CN111334580A

CN111334580A - PIK3CA gene mutation detection kit

Info

Publication number: CN111334580A
Application number: CN202010297730.8A
Authority: CN
Inventors: 蒋析文; 朱小亚; 潘翠园; 黄志文; 徐小解
Original assignee: Daan Gene Co Ltd of Sun Yat Sen University
Current assignee: Daan Gene Co Ltd of Sun Yat Sen University
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2020-06-26

Abstract

The invention provides a PIK3CA gene mutation detection kit, and particularly discloses a primer and a probe for detecting PIK3CA gene mutation and a kit comprising a primer probe mixed solution, which can detect the mutation types of a plurality of PIK3CA genes.

Description

PIK3CA gene mutation detection kit

Technical Field

The invention belongs to the field of biotechnology and tumor diagnosis, and particularly relates to a PIK3CA gene mutation detection method and a kit thereof.

Background

The phosphatidylinositol-3-kinase catalytic subunit α gene (phosphatydinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha, PIK3CA) is located on chromosome 3q26.32, 92kb long, consisting of 23 exons, and encodes the catalytic subunit p110 α of the class IA phosphatidylinositol 3 kinase (phosphatydinolitol 3-kinases, PI3Ks), whose main function is to catalyze the binding of 4,5-PIP2 to 3,4,5-PIP3 as a second messenger and to activate intracellular target proteins.

PIK3CA is an oncogene, and when the gene is mutated, the gene can continuously stimulate downstream AKT activation through a PI3K/AKT pathway, so that fibroblasts and mammary epithelial cells can be continuously proliferated, and apoptosis is inhibited, thereby being closely related to the occurrence and development of tumors.

Breast cancer is one of the most common malignancies in women, with incidence and mortality increasing year by year and becoming increasingly younger in recent years. According to research, about 8-40% of breast cancer patients have PIK3CA gene mutation, which is the most common breast cancer gene mutation besides p53 mutation and HER-2 amplification, and the main mutation hot spots are concentrated on the 9 th (E542K, E545X) and 20 th (H1047X) exons. The paraffin-embedded tissue sample is the most common sample type for clinical tumor gene mutation detection, the detection of the sample has low requirements on a detection method, but the tissue sample has high acquisition difficulty, can not be sampled for multiple times, has traumatism and has serious heterogeneity. In contrast, plasma samples can overcome the problems of difficult sampling, limited sampling times, heterogeneity and the like, and are ideal tumor gene detection sample types. However, studies show that the content of tumor free nucleic acid in plasma is lower, less than 1%, and therefore, the sensitivity requirement of the detection technology is higher.

At present, the detection commonly used for PIK3CA gene mutation comprises detection methods such as generation sequencing (Sanger sequencing), high-throughput sequencing, fluorescent quantitative PCR and the like. The methods have the defects of poor sensitivity, poor detection effect on a small amount of tumor free nucleic acid in blood plasma and the like, and only can detect paraffin-embedded tissue samples.

(1) One-generation sequencing technology, Sanger sequencing, essentially utilizes a DNA polymerase to extend primers bound to a template of a sequence to be determined until a chain terminating nucleotide is incorporated. Each sequencing consists of a set of four reactions, each containing all four deoxyribonucleoside triphosphates (dNTPs) mixed with a limited amount of a different dideoxynucleoside triphosphate (ddNTP). The extended oligonucleotide selectively terminates at G, A, T or C due to the absence of the 3-OH group required for extension of the ddNTP. The termination point is determined by the corresponding dideoxy reaction. The relative concentrations of each of dNTPs and ddNTPs can be adjusted so that a set of chain-terminating products of several hundred to several kilobases is obtained. They have a common starting point but terminate at different nucleotides and fragments of different sizes can be separated by high resolution denaturing gel electrophoresis.

Thereby obtaining a DNA base sequence. The defects are long period, high cost, easy cross contamination and low sensitivity.

(2) The high-throughput sequencing technology is a revolutionary change of the first-generation sequencing, can perform sequence determination on hundreds of thousands or even millions of DNA molecules at a time, has the characteristics of high throughput, high accuracy, high sensitivity and the like, but has higher cost and is not suitable for single-gene mutation detection.

(3) The fluorescent quantitative PCR is characterized in that a pair of primers is added and a specific fluorescent probe is added at the same time during PCR amplification, the probe is an oligonucleotide, and two ends of the probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. The 5 'end of the probe is marked with a fluorescent molecule, and the 3' end is marked with a fluorescent quenching molecule. When the probe is intact, Fluorescence Resonance Energy Transfer (FRET) occurs between the two. During PCR amplification, the probe is hydrolyzed by Taq enzyme 5'→ 3' exonuclease activity, whereby the distance between the fluorescent molecule and the quencher molecule increases, and a fluorescent signal is detected by a fluorescence monitoring system. When the fluorescent PCR is used for detecting mutation of a mutant gene, the specificity and the detection sensitivity are poor, and the fluorescent PCR is not suitable for detecting plasma free nucleic acid.

Therefore, there is an urgent need in the art to develop a technology capable of effectively detecting mutations of PIK3CA gene in plasma to meet clinical needs.

Disclosure of Invention

The invention aims to provide a PIK3CA gene mutation detection method and a kit thereof.

In a first aspect of the invention, there is provided a set of primer pairs for detecting mutations in PIK3CA gene, the set of primer pairs comprising:

a first primer pair group comprising a forward primer as set forth in SEQ ID No. 3; and, a reverse primer as set forth in SEQ ID No. 4.

In another preferred embodiment, the primer pair set further includes:

a second primer pair group, wherein the second primer pair group comprises a forward primer shown as SEQ ID No. 1; and, a reverse primer as set forth in SEQ ID No. 2.

In another preferred embodiment, the primer pair set further includes:

a third primer pair group, wherein the third primer pair group comprises a forward primer shown as SEQ ID No. 5; and, a reverse primer as set forth in SEQ ID No. 6.

In a second aspect of the present invention, there is provided a probe set for detecting a mutation in PIK3CA gene, the probe set comprising:

one or more mutant probes selected from the group consisting of: SEQ ID No.9, 11, 12, 13, 14, 15 and 16; and/or

One or more wild-type probes selected from the group consisting of: SEQ ID NO.10 and 17.

