 0.10, and wild type Ct values are about 34.00.In the mutation PCR reaction systems in tri- sites of detection G12S, G12C, G12R, the Ct values of G12S, G12C, G12R are about For 24.00 ± 0.10, G12A, G12V, G12D, G13D plasmid and wild type Ct values are about 36.00 ± 0.10.In detection G13D In the mutation PCR reaction systems of point, the Ct values of G13D are about 24.06, and G12A, G12V, G12D, G12S, G12C, G12R plasmid are equal It does not expand, wild plasmid has weaker amplification, illustrates specific amplification primer enhancing specificity.

In the super retardance PCR reaction systems in tri- sites of detection G12A, G12V, G12D, G12A, G12D, G12V mutation Type plasmid Ct values are respectively 22.01,22.12,23.08, and wild type is without amplification curve.In tri- positions of detection G12S, G12C, G12R In the super retardance PCR reaction systems of point, G12R, G12S, G12C mutant plasmids Ct values are respectively 24.25,27.14,24.88, Wild type is without amplification curve.In the super retardance PCR reaction systems in detection G13D sites, G13D mutant plasmids Ct values are about 24.11, wild type is without amplification curve.Illustrate that lock nucleic acid retardance probe adds in, further enhance reaction system specificity, be suitble to Rare mutation gene is detected under high background wild type environment.

2nd, sensitivity

1st, the G12A saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 13.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 21.89；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.24；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 28.76；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 32.06；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.94；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

2nd, the G12V saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 14.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 22.72；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 24.91；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 28.31；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 31.67；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.82；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

3rd, the G12D saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 15.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 21.66；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 24.93；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 28.34；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 31.72；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.72；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

4th, the G12S saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 16.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 21.49；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.49；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 29.06；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 32.68；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.92；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

5th, the G12C saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 17.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 22.36；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.98；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 28.89；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 33.21；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values about 35.97 (；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

6th, the G12R saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 18.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 22.02；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.29；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 29.16；Saltant type standard Product account for wild type standard items background content when being 0.1%, and Ct values are about 32.65；Saltant type standard items account for the wild type standard items back of the body When scape content is 0.01%, Ct values are about 35.78；Illustrate super retardance PCR high sensitivities, can detect down to 0.01% it is prominent Become.

7th, the G12R saltant types standard items that the wild type standard items prepared in the 3 of step 1 and various concentration are respectively adopted are made For template, super retardance PCR reactions, detection sensitivity are carried out according to the method in step 1.

As a result as shown in figure 19.The result shows that in super occlusion detection system, saltant type standard items account for the wild type standard items back of the body When scape content is 100%, Ct values are about 21.98；Saltant type standard items account for wild type standard items background content when being 10%, Ct values About 25.48；Saltant type standard items account for wild type standard items background content when being 1%, and Ct values are about 29；Saltant type standard items account for When wild type standard items background content is 0.1%, Ct values are about 32.81；Saltant type standard items account for wild type standard items background and contain When amount is 0.01%, Ct values are about 35.86；Illustrate super retardance PCR high sensitivities, the mutation down to 0.01% can be detected.

<110>Suzhou GenePharma Co., Ltd.

<120>A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation

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agggcatgag ctgca 15

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<400> 8

gcctgctgaa aatgactgaa 20

<210> 9

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 9

cctgacatac tcccaaggaa ag 22

<210> 10

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 10

aacttgtggt agttggagct gc 22

<210> 11

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 11

ataaacttgt ggtagttgga gctc 24

<210> 12

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 12

gtggtagttg gagctggaga 20

<210> 13

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 13

acttgaaacc caaggtac 18

<210> 14

<211> 13

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 14

tggagctggt ggc 13

<210> 15

<211> 319

<212> DNA

<213>People（Homo）

<400> 15

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 16

<211> 319

<212> DNA

<213>People（Homo）

<400> 16

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgctggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 17

<211> 319

<212> DNA

<213>People（Homo）

<400> 17

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgttggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 18

<211> 319

<212> DNA

<213>People（Homo）

<400> 18

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgatggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 19

<211> 319

<212> DNA

<213>People（Homo）

<400> 19

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctagtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 20

<211> 319

<212> DNA

<213>People（Homo）

<400> 20

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agcttgtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 21

<211> 319

<212> DNA

<213>People（Homo）

<400> 21

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctcgtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 22

<211> 319

<212> DNA

<213>People（Homo）

<400> 22

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctggtgac gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

Claims (10)

1. it is a kind of for detecting the primer combination of probe of the specific site in nucleic acid, for following (a1) or (a2)：

(a1) it is made of general sense primer, general reverse primer, logical inspection probe, specific amplified sense primer and retardance probe；

