CN103923973A - Digital PCR platform based gene deletion mutation detection method and kit thereof - Google Patents

Abstract

The invention relates to the field of the molecular biology, and concretely relates to gene deletion mutation detection method and a kit thereof. A PNA probe complementary to a wild non-mutation template sequence is added into a reaction system with digital PCR as a platform, and the repression effect of the PNA probe is utilized to make only deletion-mutated samples amplified; and the existence of mutant templates and the quantity and the proportion of the mutant templates are determined through fluorescence detection by utilizing a TaqMan probe as a fluorescent quantitative mark.

Description

A kind of method and test kit based on digital pcr detection of platform deletion mutant

technical field

The present invention relates to biology field and detection of nucleic acids field, be specially the method that detects deletion mutant.

Background technology

Deletion mutantion is type common in genovariation, can give microorganism, plant the biological character of animal and the mankind's physiological character is brought a lot of variations; The part or all of inactivation that comprises protein function, forms the fragment of non-activity etc., also likely changes other functions such as gene regulating of organism.The research of transgenation is significant to biological heredity, evolution and transformation.

The detection technique of genovariation mainly contains direct sequencing (also claiming Sanger sequencing) and ARMS method at present.Now respectively brief introduction under:

Sanger sequencing, i.e. Sanger(mulberry lattice) dideoxy chain termination is Frederick Sanger in invention in 1975.Order-checking process need is first done a polymerase chain reaction (PCR).In PCR process, bi-deoxyribose Nucleotide may random being added in the DNA fragmentation synthesizing.Because bi-deoxyribose Nucleotide has taken off a Sauerstoffatom more, once it is added on DNA chain, this DNA chain just can not continue to increase length.Final result is to obtain DNA fragmentation that likely obtain, different lengths.The most general state-of-the-art method, is that bi-deoxyribose Nucleotide is carried out to different fluorescent marks at present.Total DNA that PCR reaction is obtained is by capillary electrophoresis separation, and the DNA that goes to least significant end just can send fluorescence under the effect of laser.Due to ddATP, ddGTP, ddCTP, ddTTP(4 kind bi-deoxyribose Nucleotide) fluorescent mark is different, and computer can judge that according to color on this position, base is A actually automatically, T, G, which in C.Direct sequencing length consuming time and sensitivity are low, only can detect mutant proportion in more than 10%～20% sudden change.

ARMS method also claims amplification refractory mutation system (Amplification Refractory Mutation System, ARMS), allelotrope characteristic PCR(Allele Specific PCR, ASPCR) etc., be allele specific amplification method (Allele Specific Amplification, ASA) building on 1989, is the development of round pcr application.

ARMS method is mainly used in known mutations gene to detect.First design two 5 ' end primers, 3 ' tip designs of this primer is one and normal DNA complementation, and one complementary with mutant DNA.For homozygosity sudden change, add respectively these two kinds of primers and 3 ' end primer to carry out two parallel PCR, only have with the primer of the complete complementation of mutant DNA just extensible and obtain pcr amplification product.If mispairing is positioned at 3 ' end of upstream primer, primer and template DNA are unpaired, cause PCR not extend, and therefore this method is called ARMS, the wild-type that optionally increases or mutated genes.

There is following problem in above-mentioned prior art:

1. be qualitative detection, that is to say and can only provide the conclusion whether genovariation exists.But the ratio that cannot measure the DNA amount of carrying genovariation, that is to say, cannot carry out quantitative analysis detection, or quantitative analysis error is larger.

2. all provide mimic diagram result, need to manually carry out interpretation, interpretation aspect is relatively subjective.Cannot directly obtain digitized objective results.

3. remolding sensitivity is poor, and the sensitive of method is preferably 1% at present, cannot meet some highly sensitive detection demand.

Summary of the invention

The object of the present invention is to provide a kind of test kit and method based on digital pcr detection of platform deletion mutant.

Peptide nucleic acid(PNA) (Peptide Nucleic Acid, PNA) be a class using neutral amido linkage (false peptide bond) replace sugar-phosphodiester bond in DNA as the Deoxyribonucleotides of skeleton like thing, retain base position wherein.The special construction of PNA, making it to have very high stability not by proteolytic enzyme or nuclease degradation, is a kind of nonionic, achirality, is difficult for being hydrolyzed the molecule of cutting with enzyme.

