CN116219015A

CN116219015A - PCR-based detection kit and detection method for PIK3CA gene mutation in DNA

Info

Publication number: CN116219015A
Application number: CN202310067353.2A
Authority: CN
Inventors: 陈皇; 钟定荣; 王秀红; 王雅琦; 董嘉
Original assignee: Suzhou Precigenome Co ltd; China Japan Friendship Hospital
Current assignee: Suzhou Precigenome Co ltd; China Japan Friendship Hospital
Priority date: 2023-02-06
Filing date: 2023-02-06
Publication date: 2023-06-06

Abstract

The invention relates to a detection kit and a detection method for PIK3CA gene mutation in DNA based on PCR, wherein the method comprises a primer probe group designed for a preset mutation target and a wild type target; each site of the primer probe group is designed with a corresponding sequence; combining the primer probe groups at different points, and dividing the combination into detection holes with preset numbers; wherein, different detection holes are used for detecting different points. The invention can specifically detect 11 mutation sites and 4 wild type sites of PIK3CA of DNA (gDNA or cfDNA) in fresh tissue samples, paraffin embedded tissue samples or plasma samples at one time, and has the advantages of high sample utilization rate, high resolution, high sensitivity, good repeatability, simple and convenient operation, automatic interpretation and the like.

Description

PCR-based detection kit and detection method for PIK3CA gene mutation in DNA

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PCR-based detection kit and a detection method for PIK3CA gene mutation in DNA.

Background

The p110 catalytic subunit of the PIK3CA gene encoding phosphatidylinositol-3-kinases (PI 3 Ks), namely PI3Kp110a, is the catalytic subunit of PI3Ks, and PI3Ks is a lipid kinase family capable of specifically phosphorylating the 3-hydroxyl group of phosphatidylinositol to generate second messenger inositol. The PI3Ks family is divided into types I, II and III, wherein type I is divided into two subtypes IA and IB, and PIK3CA is the catalytic subunit of type IA. Mutation of the PIK3CA gene may result in abnormally enhanced catalytic activity of PI3Ks, contributing to the carcinogenesis of cells. Studies have shown that it is detectable in part colorectal, glioblastoma, gastric, breast, lung, uterine, ovarian and cervical cancers; currently, the NCCN guidelines clearly recommend gene detection of PIK3CA in breast cancer patients with HR positive HER2 negative.

Common genetic testing techniques include one-generation sequencing (Sanger sequencing), high throughput sequencing (NGS), fluorescent quantitative PCR techniques, and the like, which have certain drawbacks for high sensitivity detection of clinical samples. One generation sequencing has natural defects for mutation frequency detection below 10%; the nucleic acid information is deleted in the construction process of the NGS library, the sequencing cost is high, and the period is long; fluorescent quantitative PCR is low in cost, but is poor in specificity and sensitivity when mutation detection is performed. Whereas existing digital PCR implementations typically employ a Biorad digital PCR system. And the user moves the generated liquid drops into a PCR reaction test tube by using a liquid shifter, then the test tube is placed into a PCR thermal cycler for PCR reaction, and after the reaction is finished, the user takes out the PCR tube and places the PCR tube into a liquid drop fluorescence detector for detecting liquid drop fluorescence. The principle of detection is that liquid drop and oil are driven into a capillary tube, and the liquid drop passes through the detection individually

The position, the instrument reads the presence or absence of fluorescence in the droplet. Such a system has several problems:

1) After the system generates the liquid drops, the user needs to manually transfer the liquid drops into a 96-well plate or a PCR tube to perform the next PCR reaction, so that the liquid drops are lost or broken;

2) The whole reaction requires 2 different consumables. I.e. droplet production requires the use of a special microfluidic cartridge to generate the droplets. The PCR reaction is carried out by transferring the liquid drop manually-operated pipettor into a 96-well plate or a PCR tube, and the fluorescent signal is directly read after the PCR reaction is finished. Droplet generation cannot be achieved, and PCR reactions and signal reading are done on one consumable.