The general sense primer is general sense primer-I or general sense primer-II；The general reverse primer is general Anti-sense primer-I or general reverse primer-II；The combination of the general sense primer-I and the general reverse primer-I is used for Expand target sequence；The target sequence is the segment in the nucleic acid and contains the specific site；The general sense primer-I With the specific site is not contained in the general reverse primer-I；The general sense primer-II be by general sense primer- I has identical function by the substitution of one or several nucleotide and/or missing and/or addition and with general sense primer-I DNA molecular；The general reverse primer-II be by general reverse primer-I by one or several nucleotide substitution and/or Missing and/or addition and the DNA molecular with general reverse primer-I with identical function；

The logical inspection probe is reversely logical inspection probe or positive logical inspection probe；The reversely logical inspection probe is reversely logical inspection probe-I Or reversely lead to inspection probe-II；The reversely logical inspection probe-I and a certain section beyond specific site described in the target sequence Reverse complemental；The reversely logical inspection probe-II for will reversely logical inspection probe-I by one or several nucleotide substitution and/or Missing and/or addition and the DNA molecular with reversely logical inspection probe-I with identical function；The positive logical inspection probe is positive logical Examine probe-I or positive logical inspection probe-II；Described in the positive logical inspection probe-I and the target sequence beyond specific site A certain section is identical；The positive logical inspection probe-II is the substitution by positive logical inspection probe-I by one or several nucleotide And/or it lacks and/or adds and lead to DNA molecular of the inspection probe-I with identical function with positive；Two of the logical inspection probe Fluorophor and quenching group modification is respectively adopted in end；

The specific amplified sense primer is on specific amplified sense primer-I or specific amplified sense primer-II or specific amplified Swim primer-III；The specific amplified sense primer-I meets following three condition：With a certain section phase in the target sequence It is identical with nucleotide after mutation that the specific site, 3 ' terminal nucleotides are corresponded to, 3 ' terminal nucleotides；On the specific amplified Swim primer-II be by specific amplification primer-I by the substitution of one or several nucleotide in addition to the specific site and/ Or it lacks and/or adds and there is the DNA molecular of identical function with specific amplified sense primer-I；Draw the specific amplified upstream Object-III is that 1-5 nucleotide in specific amplified sense primer-I or specific amplified sense primer-II is carried out LNA to modify The DNA molecular arrived；

The retardance probe is retardance probe-I or retardance probe-II or retardance probe-III；The retardance probe-I meets as follows Two conditions：, some nucleotide pair identical with a certain section in the target sequence answers the specific site and the nucleotide It is identical with nucleotide before mutation；The retardance probe-II is that will block probe-I by one in addition to the specific site Or several nucleotide substitution and/or missing and/or addition and there is the DNA molecular of identical function with retardance probe-I；It is described Retardance probe-III is modify the 1-5 nucleotide progress LNA for blocking probe-I or blocking in probe-II DNA points Son；3 ' ends of the retardance probe carry out blocking modification；

(a2) it is made of general sense primer, general reverse primer, logical inspection probe, specific amplified anti-sense primer and retardance probe；

The general sense primer and general reverse primer are the general sense primer described in (a1)；

The logical inspection probe is the logical inspection probe described in (a1)；

The specific amplified anti-sense primer is under specific amplified anti-sense primer-I or specific amplified anti-sense primer-II or specific amplified Swim primer-III；The specific amplified anti-sense primer-I meets following three condition：It is reversed with a certain section in the target sequence It is complementary that complementary, 3 ' terminal nucleotides correspond to the specific site, 3 ' terminal nucleotides and nucleotide after mutation；The specific amplified Anti-sense primer-II is taking by one or several nucleotide in addition to the specific site by specific amplified anti-sense primer-I Generation and/or missing and/or addition and the DNA molecular with specific amplified anti-sense primer-I with identical function；The specific amplified Sense primer-III is that 1-5 nucleotide in specific amplified anti-sense primer-I or specific amplified anti-sense primer-II is carried out LNA Modify obtained DNA molecular；

The retardance probe is retardance probe-IV or retardance probe-V or retardance probe-VI；The retardance probe-IV meets such as Lower two conditions：The specific site is answered with a certain section reverse complemental, some nucleotide pair in the target sequence and is somebody's turn to do Nucleotide and nucleotide before mutation are complementary；The retardance probe-V passes through for that will block probe-IV in addition to the specific site One or several nucleotide substitution and/or missing and/or addition and there are DNA points of identical function with retardance probe-IV Son；The retardance probe-VI is that the 1-5 nucleotide for blocking probe-IV or blocking in probe-V is carried out LNA to modify to obtain DNA molecular；3 ' ends of the retardance probe carry out blocking modification；

The specific site is the site that target point mutation occurs in nucleic acid；On the specific site, target point mutation does not occur Nucleotide designation for mutation before nucleotide, occur target point mutation Nucleotide designation for mutation after nucleotide.