PNA and DNA profiling binding ability, higher than common oligonucleotide, can be used as repressor and are placed in before primer, and partially overlap with PCR primer; If can stop polymerase chain reaction when PNA and template are correctly matched, wild-type template cannot increase; For example, if there is mispairing (template is the DNA undergoing mutation), binding ability declines rapidly, loses derepression.Now can be used to amplification sudden change template.

Digital pcr is a kind of nucleic acid molecule absolute quantitation method, based on single-molecule PCR method, count, the main micro-fluidic or droplet method that adopts the popular research field of present analysis chemistry, nucleic acid solution after Macrodilution is dispersed in the microreactor or droplet of chip, the nucleic acid-templated number of each reactor is less than or equals 1.Through after PCR circulation, there is the reactor of a nucleic acid molecule template will provide fluorescent signal like this, do not have the reactor of template just there is no fluorescent signal.According to the volume of relative proportion and reactor, just can extrapolate the nucleic acid concentration of original solution.

The present invention be take digital pcr as platform, adds the PNA probe with the not mutated template sequence complementation of wild-type in reaction system, utilizes the effect of checking of PNA probe, makes to only have the sample that deletion mutantion has occurred just can be amplified; And utilize TaqMan probe as fluorescent quantitation mark, according to whether there being the template of sudden change in fluoroscopic examination judgement sample.

The present invention is a kind of method of highly sensitive detection deletion mutant, and step comprises:

1. prepare digital pcr mixed solution: the sample that contains gene DNA template to be detected, forward and inverse detection pcr amplification primer, mark TaqMan probe, PNA probe, forward and oppositely Quality Control PCR primer, Quality Control TaqMan probe and the mixing of PCR premixed liquid, prepare digital pcr mixed solution;

Described forward and the inverse detection pcr amplification primer gene for increasing and containing deletion mutantion; Described mark TaqMan probe is combined with mutational site downstream DNA template;

The conserved sequence of described Quality Control PCR primer for increasing on DNA profiling to be detected; Quality Control TaqMan probe is combined with the sequence that Quality Control PCR primer increases;

Mark TaqMan is the Nucleotide that is connected fluorophor and quencher group with Quality Control TaqMan probe, and both fluorophor types are different;

Preferably, fluorophor is selected from 6-Fluoresceincarboxylic acid (FAM), chlordene-6-Fluoresceincarboxylic acid (HEX), Cy5(U.S. Life Technologies), Cy3(U.S. Life Technologies), VIC(U.S. Life Technologies), quenching of fluorescence group is selected from 6-carboxyl tetramethylrhodamin (TAMARA), BHQ1(U.S. Life Technologies), BHQ2(U.S. Life Technologies) or MGB(U.S. Life Technologies).

The DNA that PNA probe and wild-type are not suddenlyd change is complementary.

2. digital pcr mixed solution is made the micro-reaction drop of PCR, then carries out pcr amplification reaction; Preferably, the method for the micro-reaction drop of digital pcr mixed solution making PCR is: digital pcr mixed solution is added to drop generator, generate 10000～20000 micro-reaction drops.

3. collect signal and carry out result judgement: the product after pcr amplification reaction is carried out to signal collection, according to the type of fluorescent signal, judge the DNA profiling that whether contains producer deletion mutantion in testing sample, also can determine wherein quantity and the content of the DNA profiling of producer deletion mutantion.

Available QuantaSoft(Biorad) software carries out data analysis, and the copy number of undergoing mutation in calculation sample and content are determined the ratio of mutant DNA sample.

Due to the effect of Quality Control PCR primer, as long as contain sample DNA template in the micro-reaction drop of PCR, no matter whether there is deletion mutantion, all can there is amplified reaction, and combine Quality Control TaqMan probe, therefore have fluorescent signal.There is the DNA profiling of deletion mutantion, added and detect after pcr amplification primer, also combined mark TaqMan probe.Therefore Quality Control is different from the fluorophor type of mark TaqMan probe, can tell in sample, whether to contain the DNA profiling that deletion mutant has occurred according to different fluorescent signals.According to having there is the fluorescent signal quantity of sudden change and total fluorescent signal quantity, can determine the DNA profiling content of deletion mutant again, carry out quantitative analysis.

By aforesaid method, can detect the deletion mutantion that is positioned at gene specified range.