Disclosure of Invention

In view of the above, the invention aims to overcome the defects of the prior art, and provides a detection kit and a detection method for PIK3CA gene mutation in DNA based on PCR, so as to solve the problems of liquid drop loss and overlong detection time in the detection of PIK3CA gene in DNA in the prior art.

In order to achieve the above purpose, the invention adopts the following technical scheme: a kit for detecting PIK3CA gene mutation in PCR-based DNA, comprising:

a primer probe set designed for a preset mutation target and a wild target; each site of the primer probe group is designed with a corresponding sequence;

combining the primer probe groups at different points, and dividing the combination into detection holes with preset numbers; wherein, different detection holes are used for detecting different points.

Further, the primer probe set includes:

a primer for H1047 locus comprising a primer consisting of PIK3CA H1047-FSEQ ID NO. 1 and a primer consisting of PIK3CA H1047-RSEQ ID NO. 2, or consisting of PIK3CA H1047-FSEQ ID NO. 1 and PIK3CA H1047-RSEQ ID NO. 2; and probes for the H1047 site: PIK3CA H1047R SEQ ID NO:7, PIK3CA H1047L SEQ ID NO:8, PIK3CA H1047YSEQ ID NO:9, and/or PIK3CA H1047-WT SEQ ID NO:10;

a primer for the C420 site comprising a primer consisting of PIK3CA C420-F SEQ ID NO:3 and a primer consisting of PIK3CA C420-RSEQ ID NO:4, or consisting of PIK3CA C420-F SEQ ID NO:3 and PIK3CA C420-R SEQ ID NO:4, and a probe for the C420 site: 11 and/or 12 of PIK3CA C420-WT SEQ ID NO;

primers for E542/E545/Q546 sites: it comprises a primer consisting of PIK3CA E542/5/6-FSEQ ID NO:5 and a primer consisting of PIK3CA E542/5/6-RSEQ ID NO:6 or consists of PIK3CA E542/5/6-FSEQ ID NO:5 and PIK3CA E542/5/6-RSEQ ID NO: 6.

Further, each of the primer-probe sets is labeled with two fluorescent dyes, one of which is a fluorescent reporter dye and the other of which is a quencher dye;

wherein the fluorescent reporter dye is selected from the group consisting of: FAM, HEX, ROX, cy5 and Cy3; the quencher dye is selected from the group consisting of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and QSY.

Further, each site of the probe is designed with a corresponding sequence, including:

PIK3CA E545K/FAM/, PIK3CA H1047R/FAM/, PIK3CA H1047L/FAM/and PIK3CA H1047Y/FAM/use FAM fluorophores to label the 5 'end, BHQ1 groups to label the 3' end;

PIK3CA H1047-WT/HEX/, PIK3CA C420-WT/HEX/, PIK3CA E545/Q546-WT/HEX/and PIK3CA E542-WT/HEX/label 5 'end with HEX fluorophore, BHQ1 group labels 3' end;

PIK3CA C420R/ROX/, PIK3CA Q546E/ROX/, PIK3CA Q546R/ROX/and PIK3CA E545A/ROX/use ROX fluorophore to label the 5 'end, BHQ2 group to label the 3' end;

PIK3CA E545G/Cy5/, PIK3CA E545D/Cy 5/and PIK3CA E542K/Cy5 labeled 5 'with a Cy5 fluorescent group, BHQ3 group labeled 3' end.

Further, the number of the detection holes is 4, and each detection hole is internally provided with a microfluidic pipeline;

the microfluidic pipeline is used for generating liquid drops and tiling;

wherein, each detection hole in the first 3 detection holes detects 4 sites, and the 4 th detection hole detects 3 sites.