2. primer combination of probe as described in claim 1, it is characterised in that：The size of the target sequence is 100-350bp.

3. primer combination of probe as claimed in claim 1 or 2, it is characterised in that：

The size of the general sense primer and the general reverse primer is 13-25nt；

The size of the logical inspection probe is 12-30nt；

The size of the specific amplified sense primer or the specific amplified anti-sense primer is 12-25nt；

The size of the retardance probe is 12-30nt.

4. the primer combination of probe as described in claims 1 to 3 is any, it is characterised in that：The specific amplified sense primer is from 3 ' 2-3 are held to introduce the base mismatch different from target sequence；The specific amplified anti-sense primer holds 2-3 introducings and target from 3 ' The not complementary base mismatch of sequence.

5. the primer combination of probe as described in Claims 1-4 is any, it is characterised in that：Described logical inspection probe one end is glimmering using FAM Light group marks, and the other end is marked using BHQ1 quenching groups；The retardance probe carries out amido modified.

6. a kind of method for detecting the specific site in nucleic acid is method A or method B；

The method A includes the following steps：(a) using determined nucleic acid as template, drawn using any described in Claims 1-4 General sense primer, general reverse primer, logical inspection probe in object probe combinations (a1) carry out quantitative fluorescent PCR；(b) with to be measured Nucleic acid is template, using the specific amplified sense primer, logical in any primer combination of probe (a1) in Claims 1-4 Quantitative fluorescent PCR is carried out with anti-sense primer, logical inspection probe and retardance probe；(c) if the quantitative fluorescent PCR of step (a) can be with Positive amplification or ct values cannot be obtained more than or equal to 37, determined nucleic acid by obtaining the quantitative fluorescent PCR of positive amplification and step (b) Described in specific site for mutation before nucleic acid, if the quantitative fluorescent PCR of step (a) can obtain positive amplification and step (b) quantitative fluorescent PCR can obtain positive amplification and ct values are less than specific site described in 37, determined nucleic acid for mutation core Acid；

The method B expands following steps：(a) using determined nucleic acid as template, drawn using any described in Claims 1-4 General sense primer, general reverse primer, logical inspection probe in object probe combinations (a2) carry out quantitative fluorescent PCR；(b) with to be measured Nucleic acid is template, using in any primer combination of probe (a2) in Claims 1-4 specific amplified anti-sense primer, General sense primer, logical inspection probe and retardance probe carry out quantitative fluorescent PCR；(c) if the quantitative fluorescent PCR of step (a) can Positive amplification cannot be obtained with the quantitative fluorescent PCR for obtaining positive amplification and step (b) or ct values are more than or equal to 37, core to be measured Specific site described in acid is nucleic acid before mutation, if the quantitative fluorescent PCR of step (a) can obtain positive amplification and step (b) quantitative fluorescent PCR can obtain positive amplification and ct values are less than specific site described in 37, determined nucleic acid for mutation core Acid；

The specific site is the site that target point mutation occurs in nucleic acid；On the specific site, target point mutation does not occur Nucleotide designation for mutation before nucleotide, occur target point mutation Nucleotide designation for mutation after nucleotide.

7. it is a kind of for detecting in nucleic acid with the presence or absence of the primer combination of probe of EGFR gene T790M mutation, drawn by general upstream Object, general reverse primer, logical inspection probe, specific amplified anti-sense primer and retardance probe composition；The general sense primer is sequence Single strand dna shown in the sequence 1 of list；The general reverse primer is the single stranded DNA point shown in the sequence 2 of sequence table Son；The specific amplified anti-sense primer is the single strand dna shown in the sequence 3 of sequence table, wherein holding the 5th nucleosides from 5 ' Acid, the 6th nucleotide and the 7th nucleotide carry out LNA modifications；The logical inspection probe is single-stranded shown in the sequence 4 of sequence table DNA molecular, 5 ' ends are marked using FAM fluorophors, and 3 ' ends are marked using BHQ1 quenching groups；The retardance probe is sequence table Sequence 5 shown in single strand dna, wherein being carried out from 5 ' the 5th nucleotide in end, the 6th nucleotide and the 7th nucleotide LNA is modified, and 3 ' ends carry out amido modified.