In another preferred example, the probe set includes:

one or more mutant probes selected from the group consisting of: SEQ ID No.7, 9, 11, 12, 13, 14, 15, 16, 18, 19 and 20; and/or

One or more wild-type probes selected from the group consisting of: SEQ ID NO.8, 10, 17 and 21.

In another preferred example, the 5' end of the mutant probe comprises a fluorescent reporter group; and/or, the 3' end of the mutant probe comprises a fluorescence quenching group.

In another preferred embodiment, the 5' end of the wild-type probe comprises a fluorescent reporter group; and/or, the 3' end of the wild-type probe comprises a fluorescence quenching group.

In another preferred embodiment, the mutant probe-labeled fluorescent reporter is different from the wild-type probe-labeled fluorescent reporter.

In a third aspect of the invention, a kit for detecting a mutation in PIK3CA gene is provided, the kit comprising the primer pair set of the first aspect of the invention.

In another preferred embodiment, the kit further comprises a set of probes according to the second aspect of the invention.

In another preferred embodiment, the kit comprises a first container, wherein the first container contains a first primer probe mixture, and the first primer probe mixture contains a primer sequence shown in SEQ ID NO: 3. 4, 16, 17.

In another preferred embodiment, the kit comprises a second container, wherein the second container contains a second primer probe mixture, and the second primer probe mixture contains a primer sequence shown in SEQ ID NO: 3. 4, 15, 17.

In another preferred embodiment, the kit comprises a third container, wherein the third container contains a third primer probe mixture, and the third primer probe mixture contains a primer sequence shown in SEQ ID NO: 3. 4, 14, 17.

In another preferred embodiment, the kit comprises a fourth container, wherein a fourth primer probe mixture is contained in the fourth container, and the fourth primer probe mixture comprises a nucleotide sequence shown in SEQ ID NO: 3. 4, 13, 17.

In another preferred example, the kit includes a fifth container, the fifth container contains a fifth primer probe mixture, and the fifth primer probe mixture contains a primer of SEQ ID NO: 3. 4, 12, 17.

In another preferred example, the kit includes a sixth container, the sixth container contains a sixth primer probe mixture, and the sixth primer probe mixture contains a nucleotide sequence represented by SEQ ID NO: 3. 4, 11, 17.

In another preferred embodiment, the kit comprises a seventh container, wherein the seventh container contains a seventh primer probe mixture, and the seventh primer probe mixture contains a primer of SEQ ID NO: 3. 4, 9, 10.

In another preferred embodiment, the kit includes an eighth container, the eighth container contains an eighth primer-probe mixture, and the eighth primer-probe mixture contains a nucleotide sequence represented by SEQ ID NO: 5. 6, 20 and 21.

In another preferred example, the kit includes a ninth container, the ninth container contains a ninth primer probe mixture, and the ninth primer probe mixture contains a primer of SEQ ID NO: 5. 6, 19 and 21.

In another preferred example, the kit includes a tenth container, the tenth container contains a tenth primer probe mixture, and the tenth primer probe mixture contains a primer of SEQ ID NO: 5. 6, 18 and 21.

In another preferred example, the kit includes an eleventh container, the eleventh container contains an eleventh primer probe mixture, and the eleventh primer probe mixture contains a primer of SEQ ID NO: 1. 2, 7 and 8.

In another preferred embodiment, the kit further comprises a twelfth container, and the twelfth container contains a PCR reaction premix. Preferably, the PCR reaction premix comprises one or more components selected from the group consisting of: hot start Taq enzyme, dNTPs, UDG enzyme, tris-hydroxymethyl aminomethane-hydrochloric acid buffer, Clarity^TMJN Solution(20×)。

In another preferred embodiment, the kit further comprises a thirteenth container comprising a positive control therein. Preferably, the positive control comprises one or more synthetic large fragments as set forth in SEQ ID nos. 29-39.

In another preferred embodiment, the kit further comprises a fourteenth container, wherein the fourteenth container contains a negative control. Preferably, the negative control is pure water.

In a fourth aspect of the present invention, there is provided a method for detecting a mutation in PIK3CA gene, the method comprising the steps of:

(1) providing a DNA sample of an object to be detected;

(2) preparing a dPCR reaction system and carrying out dPCR detection:

wherein, the dPCR reaction system comprises: the DNA sample provided in step (1), the primer pair according to the first aspect of the present invention, and the probe according to the second aspect of the present invention.

In another preferred example, the dPCR reaction system includes the DNA sample provided in step (1) and a primer-probe mixture selected from one or more of the first primer-probe mixture to the eleventh primer-probe mixture.

In another preferred example, the DNA sample may be from paraffin-embedded groups, fresh tissue, pleural effusion and plasma free nucleic acids; preferably the DNA sample may be from plasma-free nucleic acids.

In another preferred embodiment, the method is a detection method for non-diagnostic purposes.

In another preferred embodiment, the PCR reaction system further comprises a positive quality control substance, and/or a negative quality control substance.

In a fifth aspect of the invention, the use of the primer pair set of the first aspect of the invention and/or the probe set of the second aspect of the invention is provided for preparing a PCR detection kit, and the PCR detection kit is used for detecting PIK3CA gene mutation.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1: a schematic diagram of dPCR results of a negative control for detecting PIK3CA gene;

FIG. 2: schematic diagram of dPCR result of detecting PIK3CA gene wild type nucleic acid control;

FIG. 3: a schematic diagram of dPCR results of a positive control for detecting the C420R mutation site of the PIK3CA gene;

FIG. 4: a schematic diagram of dPCR results of positive control for detecting mutation sites of E542K of PIK3CA gene;

FIG. 5: a schematic diagram of dPCR results of a positive control for detecting the E545A mutation site of the PIK3CA gene;

FIG. 6: a schematic diagram of dPCR results of a positive control for detecting the E545D mutation site of the PIK3CA gene;

FIG. 7: a schematic diagram of dPCR results of a positive control for detecting the E545G mutation site of the PIK3CA gene;

FIG. 8: a schematic diagram of dPCR results of a positive control for detecting the E545K mutation site of the PIK3CA gene;

FIG. 9: a schematic diagram of dPCR results of positive control for detecting Q546E mutation site of PIK3CA gene;

FIG. 10: a schematic diagram of dPCR results of positive control for detecting Q546R mutation site of PIK3CA gene;

FIG. 11: a schematic diagram of dPCR results of a positive control for detecting H1047L mutation sites of the PIK3CA gene;

FIG. 12: a schematic diagram of dPCR results of a positive control for detecting H1047R mutation sites of the PIK3CA gene;