The embodiment of the application provides a detection and analysis method for PIK3CA gene mutation in DNA based on PCR, which comprises the following steps:

obtaining DNA to be detected;

mixing the DNA to be detected by using the primer probe set in the kit according to any one of the embodiments to obtain a PCR reaction mixture, sufficiently shaking and mixing, and centrifuging at 3000rpm for 1 minute to obtain an amplification solution;

adding the amplification solution into each PCR tube, adopting an extracted DNA sample or positive control or negative control to form a digital PCR reaction mixture, mixing and centrifuging to obtain a mixed solution;

dripping oil for the primer probe group into a preset number of detection holes, distributing the mixed solution into the detection holes, and dripping sealing oil above the mixed solution to form a kit to be detected;

during detection, a PCR thermal cycler is utilized to carry out thermal cycle on the kit to be detected so as to carry out PCR reaction;

comparing the concentration of the solution obtained after the PCR reaction with a preset threshold value, and determining mutation frequency of the corresponding site according to a comparison result; wherein, the preset threshold is divided according to the fluorescence signals of the positive quality control and the negative quality control.

Further, the obtaining the DNA to be tested includes:

extracting the DNA to be detected of the plasma sample to be detected or extracting the DNA to be detected of the tissue sample to be detected.

Further, the method further comprises the following steps: preprocessing the DNA to be detected, including:

and if the obtained concentration of the DNA to be detected is higher than the preset concentration, diluting the DNA to be detected to the preset concentration by using pure water.

Further, the preset concentration is 10 ng/. Mu.L.

By adopting the technical scheme, the invention has the following beneficial effects:

the invention provides a PCR-based PIK3CA gene mutation detection kit and a detection method, which can specifically detect 11 mutation sites and 4 wild-type sites of PIK3CA of DNA (gDNA or cfDNA) in fresh tissue samples, paraffin embedded tissue samples or plasma samples at one time by a fluorescence detection technology, and have the advantages of high sample utilization rate, high resolution, high sensitivity, good repeatability, simple and convenient operation, automatic interpretation and the like. Meanwhile, the kit provided by the application fully utilizes the advantages of multiple fluorescent channels of the digital PCR system, reduces 15 detection sites to 4 sample holes, completes all detection reactions in one microfluidic chip by one reaction, fully utilizes samples, further improves the sample utilization rate and the detection sensitivity, and has great advantages particularly for detecting plasma free DNA. Likewise, using the Qiagen FDA approved commercial detection kit product of fluorescent quantitative PCR, all 11 mutation sites and wild-type DNA of PIK3CA were required to be detected in a 6-tube reaction.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic structural diagram of a PIK3CA gene mutation detection kit in PCR-based DNA;

FIG. 2 is a layout diagram of PIK3CA 11 locus detection on a kit according to the invention;

fig. 3 is a schematic diagram of a liquid generated by the present invention in a microfluidic cartridge channel;

FIG. 4 is a schematic step diagram of a method for detecting PIK3CA gene mutation in PCR-based DNA according to the present invention

FIG. 5 is a schematic diagram of PIK3CA E545A mutation negative samples of PIK3CA E545A-T1/ROX/detection provided by the invention;

FIG. 6 is a schematic diagram of PIK3CA E545A mutation positive samples of PIK3CA E545A-T1/ROX/detection provided by the invention;

FIG. 7 is a schematic diagram of PIK3CA E545A mutation negative samples of PIK3CA E545A/ROX/detection provided by the invention;

FIG. 8 is a schematic diagram of PIK3CA E545A mutation positive samples of PIK3CA E545A/ROX/detection provided by the invention;

FIG. 9 is a graph showing the fluorescence effects of different concentrations of magnesium ions on PIK3CA 545/Q546-WT, PIK3CA E545K, PIK CA E542K and PIK3CA E545G according to the present invention;

FIG. 10 is a graph showing the fluorescence effect of Taq polymerase enzyme amounts at different concentrations provided by the invention on PIK3CA C420-WT, PIK3CA E545/Q546-WT, PIK3CA E542-WT and PIK3CA H1047-WT.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Digital PCR is a method for nucleic acid quantification based on counting and calculation by a single-molecule PCR method, and is an absolute quantification method. The principle is that a large amount of diluted nucleic acid solution is dispersed into micro-reactors or micro-droplets of a chip by a microfluidic or micro-droplet method, and the number of nucleic acid templates in each reactor is less than or equal to 1. Thus, after PCR cycles, the reactor or microdroplet with the template of the nucleic acid molecule gives a fluorescent signal, and the reactor or microdroplet without the template does not give a fluorescent signal. The nucleic acid concentration of the original solution can be calculated from the relative proportion of the reactor or micro-droplet with or without fluorescent signal and the volume of the reactor or micro-droplet.