The present invention by PNA probe for the test kit based on digital pcr detection of platform deletion mutant based on digital pcr detection of platform deletion mutant and preparation, the wild-type DNA profiling complementation that this PNA probe and gene to be detected are undergone mutation.。

The present invention is also used for mark TaqMan probe based on digital pcr detection of platform deletion mutant and preparation the test kit based on digital pcr detection of platform deletion mutant, and this mark TaqMan probe is combined with mutational site downstream DNA template.

A test kit based on digital pcr detection of platform deletion mutant, contains at least one in following material:

(1) with the PNA probe of the wild-type DNA profiling complementation of not undergoing mutation;

(2) the mark TaqMan probe of being combined with mutational site downstream DNA template;

(3) for forward and the inverse detection pcr amplification primer of the gene to be detected that increases;

(4) for the forward of conserved sequence on the testing gene place DNA profiling that increases and reverse Quality Control PCR primer;

(5) the Quality Control TaqMan probe that conserved sequence is combined on the DNA profiling of testing gene place.

This test kit can be used for detecting the deletion mutantion in genome specified range, both can carry out qualitative analysis, and sensitivity can reach 1/2500, can carry out quantitative analysis again, even extremely low at sudden change sample content, also has very high accuracy of detection.

Compared with the prior art, the invention has the advantages that:

1. result interpretation mode: the interpretation of former technological method result needs artificial participation, and naked eyes are made last judgement according to relational graph, and the mode of this interpretation seriously relies on experience, speed is slow and be easy to produce false positive and false-negative sentence read result.What our technical approach provided is data message, can carry out result interpretation by software is full automatic, thereby accelerate the speed of data analysis, has also reduced the possibility that produces false negative and false positive interpretation.

2. the mode that result is described: former technical approach is mode qualitatively to the description of genovariation, that is to say, just describes certain special genovariation and whether is present in detection sample.This technological method is the mode of absolute quantitation to the description of genovariation, can provide absolute quantity and the ratio of entrained certain specific gene abnormal dna of sample.And even in the situation that sudden change sample content is extremely low, the error of quantitative analysis is also very little.

3. detection sensitivity (numerical value more sluggishness is better): the sensitivity of former technological method is generally between 1%-50%, and that the technological method that we propose promotes the detection sensitivity of genovariation is very obvious, is 1/2500.Can in the wild DNA of very high background, detect the extremely DNA that carries genovariation of trace.

Accompanying drawing explanation

Fig. 1 is in embodiment 1, the fluoroscopic examination result of different content sudden change sample.

Embodiment

Embodiment 1

(1) prepare DNA sample to be measured: the sample (wherein mutant is respectively 1/1,1/100,1/1000,1/2500 with the content ratio of wild-type) that the positive DNA of sudden change of the DNA that contains Wild type EGFR gene, EGFR exons 19 disappearances mixes with wild-type DNA.DNA source can be serum, blood plasma, peripheral blood, oral mucosa, hydrothorax, body fluid or tissue etc.Mutant DNA derives from the positive cell line that carries EGFR19 sudden change.Wherein c.2238-c.2255 position deletion mutantion on EGFR19 exon.

(2) room temperature is melted forward and inverse detection pcr amplification primer and corresponding mark TaqMan probe and the PNA probe for detection of EGFR exons 19 disappearances, and forward and reverse Quality Control PCR primer and Quality Control TaqMan probe.

The sequence of forward and inverse PCR amplimer is:

F1:5’-GAGAAAGTTAAAATTCCCGTCGCT-3’

R1:5’-ATGGACCCCCACACAGCA-3’

The sequence of mark TaqMan probe comprises fluorophor, nucleotide segment and quencher group, fluorophor and quencher group are respectively FAM and MGB, and nucleotide segment, as shown in SEQ ID No.11, is P1:5 '-ACTCACATCGAGGATTTCC-3 '.

The sequence of PNA as shown in SEQ ID No.16, PNA1:5 '-GAATTAAGAGAAGCA-3 ';

The Quality Control PCR primer EGFR exon 2 that is used for increasing, forward and the oppositely sequence of Quality Control PCR primer are:

CF2:5’-GCCAAGGCACGAGTAACAAGC-3’

CR2:5’-CCTCCTCTGGAGGCTGAGAAAAT-3’

Fluorophor and the quencher group of Quality Control TaqMan probe are respectively VIC and MGB, and nucleotide segment is as shown in SEQ ID No.26, and Quality Control TaqMan probe is:

CP2:VIC-ACGCAGTTGGGCACTT–MGB。

(3) according to following proportioning, prepare PCR reaction solution: 2 * digital pcr premixed liquid (Biorad, #186-3022) with detection pcr amplification primer and Quality Control PCR primer (600nM), mark and Quality Control TaqMan probe (300nM) and PNA probe (300nM) and DNA(10ng to be detected) be mixed with digital pcr mixed solution, with distilled water, complementing to final volume is 20 μ L.In the digital pcr mixed solution of preparation, forward and inverse detection pcr amplification primer content are respectively 600nM, and mark TaqMan probe content is 300nM, PNA probe content 300nM, forward and oppositely Quality Control PCR primer content are respectively 600nM, and Quality Control TaqMan probe content is 300nM.

(4) the 20 μ L digital pcr mixed solutions that prepare join the drop in 8-road and make in plate, then add 60 μ L drops to make oil to making plate, then put into QX200 drop generator, for the preparation of the micro-reaction drop of PCR.

(5) the micro-reaction drop of the PCR preparing is transferred to 96 hole Sptting plates, and seals with shrouding film, micro-reaction amount of droplets of generation is 10000～20000.

(6) 96 hole PCR plates are put into PCR instrument and are carried out amplified reaction according to condition below:

94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 10 seconds, 65 ℃ of annealing 10 seconds, 56 ℃ are extended 45 seconds, carry out altogether 32 circulations, 10 ℃ of termination reactions.

(7) after pcr amplification reaction, PCR Sptting plate is positioned in the micro-reaction drop of PCR signal-obtaining instrument (QX200 droplet fluorescent signal gathering system) and carries out signal collection, result is as Fig. 1.With QuantaSoft(Biorad) software carries out data analysis, draws the absolute content of mutant DNA in sample and the ratio of all DNA relatively.

In the micro-reaction drop that contains mutant DNA template, there is FAM and VIC fluorescent signal simultaneously, be designated as a number of signals; In micro-reaction drop containing the wild-type DNA profiling of not undergoing mutation, only there is VIC fluorescent signal.By above-mentioned steps, detect, the in the situation that of sudden change sample content 1/2500, also can detect sudden change.

Detection by quantitative result, in the different sample of mutant DNA template content, sudden change positive signal number (being FAM number of signals) and resultant signal number are that total micro-quantitative proportion that reacts drop is as follows:

1/1 sample 6477/13028 calculated value sudden change/wild=0.989/1, error 1.2%

1/100 sample 186/18700 calculated value sudden change/wild=0.01/1, error 0.2%

1/1000 sample 23/19854 calculated value sudden change/wild=0.00116/1, error 16%

1/2500 sample 11/19928 calculated value sudden change/wild=0.00055/1, error 37%

Mutant DNA template and wild-type DNA profiling ratio are 1/1～1/100 o'clock, and detection by quantitative error is lower than 2%.

While selecting the forward of one of following group and inverse PCR primer, by above-mentioned steps, detect, the in the situation that of sudden change sample content 1/2500, also can detect sudden change:

(1) forward F2:5 '-GAAAGTTAAAATTCCCGTCGCTAT-3 '

Reverse R2:5 '-GGACCCCCACACAGCAAA-3 '

(2) forward F3:5 '-AAGTTAAAATTCCCGTCGCTATCA-3 '

Reverse R3:5 '-ACCCCCACACAGCAAAGC-3 '

(3) forward F4:5 '-GGTGAGAAAGTTAAAATTCCCGTC-3 '

Reverse R4:5 '-CCCCACACAGCAAAGCAG-3 '

(4) forward F5:5 '-AAGGTGAGAAAGTTAAAATTCCCG-3 '

Reverse R5:5 '-CCACACAGCAAAGCAGAA-3 '.

While using the PNA probe of one of following sequence instead (SEQ ID No.17～20), by above-mentioned steps, detect, the in the situation that of sudden change sample content 1/2500, also can detect sudden change:

（1）PNA2:5’-ATTAAGAGAAGCAAC-3’

（2）PNA3:5’-TAAGAGAAGCAACAT-3’

（3）PNA4:5’-AGAGAAGCAACATCT-3’

（4）PNA5:5’-AGAAGCAACATCTCC-3’。

When the nucleotide segment of mark TaqMan probe is used one of following sequence instead, the in the situation that of sudden change sample content 1/2500, also can detect sudden change:

（1）P2:5’-TCACATCGAGGATTTCCTT-3’

（2）P3:5’-ACATCGAGGATTTCCTTGT-3’

（3）P4:5’-ATCGAGGATTTCCTTGTTG-3’

（4）P5:5’-AGGATTTCCTTGTTGGCTT-3’。

C.2238 the DNA sample of saltant type is changed on EGFR19 exon-DNA of position deletion mutantion c.2250, with aforesaid method, detects, detection limit also can reach 1/2500, and the error of quantitative analysis is lower than 5%.