FIG. 13: a schematic diagram of dPCR results of a positive control for detecting H1047Y mutation sites of the PIK3CA gene;

FIG. 14: schematic diagram of dPCR result of detecting PIK3CA gene C420R mutation sample DNA template;

FIG. 15: schematic diagram of dPCR result of detecting PIK3CA gene E542K mutation sample DNA template;

FIG. 16: schematic diagram of dPCR result of detecting PIK3CA gene E545A mutation sample DNA template;

FIG. 17: schematic diagram of dPCR result of detecting PIK3CA gene E545D mutation sample DNA template;

FIG. 18: schematic diagram of dPCR result of detecting PIK3CA gene E545G mutation sample DNA template;

FIG. 19: schematic diagram of dPCR result of detecting PIK3CA gene E545K mutation sample DNA template;

FIG. 20: schematic diagram of dPCR result of detecting PIK3CA gene Q546E mutation sample DNA template;

FIG. 21: schematic diagram of dPCR result of detecting PIK3CA gene Q546R mutation sample DNA template;

FIG. 22: schematic diagram of dPCR result of detecting PIK3CA gene H1047L mutation sample DNA template;

FIG. 23: schematic diagram of dPCR result of detecting PIK3CA gene H1047R mutation sample DNA template;

FIG. 24: schematic diagram of dPCR result of detecting PIK3CA gene H1047Y mutation sample DNA template;

FIG. 25: schematic diagram of dPCR results of control universal primer pair 1 for detecting E542K, E545A, E545D, E545G, E545K, Q546E, and Q546R 7 mutation sites of PIK3CA gene;

FIG. 26: schematic diagram of dPCR results of control universal primer pair 2 for detecting E542K, E545A, E545D, E545G, E545K, Q546E, and Q546R 7 mutation sites of PIK3CA gene;

FIG. 27 is a schematic view showing: a schematic diagram of dPCR results of a control mutant probe for detecting the Q546E mutant site of the PIK3CA gene;

FIG. 28: schematic diagram of dPCR result of detecting PIK3CA gene Q546R mutation site control mutation type probe.

Detailed Description

The inventor obtains a kit and a method for multiplex detection of PIK3CA gene mutation through extensive and intensive research, can simultaneously detect 11 mutation types of PIK3CA gene, and has the advantages of simple detection process, more detectable mutation types, high sensitivity, small sample dependence and the like.

The invention provides a primer, a probe, a kit and a primer distribution mode for detecting multiple lung cancer gene mutations with high specificity and high sensitivity. The invention uses the fresh tissue sample, the paraffin embedded tissue sample, the hydrothorax ascites or the plasma sample to detect the gene mutation by the fluorescence detection technology, has the advantages of convenient material taking, high specificity, high sensitivity, good repeatability of the detection result, quick and simple operation and the like, can detect 11 mutations of the tumor hotspot gene mutation PIK3CA gene mutation at one time, greatly shortens the detection time, improves the sensitivity and the accuracy, and provides a reliable and convenient detection method.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Digital PCR (digital PCR, dPCR)

The dPCR can realize absolute quantification of nucleic acid molecules, and has higher sensitivity and specificity when detecting PIK3CA gene mutation. The method comprises the steps of distributing a fluorescent PCR reaction system into tens of thousands of tiny reactors by means of physical or chemical division, and carrying out single-molecule template PCR amplification by containing or not containing 1 or more copies of target nucleic acid molecules in each microreactor. After the amplification is finished, the copy number of the target gene in the original sample is calculated through the number of positive reaction units and a statistical method. The method has the advantages of high sensitivity, strong anti-interference capability, absolute quantification independent of a standard curve and the like.

The method has the advantages that the digital PCR technology is utilized to detect the mutation of the PIK3CA gene of the plasma sample for developing the breast cancer, the method has higher sensitivity, and can be used for detecting the limitations of large trauma, heterogeneity, low sensitivity and the like of the traditional detection method without depending on a tissue sample and can carry out real-time sampling for detection.

Because the sensitivity of the digital PCR is extremely high, the requirement on the specificity of the primer probe combination in a detection system is very high, and the design difficulty of the primer probe combination is higher.

The invention provides a method, a primer, a probe and a kit for qualitatively detecting 11 mutation sites of PIK3CA gene in a plasma sample of a breast cancer patient based on a digital PCR platform, and has the advantages of complete detection sites, simple operation, high absolute quantitative sensitivity, strong specificity, no wound and the like.

The technical scheme of the invention is as follows:

a method, primers, probes and a kit for detecting plasma sample PIK3CA gene mutation based on a digital PCR platform. Provides specific primers, probes and kit premix liquid required for detecting 11 sites of the PIK3CA gene.

The invention discloses a primer and a probe capable of detecting 11 mutations of PIK3CA gene in a breast cancer plasma sample, which are as follows:

the primers comprise primers for detecting 11 mutations of the PIK3CA gene:

wherein, the nucleotide sequence of the upstream primer for detecting the C420R mutation of the PIK3CA gene is shown as SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting the C420R mutation of the PIK3CA gene is shown as SEQ ID NO: 2 is shown in the specification;

the sequences of the universal upstream primers for detecting mutations of the PIK3CA genes E542K, E545A, E545D, E545G, E545K, Q546E and Q546R are shown as SEQ ID NO: 3, and the sequences of the universal downstream primers for detecting mutations of the PIK3CA genes E542K, E545A, E545D, E545G, E545K, Q546E and Q546R are shown as SEQ ID NO: 4 is shown in the specification;

the sequences of the universal upstream primers for detecting the mutations of the PIK3CA genes H1047L, H1047R and H1047Y are shown as SEQ ID NO: 5, the sequences of the universal downstream primers for detecting H1047L, H1047R and H1047Y mutations of the PIK3CA genes are shown as SEQ ID NO: 6 is shown in the specification;

the probes include probes for detecting 11 mutations in the PIK3CA gene:

wherein, the nucleotide sequence of the mutant probe for detecting the C420R mutation site of the PIK3CA gene is shown as SEQ ID NO: 7, and the nucleotide sequence of the wild-type probe for detecting the C420R mutation site of the PIK3CA gene is shown as SEQ ID NO: 8 is shown in the specification;

detecting a mutant probe nucleotide sequence aiming at the E542K mutant site of the PIK3CA gene as shown in SEQ ID NO: 9, and the nucleotide sequence of the wild-type probe for detecting the E542K mutation site of the PIK3CA gene is shown as SEQ ID NO: 10 is shown in the figure;