The PIK3CA gene is located at 3q26.3, length 34 kb, containing 21 exons.

The PIK3CA mutations occur mainly in the two hot spot regions of the Helical (helix, exon 9) and Kinase (Kinase, exon 20), the three most common mutations being H1047R located in exon20, E542K and E545K located in exon 9.

The tissue sample (FFPE or fresh tissue) is a sample type commonly used for clinical tumor gene mutation detection, and the nucleic acid extracted from the sample has high concentration and good quality, so that the requirement on a detection method is low, but the tissue sample is difficult to obtain, difficult to repeatedly sample, traumatic and severely heterogeneous. In comparison, the plasma sample can overcome the problems of difficult sampling, limited sampling times, heterogeneity and the like, is almost noninvasive, and is an ideal tumor gene detection sample type. However, the study shows that the free nucleic acid of tumor in blood plasma is relatively low, so that the sensitivity requirement on the detection technology is relatively high.

The following describes a specific PCR-based detection kit and a specific detection method for PIK3CA gene mutation in DNA according to the embodiments of the present application with reference to the accompanying drawings.

As shown in fig. 1, the kit for detecting PIK3CA gene mutation in PCR-based DNA provided in the embodiments of the present application includes:

It can be understood that the PCR-based DNA PIK3CA gene mutation detection kit provided by the application is provided with primer probe groups designed for a preset mutation target and a wild type target, and then the primer probe groups at different points are combined and divided into detection holes with preset numbers; that is to say that the kit provided in the present application has 4 detection wells, different detection wells can be used for detecting different spots.

In some embodiments, the primer probe set comprises:

In some embodiments, each of the primer-probe sets is labeled with two fluorescent dyes, wherein one dye is a fluorescent reporter dye and the other dye is a quencher dye;

Each site of the probe is designed with a corresponding sequence, comprising:

PIK3CA E545G/Cy5/, PIK3CA E545D/Cy 5/and PIK3CA E542K/Cy5 labeled 5 'with a Cy5 fluorescent group, BHQ3 group labeled 3' end. Specifically, the results are shown in Table 1.

Table 1 primer probe set designed for mutation target and wild target

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In some embodiments, as shown in fig. 2, the number of the detection holes includes 4, and each detection hole is provided with a microfluidic pipeline;

the microfluidic pipeline is used for generating and tiling liquid drops. As shown in fig. 3, a schematic diagram of the liquid in the microfluidic channel after the liquid is generated is shown.

Wherein, each detection hole in the first 3 detection holes detects 4 sites, and the 4 th detection hole detects 3 sites. As shown in the detail of the table 2,

TABLE 2 combinations of primer probes at different sites

The microfluidic chip can be basically prepared, so that detection of all 11 PIK3CA mutation sites and relevant 4 wild type sites can be completed in one reaction on one microfluidic chip. The microfluidic chip is placed in the detection well.

As shown in fig. 4, an embodiment of the present application provides a method for detecting and analyzing PIK3CA gene mutation in PCR-based DNA, including:

s101, obtaining DNA to be detected;

in some embodiments, the test DNA of the test plasma sample is extracted, or the test DNA of the test tissue sample is extracted.

Further comprises: preprocessing the DNA to be detected, including:

Specifically, firstly, a plasma sample is obtained, 4-10ml of blood is collected by using a STRECK Cell-Free DNA BCT blood collection tube, a PAXgene Blood ccfDNA blood collection tube or an EDTA blood collection tube, and 2-5ml of plasma is separated from the blood sample for cfDNA extraction. The blood collected by the EDTA blood collecting tube is separated from the blood plasma within 8 hours as much as possible; blood collected by STRECK Cell-Free DNA BCT blood collection tube and PAXgene Blood ccfDNA blood collection tube can be separated into blood plasma in 7 natural days, and the separated blood plasma can be preserved for 1 year below-70 ℃.