By aforesaid method, can detect c.2230-c.2260 position deletion mutantion on EGFR19 exon, detection limit reaches 1/2500.

Forward and reverse Quality Control PCR primer, and Quality Control TaqMan probe can use following sequence instead, effect is identical.

Forward Quality Control PCR primer CF1:5 '-GTTTGCCAAGGCACGAGTAAC-3 '

Reverse Quality Control PCR primer CR1:5 '-TCCTCTGGAGGCTGAGAAAATGA-3 '

Quality Control TaqMan probe CP1VIC-TCACGCAGTTGGGCAC-MGB.

Use the method for quantitative fluorescent PCR to detect, detect and be limited to 1/100, and in the situation that sudden change sample content is greater than 1%, the error of quantitative analysis is greater than 10%.

Claims (8)

1. the method based on digital pcr detection of platform deletion mutant, is characterized in that, comprises the steps:

(1) prepare digital pcr mixed solution: the sample that contains gene DNA template to be detected, forward and inverse detection pcr amplification primer, mark TaqMan probe, PNA probe, forward and oppositely Quality Control PCR primer, Quality Control TaqMan probe and the mixing of PCR premixed liquid, prepare digital pcr mixed solution;

Described forward and the inverse detection pcr amplification primer gene for increasing and containing deletion mutantion; Described mark TaqMan probe is combined with mutational site downstream DNA template;

Described Quality Control PCR primer is for the conserved sequence of the DNA profiling to be detected that increases; Quality Control TaqMan probe is combined with the sequence that Quality Control PCR primer increases;

Mark TaqMan is the Nucleotide that is connected fluorophor and quencher group with Quality Control TaqMan probe, and both fluorophor types are different;

(2) digital pcr mixed solution is made the micro-reaction drop of PCR, then carries out pcr amplification reaction;

(3) collect signal and carry out result judgement: the product after pcr amplification reaction being carried out to signal collection, according to the type of fluorescent signal, judge DNA profiling and quantity and the content that whether contains producer deletion mutantion in testing sample.

2. the method based on digital pcr detection of platform deletion mutant described in claim 1, it is characterized in that, described fluorophor is selected from 6-Fluoresceincarboxylic acid, chlordene-6-Fluoresceincarboxylic acid, Cy5, Cy3 or VIC, and quencher group is selected from 6-carboxyl tetramethylrhodamin, BHQ1, BHQ2 or MGB.

3. the method based on digital pcr detection of platform deletion mutant described in claim 1, is characterized in that, the wild-type DNA profiling that described PNA probe and gene to be detected are undergone mutation is complementary.

4.PNA probe, for the preparation of the test kit based on digital pcr detection of platform deletion mutant, is characterized in that, the wild-type DNA profiling that described PNA probe and gene to be detected are undergone mutation is complementary.

5.PNA probe for based on digital pcr detection of platform deletion mutant, is characterized in that, the wild-type DNA profiling complementation that described PNA probe and gene to be detected are undergone mutation.

6. mark TaqMan probe, for the preparation of the test kit based on digital pcr detection of platform deletion mutant, is characterized in that, described mark TaqMan probe is combined with mutational site downstream DNA template.

7. mark TaqMan probe is used for based on digital pcr detection of platform deletion mutant, and described mark TaqMan probe is combined with mutational site downstream DNA template.

8. the test kit based on digital pcr detection of platform deletion mutant, is characterized in that, comprises at least one in following material:

(1) with the PNA probe of the wild-type DNA profiling complementation of not undergoing mutation;

(2) the mark TaqMan probe of being combined with mutational site downstream DNA template;

(3) for forward and the inverse PCR amplimer of the gene to be detected that increases;

(4) for the forward of conserved sequence on the testing gene place DNA profiling that increases and reverse Quality Control PCR primer;

(5) the Quality Control TaqMan probe that conserved sequence is combined on the DNA profiling of testing gene place.