detecting that the nucleotide sequence of a mutant probe aiming at the E545A mutant site of the PIK3CA gene is shown as SEQ ID NO: 11, and the nucleotide sequence of the mutant probe for detecting the E545D mutant site of the PIK3CA gene is shown as SEQ ID NO: 12, and the nucleotide sequence of the mutant probe for detecting the E545G mutant site of the PIK3CA gene is shown as SEQ ID NO: 13, and the nucleotide sequence of the mutant probe for detecting the E545K mutant site of the PIK3CA gene is shown as SEQ ID NO: 14, and the nucleotide sequence of the mutant probe for detecting the Q546E mutant site of the PIK3CA gene is shown as SEQ ID NO: 15, and the nucleotide sequence of the mutant probe for detecting the Q546R mutant site of the PIK3CA gene is shown as SEQ ID NO: 16, and the nucleotide sequences of wild-type probes for detecting mutation sites of E545A, E545D, E545G, E545K, Q546E and Q546R of the PIK3CA genes are shown as SEQ ID NO: 17 is shown;

and (3) detecting the nucleotide sequence of a mutant probe aiming at the H1047L mutant site of the PIK3CA gene as shown in SEQ ID NO: 18, and the nucleotide sequence of the mutant probe for detecting the H1047R mutant site of the PIK3CA gene is shown as SEQ ID NO: 19, and the nucleotide sequence of the mutant probe for detecting the H1047Y mutant site of the PIK3CA gene is shown as SEQ ID NO: 20, and the nucleotide sequences of wild-type probes for detecting the mutation sites of the PIK3CA genes H1047L, H1047R and H1047Y are shown as SEQ ID NO: shown at 21.

Further, the SEQ ID NO: 7. 9, 11, 12, 13, 14, 15, 16, 18, 19, 20 nucleotide sequence is labeled with FAM at 5 'end and MGB at 3' end, and the nucleotide sequence of SEQ ID NO: 8. the nucleotide sequences 10, 17 and 21 are marked with VIC at the 5 'end and MGB at the 3' end.

Preferably, the final concentration of the upstream primer in the reaction system is 1 mu mol/L, the final concentration of the downstream primer in the reaction system is 1 mu mol/L, the final concentration of the mutant probe in the reaction system is 0.2 mu mol/L, and the final concentration of the wild-type probe in the reaction system is 0.2 mu mol/L;

the primer probe sequences of the 11 mutations of the PIK3CA gene are shown in the table below.

Table 1. detection of primer probe nucleotide sequence information for 11 mutations of PIK3CA gene:

the specific primer and the probe can accurately detect whether C420R, E542K, E545A, E545D, E545G, E545K, Q546E, Q546R, H1047L, H1047R and H1047Y mutation occurs in the PIK3CA gene, and the gene mutation ratio can be directly obtained while the mutation is detected. The mutant probe is adopted to detect the PIK3CA gene mutation copy number and the wild probe is adopted to detect the PIK3CA non-mutant gene copy number, and whether the PIK3CA has gene mutation and mutation degree are determined according to the copy number ratio.

The kit prepared by the specific primers and the probes can detect 11 mutation types of the PIK3CA gene based on the dPCR platform, provides reference for whether a patient needs targeted therapy or not, and can also be used for high-sensitivity early detection and curative effect monitoring of malignant tumor patients.

In a preferred embodiment of the invention, the invention provides a kit for detecting free nucleic acid PIK3CA gene mutation in breast cancer plasma, which comprises a primer probe mixed solution for preparing a dPCR reaction solution, a PCR reaction premixed solution, a control sample and RNase-free water, wherein the primer probe mixed solution comprises the following components as shown in Table 2,

TABLE 2 primer Probe mix

Preferably, the amino acid sequence of SEQ ID NO: 7. 9, 11, 12, 13, 14, 15, 16, 18, 19, 20 nucleotide sequence is labeled with FAM at 5 'end and MGB at 3' end, and the nucleotide sequence of SEQ ID NO: 8. the nucleotide sequences 10, 17 and 21 are marked with VIC at the 5 'end and MGB at the 3' end.

When PIK3CA gene is mutated, the mutant probe and the wild probe are combined with the target segment amplified by the upstream primer and the downstream primer to release FAM fluorescent signal and VIC fluorescent signal; when the PIK3CA gene is not mutated, the mutant probe cannot be combined with the amplified target fragment and has no fluorescent signal;

the PCR reaction premix contained the following ingredients, as shown in Table 3,

TABLE 3

The control included the following ingredients, as shown in table 4,

TABLE 4 control samples

Preferably, the wild type nucleic acid control cell strain DNA mixture contains fragmented Caco2 cell strain nucleic acid;

the artificially synthesized DNA mixture in the positive control contains:

artificially synthesized DNA fragment 1 for detecting C420R mutation site:

ATGGGGGTGTTTCTTCATTCTTTTTTTCTTACTGGTTTTTACTTTTTAAATTTGAAAGCTTTGCAGGGATCATAAGGATCTGTTCAGGCAAAGAACATGAAAGGGTTTACATTTTTATCATTTTAGTGTTTCTTATTCTCTATATCAAAAACATTCACAGATAAGTTAACAAGATCCTCATCAGGAGGAAAAGTAAATTGTTCACTACCATCCTCTAGTATCCTAACCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAA(SEQ ID NO.:28)

artificially synthesized DNA fragment 2 for detecting E542K mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTAAAATCACTGAGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:29)

artificially synthesized DNA fragment 3 for detecting E545A mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGCGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:30)

artificially synthesized DNA fragment 4 for detecting E545D mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGATCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:31)

artificially synthesized DNA fragment 5 for detecting E545G mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGGGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:32)

artificially synthesized DNA fragment 6 for detecting E545K mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTAAGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:33)

artificially synthesized DNA fragment 7 for detecting Q546E mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGAGGAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTT(SEQ ID NO.:34)

artificially synthesized DNA fragment 8 for detecting Q546R mutation site:

CCTAATCTGGTCTTGTTGTTGGCTAACTTCAGCAGTTACTATTCTGTGACTGGTGTAATATTAACCAAATAAATTACTGGATTTGTTCTACAAATATTATGTCTTAGATTGGTTCTTTCCTGTCTCTGAAAATAAAGTCTTGCAATGAAAATAAATTATTTTACAACAGTTAATTAGCAATGTAAAATTTATTGAAAATGTATTTGCTTTTTCTGTAAATCATCTGTGAATCCAGAGGGGAAAAATATGACAAAGAAAGCTATATAAGATATTATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGAGCGGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAGATTCTCTGTTTCTTTTTCTTTATTACAGAAAAAATAACTGAATTTGGCTGATCTCAGCATGTTTTTACCATACCTATTGGAATAAATAAAGCAGAATTTACATGATTTTTAAACTATAAACATTGCCTTTTTAAAAACAATGGTTGTAAATTGATATTTGTGGAAAATCATACTACATTGGTAGTTGGCACATTAAATGCTTTTT(SEQ ID NO.:35)

artificially synthesized DNA fragment 9 for detecting H1047L mutation site:

AGAACTACAATCTTTTGATGACATTGCATACATTCGAAAGACCCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCACTTCATGGTGGCTGGACAACAAAAATGGATTGGATCTTCCACACAATTAAACAGCATGCATTGAACTGAAAAGATAACTGAGAAAATGAAAGCTCACTCTGGATTCCACACTGCACTGTTAATAACTCTCAGCAGGCAAAGACCGATTGCATAGGAATTGCACAATCCATGAACAGCATTAGAATTTACAGCAAG(SEQ ID NO.:36)

artificially synthesized DNA fragment 10 for detecting H1047R mutation site:

AGAACTACAATCTTTTGATGACATTGCATACATTCGAAAGACCCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCACGTCATGGTGGCTGGACAACAAAAATGGATTGGATCTTCCACACAATTAAACAGCATGCATTGAACTGAAAAGATAACTGAGAAAATGAAAGCTCACTCTGGATTCCACACTGCACTGTTAATAACTCTCAGCAGGCAAAGACCGATTGCATAGGAATTGCACAATCCATGAACAGCATTAGAATTTACAGCAAG(SEQ ID NO.:37)

artificially synthesized DNA fragment 11 for detecting H1047Y mutation site:

AGAACTACAATCTTTTGATGACATTGCATACATTCGAAAGACCCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCATATCATGGTGGCTGGACAACAAAAATGGATTGGATCTTCCACACAATTAAACAGCATGCATTGAACTGAAAAGATAACTGAGAAAATGAAAGCTCACTCTGGATTCCACACTGCACTGTTAATAACTCTCAGCAGGCAAAGACCGATTGCATAGGAATTGCACAATCCATGAACAGCATTAGAATTTACAGCAAG(SEQ ID NO.:38)

the kit provided by the invention is suitable for the sample which is plasma free nucleic acid.

The kit provided by the invention is used for judging the detection effectiveness according to the following standards: and a negative control group, a wild type nucleic acid control group and a positive control group are arranged in each detection, and when the positive control group of the detection result is positive and the negative control group and the wild type nucleic acid control group are negative, the experimental result is effective. The detection sensitivity of the kit can reach 0.05%.

The invention also discloses a method for detecting the mutation number of the free nucleic acid PIK3CA gene in the breast cancer plasma, which comprises the following steps:

1. processing a sample to be detected and extracting a sample DNA template; preferably, the sample type to be tested is plasma;

2. preparing a dPCR reaction system, and mixing a DNA template of a sample to be detected, a specific primer probe mixed solution A1-A11, a PCR reaction premixed solution B and RNase-free water to prepare a dPCR reaction solution; the composition of the dPCR reaction solution is shown in Table 5,

TABLE 5.dPCR reaction solution

Specific primer probe mixture A1-A11	Each 1 mu L
		DNA template	5μL
PCR reaction premix B	8.25μL
		RNase-free water	Make up to 15. mu.L

3. Opening a DAAN Starry SKY sample pretreatment system, and taking out the eight-tube chip and the sample pretreatment auxiliary device from the chip box;

4. opening a cover of the eight-tube chip, inserting a sample combing plate in the sample pretreatment assistor into the eight-tube chip, and fixing the sample combing plate to a DAAN Starry SKY sample pretreatment system micro-reaction preparation module;

5. arranging a sample injector in the sample pretreatment assistor on a micro-reaction preparation module of the DAAN Starry SKY sample pretreatment system, and closing a latch;

6. sucking 15 mu L of PCR reaction mixed liquor to the triangular tip of the sample injector, and paying attention to avoid generating bubbles;

7. pressing a start button to slightly push the PCR reaction mixed solution onto the chip by the sample pusher;

8. releasing the latch, taking down the sample pushing device and the sample combing flat plate, taking out the chip, and observing and confirming whether the PCR reaction solution is uniformly smeared on the chip in the eight-way tube;

9. opening a DAAN Starry SKY sample pretreatment system closed module, inserting the prepared eight-tube chip carrying the PCR reaction liquid into a DAAN Starry SKY sample pretreatment closed module clamping groove, closing a DAAN Starry SKY sample pretreatment system closed module bin gate, and pressing a start button to enable the PCR reaction mixed liquid remained on the surface of the chip to completely enter a micro-reaction chamber to finish the preparation of the chip;

10. after the preparation of the chip is finished, taking out the eight-tube chip, observing whether liquid residue exists on the surface of the chip, and repeating for 9 times if the liquid residue exists;

11. the prepared chip is taken out and added with 235 mu L of confining liquid, the tube cover is tightly covered, and the chip is placed into a PCR instrument for amplification.

12. PCR amplification was set up with the hot lid temperature set to: the volume was 50. mu.L at 90 ℃. Conditions are as follows:

13. analyzing results by using a DAAN Starry SKY reader and software, and calculating the copy number of the target molecules in each sample based on a Poisson distribution statistical principle;

14. and judging whether the sample to be detected mutates according to the intensity and the proportion of each fluorescence signal, and further calculating the mutation rate.

The principles of dPCR detection employed in the present application are as follows:

the dPCR technology divides the reaction reagent in a single PCR tube into about ten thousand micro-reactions, each micro-reaction contains no nucleic acid target molecules to be detected or contains 1 to a plurality of nucleic acid target molecules to be detected, and each micro-reaction is used as an independent PCR reaction unit. After the PCR process is finished, the micro-reactions are subjected to fluorescence detection one by one, and negative/positive reactions are identified. The microreaction containing different DNA templates releases different fluorescent signals, and the microreaction without templates does not generate fluorescent signals. And finally, calculating the copy number of the target molecule to be detected according to the Poisson distribution statistical principle and the proportion of positive micro-reaction. Because the judgment of the dPCR result only judges whether amplification exists or not and does not depend on the Ct value, the tolerance capability to the PCR reaction inhibitor is greatly improved, and accurate quantification can be realized without a reference substance and a standard curve, thereby providing a brand-new technical idea and means for the patent.