For plasma samples, extraction of cfDNA using QIAamp Circulating Nucleic Acid Kit (Qiagen) was suggested. Since there is a certain difference in cfDNA content in different blood samples, studies have shown that in normal individuals, the cfDNA extraction amount is typically 1.8-44ng/ml, but will vary correspondingly for different cancer species and different period of patients.

The volume of elution was 65ul, and cfDNA obtained was quantified using Qubit, and the cfDNA concentration was controlled to be not higher than 10 ng/. Mu.l. If the concentration of the extracted cfDNA is higher than 10 ng/. Mu.L, it can be diluted to 10 ng/. Mu.L with pure water.

For tissue samples, including but not limited to FFPE, fresh tissue samples, etc., it is suggested to use Qiagen-related kits for extraction. Wherein tissues and cells were subjected to DNA extraction using QIAamp DNA Mini Kit (Qiagen), and FFPE was subjected to DNA extraction using QIAamp DSP DNA FFPE Tissue Kit (Qiagen).

The volume of elution was 65ul, and the obtained gDNA was quantified using Qubit, and the gDNA concentration was controlled to not higher than 10 ng/. Mu.L. If the concentration of the extracted gDNA is higher than 10 ng/. Mu.L, it can be diluted to 10 ng/. Mu.L with pure water.

S102, mixing the DNA to be detected by using the primer probe set in the kit according to any one of claims 1 to 4 to obtain a PCR reaction mixture, sufficiently shaking and mixing, and centrifuging at 3000rpm for 1 minute to obtain an amplification solution;

specifically, in 4 test tubes, according to the requirements of Table 3, digital PCR premix (containing 2-200mM Tris-HCl,0.1-8mM dNTPs, 2-20mM Mg2+, 0.5-10U/reaction Taq DNA polymerase), PIK3CA mutation primer probe mix A (PIK 3CA E542/5/6-F, PIK3CA E542/5/6-R, PIK3CA H1047-F, PIK3CA H1047-R, PIK3CA 545K/FAM/, PIK3CA H1047-WT/HEX/, PIK3CA Q546R/ROX/, PIK3CA E542K/5 /), PIK3CA mutation primer mix B (PIK 3CA E542/5/6-R, PIK3CA H1047-F, PIK3CA H1047-R, PIK3CA C420-F, PIK3CA C420-R, PIK3CA H1047R/FAM/, PIK3CA C420-WT/HEX/, PIK3CA Q546E/ROX/, PIK3CA E545G/Cy5 /), PIK3CA mutation primer mix C (PIK 3CA E542/5/6-F, PIK3CA 542/5/6-R, PIK3CA H1047-F, PIK3CA H1047-R, PIK3CAC420-F, PIK3CA C420-R, PIK3CA H1047Y/FAM/, PIK3CA E542-WT/HEX/, PIK3CA C420R/ROX/, PIK3CA E545D/Cy5 /), PIK3CA mutation primer mix D (PIK 3CA 542/5/6-F, PIK3CA 542/5/6-R, PIK3CA 542/6-R, PIK3CA C420-R, PIK3CA H1047-K3 CA H1047-F, PIK3CA L5-F, PIK3CA L-K3 CA L, PIK3CA L-K420-WT/FAM/, PIK3CAE545/Q546-WT/HEX/, PIK3CA E545A/ROX /), a digital PCR reaction mixture was prepared, shaken well and mixed, and centrifuged at 3000rpm for 1 minute.

TABLE 3 preparation ratio of digital PCR mother liquor and PIK3CA mutation detection mixture

S103, adding the amplification solution into each PCR tube, adopting an extracted DNA sample or positive control or negative control to form a digital PCR reaction mixture, mixing and centrifuging to obtain a mixed solution;

specifically, the present application adds 6 μl of the above mixed solution, 14 μl of the extracted cfDNA sample or positive control or negative control to each PCR tube, forms 20 μl of digital PCR reaction mixture, caps the tube, mixes and centrifugates, each sample is added to 4 PCR tubes, and the 4 solutions are mixed in the PCR tubes.