8. it is a kind of for detecting the primer combination of probe that whether there is K-ras gene mutations in nucleic acid, by general sense primer, lead to It is formed with anti-sense primer, logical inspection probe, specific amplified sense primer and retardance probe；

The general sense primer is as shown in the sequence 8 of sequence table；

The general reverse primer is as shown in the sequence 9 of sequence table；

The specific amplified sense primer is following (b1) and/or (b2) and/or (b3)：

(b1) single strand dna shown in the sequence 10 of sequence table；

(b2) single strand dna shown in the sequence 11 of sequence table；

(b3) single strand dna shown in the sequence 12 of sequence table；

It is described it is logical inspection probe as shown in the sequence 13 of sequence table, wherein from 5 ' end the 7th nucleotide, the 8th nucleotide, the 11st Nucleotide, the 12nd nucleotide and the 14th nucleotide carry out LNA modifications；14 institute of sequence of the retardance probe such as sequence table Show, wherein carrying out LNA modifications from 5 ' the 6th nucleotide in end, the 9th nucleotide and the 10th nucleotide, 3 ' ends carry out amino and repair Decorations.

9. a kind of detect in nucleic acid with the presence or absence of the method for EGFR gene T790M mutation, include the following steps：(1) with core to be measured Acid is template, and fluorescent quantitation is carried out using general sense primer, general reverse primer, the logical inspection probe described in claim 7 PCR；(2) using determined nucleic acid as template, using the specific amplified anti-sense primer, general sense primer, logical described in claim 7 It examines probe and retardance probe carries out quantitative fluorescent PCR；(3) if the quantitative fluorescent PCR of step (1) can obtain positive amplification simultaneously And the quantitative fluorescent PCR of step (2) cannot obtain positive amplification or ct values more than or equal to there is no EGFR bases in 37, determined nucleic acid Because T790M is mutated, if the quantitative fluorescent PCR of step (1) can obtain positive amplification and the quantitative fluorescent PCR of step (2) It can obtain positive amplification and ct values are less than there are EGFR gene T790M mutation in 37, determined nucleic acid.

It is method first or method second or method 10. detecting with the presence or absence of the method for 2 exon of K-ras genes mutation in nucleic acid Third；

The method first includes the following steps：(1) using determined nucleic acid as template, using the general upstream described in claim 8 Primer, general reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, using in claim 8 Specific amplified sense primer (b1), general reverse primer, logical inspection probe and the retardance probe carries out quantitative fluorescent PCR；(3) If the quantitative fluorescent PCR of step (1) can obtain positive amplification and the quantitative fluorescent PCR of step (2) cannot obtain the positive Amplification or ct values, which are more than or equal in 37, determined nucleic acid, there is no 2 exon G12A of K-ras genes is mutated and is not present K-ras 2 exon G12V of gene, which is mutated, and there is no 2 exon G12D of K-ras genes to be mutated, if the fluorescent quantitation of step (1) The quantitative fluorescent PCR that PCR can obtain positive amplification and step (2) can obtain positive amplification and ct values are less than 37, to be measured There are 2 exon G12A of K-ras genes mutation or 2 exon G12V of K-ras genes mutation or K-ras genes 2 in nucleic acid Exon G12D is mutated；

Method second includes the following steps：(1) using determined nucleic acid as template, using described in claim 8 general sense primer, General reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, using described in claim 8 Specific amplified sense primer (b2), general reverse primer, logical inspection probe and retardance probe carry out quantitative fluorescent PCR；(3) if The quantitative fluorescent PCR of step (1) can obtain positive amplification and the quantitative fluorescent PCR of step (2) cannot obtain positive amplification Or ct values are more than or equal in 37, determined nucleic acid there is no 2 exon G12S of K-ras genes mutation and there is no K-ras genes 2 Exon G12C, which is mutated, and there is no 2 exon G12R of K-ras genes to be mutated, if the quantitative fluorescent PCR of step (1) can Positive amplification can be obtained with the quantitative fluorescent PCR for obtaining positive amplification and step (2) and ct values are less than in 37, determined nucleic acid There are 2 exon G12S of K-ras genes mutation or 2 exon G12C of K-ras genes mutation or 2 extra of K-ras genes to show Sub- G12R mutation；

Method third includes the following steps：(1) using determined nucleic acid as template, using described in claim 8 general sense primer, General reverse primer, logical inspection probe carry out quantitative fluorescent PCR；(2) using determined nucleic acid as template, using described in claim 8 Specific amplified sense primer (b3), general reverse primer, logical inspection probe and retardance probe carry out quantitative fluorescent PCR；(3) if The quantitative fluorescent PCR of step (1) can obtain positive amplification and the quantitative fluorescent PCR of step (2) cannot obtain positive amplification Or ct values are more than or equal in 37, determined nucleic acid there is no 2 exon G13D of K-ras genes mutation, if the fluorescence of step (1) The quantitative fluorescent PCR that quantitative PCR can obtain positive amplification and step (2) can obtain positive amplification and ct values are less than 37, There are 2 exon G13D of K-ras genes mutation in determined nucleic acid.