In a preferred embodiment of the invention, the method for detecting the mutation of the free nucleic acid PIK3CA gene in the breast cancer plasma comprises the following steps:

step one, extracting a DNA template of a sample to be detected

Extracting 8-10mL of peripheral blood of a breast cancer patient, placing the breast cancer patient in a blood collection tube of EDTA or a sodium citrate anticoagulant, marking to ensure that label information is correct, and storing the breast cancer patient at 4 ℃. After plasma is centrifugally separated, nucleic acid extraction or purification reagent of Daan Gen corporation of Zhongshan university is used, and the nucleic acid extraction is carried out according to the instruction of a kit; it is suggested that a micro-spectrophotometer (such as INVITROGEN QUBIT 4.0Fluorometer REF Q32866) is adopted to detect the extracted nucleic acid, the purity of the nucleic acid is required to meet the condition that the ratio of A260/A280 is in the range of 1.8-2.2, the concentration is not lower than 1 ng/mu L, and the template nucleic acid can be directly used for subsequent experiments or stored at-80 ℃ for standby, so that repeated freezing and thawing are avoided.

Step two, preparation of dPCR system

Preparation before preparation of the dPCR system: taking out primer probe mixed solution A1-A11, PCR reaction premixed solution B, RNase-free water and the like in the kit, melting at room temperature, vortexing, shaking uniformly, centrifuging for 10 seconds, and preparing a dPCR system; the dPCR system is constructed as shown in table 6:

TABLE 6 dPCR System

Primer probe mixture A1-A11	Each 1 mu L
		PCR reaction premix B	8.25μL
RNase-free water	Make up to 10. mu.L

The nucleotide sequence information of the primer probes is as described above;

step three, sample adding

Taking 5 mu L of the sample DNA template prepared in the step one and 5 mu L of each control sample in the kit, and adding the samples into eight connecting tubes of the dPCR reaction system prepared in the step two to ensure that the total volume of the dPCR reaction solution in each tube is 15 mu L; tightly covering the eight-connecting-tube cover, fully mixing uniformly, and centrifuging at a high speed for 10 seconds for preparing the micro-reaction; control samples of the kit are shown in table 7:

TABLE 7 control samples of the kit

The wild type nucleic acid control sample is a wild type nucleic acid sample containing PIK3CA gene and is derived from Caco2 cell line DNA; the artificially synthesized DNA fragments in the positive control are artificially synthesized DNA fragment 1 containing the C420R mutant gene, artificially synthesized DNA fragment 2 containing the E542K mutant gene, artificially synthesized DNA fragment 3 containing the E545A mutant gene, artificially synthesized DNA fragment 4 containing the E545D mutant gene, artificially synthesized DNA fragment 5 containing the E545G mutant gene, artificially synthesized DNA fragment 6 containing the E545K mutant gene, artificially synthesized DNA fragment 7 containing the Q546E mutant gene, artificially synthesized DNA fragment 8 containing the Q546R mutant gene, artificially synthesized DNA fragment 9 containing the H1047L mutant gene, artificially synthesized DNA fragment 10 containing the H1047R gene mutation, artificially synthesized DNA fragment 11 containing the H1047Y gene mutation, and the cell strain DNA fragments are derived from Caco2 cell strains, respectively; the negative control sample was RNase-free water.

Step four, preparing micro reaction and PCR amplification

Taking 15 mu L of prepared dPCR reaction solution, automatically and uniformly subdividing the dPCR reaction solution into 10000 nano-scale reaction units by means of a DAAN Starry SKY sample pretreatment system instrument, and directly carrying out PCR amplification. Reaction conditions for PCR amplification:

step five, reading and analyzing results

The eight-tube chip is used for detecting the amplification result by using 'biochip reader' software, and information such as experiment naming, fluorescent dye, gene name, sample name and the like is set. After the result detection is finished, opening data analysis in a software interface to analyze the result and checking the values of fluorescence signals of the FAM channel and the VIC channel; the threshold value of the microreaction signal can be adjusted by the reference of the negative control sample, the positive control sample and the non-template control sample and the distribution of the fluorescence signal.

In other embodiments, the fluorophore may be any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY 5. The quenching group can be selected from any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

The invention selects specific primers and probes through a large number of tests, and combines, optimizes and verifies the specific primers and probes to finally and preferably select the optimal probe combination which can synchronously amplify, has high amplification efficiency and good specificity.

In the invention, common upstream and downstream primers are designed, and the least primers are used while a plurality of mutation sites are identified, so that the cost is saved.

The sequence combination comprises a plurality of primers and probes required by gene mutation region detection, and can complete the detection of 11 gene loci at one time.

The invention has the beneficial effects that:

the invention provides a method, a primer, a probe and a kit for detecting 11 mutations of free nucleic acid PIK3CA gene in breast cancer plasma.

By using the specific primer and the probe provided by the invention, whether PIK3CA gene mutation exists in a breast cancer patient can be detected quickly and highly sensitively.

The invention can detect the mutation rate of 0.05% and has high sensitivity. The method has the advantages of noninvasive and simple process operation, low concentration of required samples, stable and efficient performance, high accuracy, high specificity and the like, can distinguish the difference of single copies, realizes absolute quantification in the true sense, is automatic in data analysis, and can observe results in real time.

The invention is suitable for monitoring the mutation state of the PIK3CA gene of a breast cancer patient and guiding the use of a targeted drug. Dynamic tracking of therapeutic effect is achieved, thereby early identification of drug resistance or disease progression and guidance of interventional therapy. The method is a feasible way for exploring early diagnosis and high-efficiency treatment of the breast cancer, and is worthy of popularization and application.