S104, dripping oil for the primer probe group into a preset number of detection holes, distributing the mixed solution into the detection holes, and dripping sealing oil above the mixed solution to form a kit to be detected;

the microfluidic chip is placed on a chip holder in this application. Then 75 μl of the first generation oil for the probe was dispensed into the oil well on the kit. Then, 20. Mu.L of each tube prepared in step S103 was dispensed into the sample well, and after loading the digital PCR reaction mixture, 10. Mu.L of sealing oil was added to the reaction mixture. And covering the microfluidic cartridge with a sealing cover to obtain the prepared kit to be detected.

S105, performing thermal cycling on the kit to be detected by using a PCR thermal cycler during detection so as to perform PCR reaction;

in particular, the application transferred the clamps with all the kits into the PCR thermocycler SG32-3000 (Su Xie standard: 20232220073). The procedure for digital PCR thermal cycle amplification of the PIK3CA mutant was set as follows. Then click the "run" button, start the droplet generation and perform the thermal cycle. The specific steps, temperatures, times, heating rates and cycle numbers are shown in Table 4,

TABLE 4 detection of specific parameters

S106, comparing the concentration of the solution obtained after the PCR reaction with a preset threshold value, and determining mutation frequencies of corresponding sites according to comparison results; wherein, the preset threshold is divided according to the fluorescence signals of the positive quality control and the negative quality control.

Specifically, after the PCR reaction is completed, the digital PCR chip is placed in a Dscanner4-1000 (Su Xie standard: 20202220818) digital PCR device, and the device divides the threshold line according to the fluorescence signals of the positive quality control and the negative quality control. And the defined threshold line is suitable for a detected sample, the GeneCount (soft-copy logon No. 8251970) is analyzed by using the software of Dscanner4-1000 (Su Xie standard: 20202220818), the copy number concentration of different sites can be obtained, and the mutation frequency of the corresponding sites can be obtained through simple calculation.

The PIK3CA gene mutation detection kit in the PCR-based DNA provided by the invention can overcome the defects of a biorad digital PCR system, has advantages compared with the traditional fluorescence quantitative PCR (qPCR), and can realize an integrated system for generating liquid drops and realizing rapid PCR reaction on the same microfluidic chip. The method provided by the application has the following characteristics: 1) The user does not need to carry out manual operation of transferring the liquid drops after the liquid drops are generated; 2) The method fully utilizes the characteristics of small sample quantity of microfluidic treatment, adoption of film bonding (small heat capacity of a heating object) of a microfluidic chip and the like, and can realize rapid temperature rise and fall of the sample. The highest temperature rise and drop rate of the system can be set to be 1.6-10 ℃/s, which is the fastest in the current similar products, and the whole digital PCR reaction can be completed within 90 minutes; 3) The PIK3CA gene mutation detection kit is matched with the detection method provided by the invention, so that 0.1% of gene mutation can be detected from plasma free DNA; 4) By using the method, the free ctDNA in the blood plasma can be utilized to the greatest extent, the loss of the ctDNA is reduced, and the detection resolution is improved; 5) According to the invention, by judging and reading the negative and positive quality control in the kit, the negative and positive fluorescent droplet groups in the detection sample in the same batch of reaction are calibrated automatically, so that the mutation types in the sample are read automatically.

Specific test data are provided for testing using plasma samples from patients diagnosed with breast cancer, as compared to reagent results based on fluorescent quantitative PCR techniques, as shown in table 5,

TABLE 5 detection of plasma samples from patients diagnosed with breast cancer, comparison with the reagent results based on fluorescent quantitative PCR technique

Wherein N.A indicates undetected.

Four of the positive samples confirmed by the digital PCR test were selected, retested using NGS (high throughput sequencing method) and the results are shown in Table 6,

TABLE 6 retesting of positive samples confirmed by digital PCR detection

From the above experimental data, it is known that the digital PCR and NGS detection results have high consistency when the sample mutation frequency is 1-10%, and that the digital PCR exhibits higher sensitivity than the fluorescent quantitative PCR and NGS when the sample mutation frequency is 0.1-1%. NGS and fluorescent quantitative PCR were both detected for samples with mutation frequencies below 1% and judged negative.