In addition, the method of the present invention is also applicable to non-diagnostic purposes, for example, in the development process of new drugs, gene mutation information used as an intermediate result is obtained by using the detection method of the present invention, and the gene mutation information can be used as the requirement of public health management, and can also be used for the research of PIK3CA gene mutation and the development of targeted new drugs.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1: reagent kit

The components, packages and quantities (20 reaction/box) of the kit for detecting the mutation of the breast cancer plasma free nucleic acid PIK3CA gene provided by the embodiment are shown in Table 8,

TABLE 8 kit Components, packaging and quantities

Example 2: sensitivity detection and minimum detection rate experiment

The wild-type nucleic acid control sample is a wild-type nucleic acid containing fragmented PIK3CA gene and is derived from Caco2 cell line DNA; the sensitivity reference substance is prepared by respectively mixing 11 kinds of PIK3CA mutant gene template DNA and Caco2 cell strain DNA according to a certain proportion, wherein the mutation rates (%) of the mixed liquid are respectively 1%, 0.1% and 0.05%; wherein, 11 kinds of PIK3CA mutant gene template DNA are respectively derived from artificially synthesized DNA segments 1-11; negative control is RNase-free water;

taking 5 mu L of each of the negative control, the wild type nucleic acid control and the sensitivity reference substance, and adding the sample into eight connecting tubes of the dPCR reaction system prepared in the second step to ensure that the total volume of the dPCR reaction solution in each tube is 15 mu L; tightly covering the eight-connecting-tube cover, fully mixing uniformly, and centrifuging at a high speed for 10 seconds for the preparation of the micro-reaction;

taking 15 mu L of prepared dPCR reaction solution, automatically and uniformly subdividing the dPCR reaction solution into 10000 nano-scale reaction units by means of a DAAN Starry SKY sample pretreatment system instrument, and directly carrying out PCR amplification.

Reaction conditions for PCR amplification:

the eight-tube chip is used for detecting the amplification result by using 'biochip reader' software, and information such as experiment naming, fluorescent dye, gene name, sample name and the like is set. After the result detection is finished, opening data analysis in a software interface to analyze the result and checking the values of fluorescence signals of the FAM channel and the VIC channel; the threshold value of the micro-reaction signal can be adjusted through the reference of the negative control sample, the positive control sample and the non-template control sample and the distribution of the fluorescence signal;

the sensitivity and the minimum detection rate of the present invention were measured by a dPCR system, and when the theoretical mutation rates (%) of the mixed solution of the above-mentioned positive control samples were 1%, 0.1% and 0.05%, respectively, the actually measured mutation rates (%) were as shown in Table 9,

TABLE 9 results of sensitivity detection

The sensitivity detection result of the kit conforms to a theoretical value, and the primer and the probe have better specificity and good sensitivity detection; when the mutation rate of a positive sample mixed by the copy number of 11 PIK3CA mutation sites and the DNA copy number of a Caco2 cell line is 0.05%, the dPCR system can stably detect the corresponding mutation positivity, so that the detectable mutation rate (%) of the invention is 0.05%.

Example 3: accuracy detection

Preparing an accuracy reference product according to the copy number of the measured control sample

Respectively mixing 11 kinds of PIK3CA mutant gene template DNA and Caco2 cell strain DNA according to a certain proportion to prepare mixed liquid with mutation rates (%) of 1 percent respectively; wherein, 11 kinds of PIK3CA mutant gene template DNA are respectively derived from artificially synthesized DNA segments 1-11;

2 repeated experiments are carried out on each accuracy reference product, and the total number is 22; taking 5 mu L of C420R mutation site accuracy reference product, 5 mu L, E545A mutation site accuracy reference product, 5 mu L, E545D mutation site accuracy reference product, 5 mu L, E545G mutation site accuracy reference product, 5 mu L, E545K mutation site accuracy reference product, 5 mu L, Q546E mutation site accuracy reference product, 5 mu L, Q546R mutation site accuracy reference product, 5 mu L, H104L mutation site accuracy reference product, 5 mu L, H1047R mutation site accuracy reference product, 5 mu L, H1047Y mutation site accuracy reference product, and adding the 5 mu L of the mutation site accuracy reference product into an eight-connected tube of the dPCR reaction system prepared in the second step, so that the total volume of the dPCR reaction solution is 15 mu L; tightly covering the eight-connecting-tube cover, fully mixing uniformly, and centrifuging at a high speed for 10 seconds for the preparation of the micro-reaction;

Reaction conditions for PCR amplification:

the accuracy of the kit of the invention was tested using the dPCR system to obtain the results as shown in Table 10,

table 10: accuracy test results

According to the results shown in the table, the positive rate of the detection result of the accuracy of each quality control product is 100%, and the accuracy detection of the kit meets the requirements.

Example 4: clinical application experiment

5-10mL of peripheral blood of 30 breast cancer patients is extracted and placed in blood collection tubes of EDTA or sodium citrate anticoagulant, and tissue samples of 30 patients providing blood samples are subjected to first-generation sequencing detection and are definitely PIK3CA gene mutation types; the samples are marked and the label information is ensured to be correct, and the samples are stored at 4 ℃. After centrifugally separating plasma within 3h, extracting nucleic acid by using a nucleic acid extraction or purification reagent of Daan Gen corporation of Zhongshan university according to a kit instruction; it is suggested that a micro-spectrophotometer (such as a Qubit 2.0 nucleic acid protein quantifier) is adopted to detect the extracted nucleic acid, the purity of the nucleic acid is required to meet the condition that the ratio of A260/A280 is in the range of 1.8-2.2, the concentration is not lower than 1 ng/mu L, and the template nucleic acid can be directly used for subsequent experiments or stored at-80 ℃ for standby, so that repeated freeze thawing is avoided.

Taking 5 mu L of DNA template of each sample and 5 mu L of each control sample in the kit, and adding the DNA template and the control samples into eight connecting tubes of the dPCR reaction system prepared in the second step to ensure that the total volume of the dPCR reaction solution of each tube is 15 mu L; tightly covering the eight-connecting-tube cover, fully mixing uniformly, and centrifuging at a high speed for 10 seconds for the preparation of the micro-reaction;

Reaction conditions for PCR amplification:

the detection result is as follows: of the 30 samples, 8 samples were positive for the PIK3CA mutation, and the others were negative, and the consistency of the detected result and the result of the first-generation sequencing detection was 100%.

Comparative example 1

In the research process, dozens of groups of PCR primer and probe combinations are screened aiming at target sequences of all mutation sites, and finally, primer and probe combinations with sensitivity and specificity capable of being used for dPCR detection are screened.

For example, the present inventors expect to be able to design common primers for the E542K, E545A, E545D, E545G, E545K, Q546E, and Q546R mutation sites, and to be able to secure excellent sensitivity and specificity.