To more precisely illustrate the effects of the present application, the present application is illustrated using the following comparative examples.

In the first comparative example, during the development of the method, the probe PIK3CA E545A-T1/ROX/(shown in the following Table 7) designed previously for detecting E545A was found to have a serious non-specific amplification in the actual detection amplification, and the effect of detecting E545A mutation could not be achieved.

TABLE 7 PIK3CA E545A-T1/ROX/Probe information

Mutant target spot

Specific mutations

Sequence number

Primer probe name

Sequence (5 'to 3')

5' modification

3' modification

E545

E545A

22

PIK3CA E545A-T1/ROX/

CTGCGCAGGAGA

ROX

BHQ1

Table 8 two sets of different probe comparison configuration tables

The PIK3CA E545A-T1/ROX/detection results are shown in FIG. 5 and FIG. 6. Wherein, FIG. 5 is a PIK3CA E545A mutation negative sample, and FIG. 6 is a PIK3CA E545A mutation positive sample.

The PIK3CA E545A/ROX/detection results are shown in FIGS. 7 and 8. Wherein, FIG. 7 is a PIK3CA E545A mutation negative sample, and FIG. 8 is a PIK3CA E545A mutation positive sample.

From the comparison results, when PIK3CA E545A-T1/ROX is used as a detection probe for detecting E545A, PIK3CA E545A mutation negative samples can non-specifically amplify a certain liquid drop which meets the fluorescence signal intensity of a positive liquid drop; in contrast, when PIK3CA E545A/ROX is used as a detection probe for detecting E545A, PIK3CAE545A mutation negative samples have no ROX fluorescent signal positive droplets and can be completely distinguished from PIK3CA E545A mutation positive samples.

In a second comparative example, in which Mg2+ in the digital PCR premix was adjusted, it was found that the signal effect of Mg2+ on the positive droplet cluster was very pronounced in this system and that the adaptation concentration of the primer probe was not the same for different sites, positive droplets could be substantially separated from negative droplets at Mg2+ concentrations of 5-14mM, but the degree of separation of the yin and yang droplets was much different at different concentrations.

Specifically, as shown in the comparison chart of FIG. 9, the fluorescence effect charts of PIK3CA 545/Q546-WT, PIK3CA E545K, PIK CA E542K and PIK3CA E545G are sequentially shown from top to bottom, and as can be seen from the comparison result, the PIK3CA E545/Q546-WT has better Mg2+ effect by using 8-10mM under the system; the PIK3CA E545K has better effect by using 5-14mM Mg2+ under the system; under the system, the PIK3CA E542K has better effect by using 8-12mM Mg2+; PIK3CA E545G showed better effect with 6-12mM Mg2+. To combine the best results, 8-10mM Mg2+ was considered for use in this system.

In the third comparative example, in the case of performing an example test, the amount of Taq DNA polymerase in the digital PCR premix was adjusted, and it was found that under this system, the signal effect of the amount of Taq DNA polymerase on the positive droplet cluster was very remarkable, and the amount of the adaptation enzyme for the primer probe at different sites was not the same, and at 3.75 to 10U of Taq DNA polymerase amount, positive droplets could be separated from negative droplets basically, but the degree of separation of the positive and negative droplets was quite different in the case of different enzyme amounts.

Specifically, as shown in the comparison chart of FIG. 10, the fluorescence effect charts corresponding to PIK3CA C420-WT, PIK3CA E545/Q546-WT, PIK3CA E542-WT and PIK3CA H1047-WT are sequentially shown from top to bottom, and as can be seen from the comparison result, the PIK3CA C420-WT has better effect by using 3.75U-10U Taq DNA polymerase under the system; under the system, the PIK3CA E545/Q546-WT has better effect by using 2.5U-10U Taq DNA polymerase; under the system, the PIK3CA E542-WT has better effect by using 1.25U-10U Taq DNA polymerase; PIK3CA H1047-WT uses 3.75U-10U Taq DNA polymerase with better effect under this system. To combine the best results, the test was performed under this system with Taq DNA polymerase considered with U at 3.75-10 mM.

The PIK3CA gene mutation detection kit in the PCR-based DNA provided by the application can use an extracted sample as much as possible under the premise of ensuring the elution efficiency by means of high sensitivity of digital PCR and large-volume loading (14 ul/hole, 56 ul/reaction) of the kit design. All mutation and wild type DNA can be detected on one microfluidic chip at a time, compared with a detection method of single reaction of a fluorescent quantitative 6-tube, the detection efficiency is improved, less sample size can be used, and the detection sensitivity can be improved by about 1 order of magnitude. The lower limit of fluorescence quantitative detection is 1% mutation frequency, and the lower limit of digital PCR detection is 0.1% mutation frequency.

In summary, the invention provides a PCR-based detection kit and a detection method for PIK3CA gene mutation in DNA, which can specifically detect 11 mutation sites and 4 wild type sites of PIK3CA of DNA (gDNA or cfDNA) in fresh tissue samples, paraffin embedded tissue samples or plasma samples at one time, and has the advantages of high sample utilization rate, high resolution, high sensitivity, good repeatability, simple and convenient operation, automatic interpretation and the like.

It can be understood that the above-provided method embodiments correspond to the above-described apparatus embodiments, and corresponding specific details may be referred to each other and will not be described herein.

It will be appreciated by those skilled in the art that embodiments of the present application may be provided as a method, system, or computer program product. Accordingly, the present application may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present application may take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, magnetic disk storage, optical storage, and the like) having computer-usable program code embodied therein.

The present application is described with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems) and computer program products according to embodiments of the application. It will be understood that each flow and/or block of the flowchart illustrations and/or block diagrams, and combinations of flows and/or blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.

These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means which implement the function specified in the flowchart flow or flows and/or block diagram block or blocks.

These computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A kit for detecting PIK3CA gene mutation in PCR-based DNA, comprising:

2. The kit of claim 1, wherein the primer probe set comprises:

a primer for H1047 locus comprising a primer consisting of PIK3CA H1047-FSEQ ID NO. 1 and a primer consisting of PIK3CA H1047-RSEQ ID NO. 2, or consisting of PIK3CA H1047-FSEQ ID NO. 1 and PIK3CA H1047-RSEQ ID NO. 2; and probes for the H1047 site: PIK3CA H1047R SEQ ID NO:7, PIK3CA H1047L SEQ ID NO:8, PIK3CA H1047Y SEQ ID NO:9, and/or PIK3CA H1047-WT SEQ ID NO:10;

a primer for the C420 site comprising a primer consisting of PIK3CA C420-F SEQ ID NO:3 and a primer consisting of PIK3CA C420-R SEQ ID NO:4, or consisting of PIK3CA C420-F SEQ ID NO:3 and PIK3CA C420-R SEQ ID NO:4, and a probe for the C420 site: 11 and/or 12 of PIK3CA C420-WT SEQ ID NO;

3. The kit of claim 1, wherein each of said primer-probe sets is labeled with two fluorescent dyes, one of which is a fluorescent reporter dye and the other of which is a quencher dye;

4. A kit according to claim 3, wherein each site of the probe is designed with a corresponding sequence comprising:

5. The kit according to claim 1, wherein,

the number of the detection holes is 4, and each detection hole is internally provided with a microfluidic pipeline;

the microfluidic pipeline is used for generating liquid drops and tiling;

6. The method for detecting and analyzing the PIK3CA gene mutation in the DNA based on the PCR is characterized by comprising the following steps:

obtaining DNA to be detected;

mixing the DNA to be detected by using the primer probe set in the kit according to any one of claims 1 to 4 to obtain a PCR reaction mixture, shaking and mixing thoroughly, and centrifuging at 3000rpm for 1 minute to obtain an amplification solution;

7. The method of claim 6, wherein the obtaining the test DNA comprises:

8. The method as recited in claim 6, further comprising: preprocessing the DNA to be detected, including:

9. The method of claim 8, wherein the step of determining the position of the first electrode is performed,

the preset concentration is 10 ng/. Mu.L.