For the above 3 mutation sites, the inventors designed some typical primer sequences as follows:

control universal primer pair 1:

a universal upstream primer: 5'GCTCAAAGCAATTTCTACACG 3' (SEQ ID NO: 22)

A universal downstream primer: 5'GAATCTCCATTTTAGCACTTACCT 3' (SEQ ID NO: 23)

Control universal primer pair 2:

a universal upstream primer: 5'CTAGAGACAATGAATTAAGGGAA 3' (SEQ ID NO: 24)

A universal downstream primer: 5'CTCCATTTTAGCACTTACCTGT 3' (SEQ ID NO: 25)

The specific detection steps, detection conditions and probe sequences are the same as those in the above examples, and each primer pair is used for separate detection and test.

The detection result using the control universal primer set 1 is shown in FIG. 25, and the detection result shows that the primer set has poor discrimination effect. The detection results using the control universal primer set 2 are shown in FIG. 26, and indicate that the primer set does not amplify positively.

Comparative example 2

In the research process, the inventor needs to optimize specific probes in addition to the primers. In experiments, it is found that sometimes a difference of one base in a probe may cause a significant change in detection results.

For example, typical probes tested for the Q546E, and Q546R mutation sites include:

control mutant probe 1 for Q546E mutation site:

5'GAAATCACTGAGGAGGAGA 3'(SEQ ID NO.:26)

control mutant probe 2 for Q546R mutation site:

5'AATCACTGAGCGGGAG 3'(SEQ ID NO.:27)

the results of the detection by using the control mutant probes respectively show that the Q546E mutation site can be normally detected in the dPCR detection system by using the SEQ ID NO. 26, but the Q546R mutation site can also be detected at the same time, which indicates that the specificity of the SEQ ID NO. 26 is poor, as shown in FIG. 27; 27 in the dPCR detection system, the Q546R mutation site can be normally detected, but the distinguishing effect is poor, as shown in FIG. 28. In addition, it was found that some probes in the dPCR detection system can normally detect the target mutation site, but strong non-specific amplification occurs when detecting a wild-type sample.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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ttgcagggat cataaggatc tgttcaggca aagaacatga aagggtttac atttttatca 120

ttttagtgtt tcttattctc tatatcaaaa acattcacag ataagttaac aagatcctca 180

tcaggaggaa aagtaaattg ttcactacca tcctctagta tcctaacctg gtcttgttgt 240

tggctaactt cagcagttac tattctgtga ctggtgtaat attaaccaaa taaattactg 300

gatttgttct acaaatatta tgtcttagat tggttctttc ctgtctctga aaataaagtc 360

ttgcaatgaa aataaattat tttacaacag ttaattagca atgtaaaatt tattgaaaat 420

gtatttgctt tttctgtaaa tcatctgtga a 451

<210>29

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>29

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc taaaatcact gagcaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>30

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>30

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact gcgcaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>31

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>31

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact gatcaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>32

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>32

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact gggcaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>33

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>33

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact aagcaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>34

<211>634

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>34

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact gaggaggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc tttt 634

<210>35

<211>635

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>35

cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 60

ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 120

tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 180

tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 240

aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 300

ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 360

atcctctctc tgaaatcact gagcgggaga aagattttct atggagtcac aggtaagtgc 420

taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 480

atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 540

aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 600

tactacattg gtagttggca cattaaatgc ttttt 635

<210>36

<211>301

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>36

agaactacaa tcttttgatg acattgcata cattcgaaag accctagcct tagataaaac 60

tgagcaagag gctttggagt atttcatgaa acaaatgaat gatgcacttc atggtggctg 120

gacaacaaaa atggattgga tcttccacac aattaaacag catgcattga actgaaaaga 180

taactgagaa aatgaaagct cactctggat tccacactgc actgttaata actctcagca 240

ggcaaagacc gattgcatag gaattgcaca atccatgaac agcattagaa tttacagcaa 300

g 301

<210>37

<211>301

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>37

agaactacaa tcttttgatg acattgcata cattcgaaag accctagcct tagataaaac 60

tgagcaagag gctttggagt atttcatgaa acaaatgaat gatgcacgtc atggtggctg 120

gacaacaaaa atggattgga tcttccacac aattaaacag catgcattga actgaaaaga 180

taactgagaa aatgaaagct cactctggat tccacactgc actgttaata actctcagca 240

ggcaaagacc gattgcatag gaattgcaca atccatgaac agcattagaa tttacagcaa 300

g 301

<210>38

<211>301

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>38

agaactacaa tcttttgatg acattgcata cattcgaaag accctagcct tagataaaac 60

tgagcaagag gctttggagt atttcatgaa acaaatgaat gatgcatatc atggtggctg 120

gacaacaaaa atggattgga tcttccacac aattaaacag catgcattga actgaaaaga 180

taactgagaa aatgaaagct cactctggat tccacactgc actgttaata actctcagca 240

ggcaaagacc gattgcatag gaattgcaca atccatgaac agcattagaa tttacagcaa 300

g 301

Claims

1. A set of primer pairs for detecting a mutation in PIK3CA gene, the set of primer pairs comprising:

2. The set of primer pairs of claim 1, further comprising:

3. The set of primer pairs of claim 1, further comprising:

4. A probe set for detecting mutations in PIK3CA gene, the probe set comprising:

5. The probe set of claim 4, wherein the 5' end of the mutant probe comprises a fluorescent reporter group; the 3' end of the mutant probe comprises a fluorescence quenching group; and/or

The 5' end of the wild-type probe comprises a fluorescent reporter group; the 3' end of the wild-type probe contains a fluorescence quenching group.

6. A kit for detecting a mutation in PIK3CA gene, comprising the primer set of claim 1.

7. The kit of claim 6, further comprising the probe set of claim 4.

8. The kit of claim 6, comprising a first container, wherein the first container comprises a first primer probe mixture comprising the nucleic acid sequence of SEQ ID NO: 3. 4, 16, 17; and/or

The kit comprises a second container, wherein a second primer probe mixed solution is contained in the second container, and the second primer probe mixed solution contains a nucleotide sequence shown by SEQ ID NO: 3. 4, 15, 17;

the kit comprises a third container, wherein a third primer probe mixed solution is contained in the third container, and the third primer probe mixed solution contains a nucleotide sequence shown by SEQ ID NO: 3. 4, 14, 17; and/or

The kit comprises a fourth container, wherein a fourth primer probe mixed solution is contained in the fourth container, and the fourth primer probe mixed solution contains a nucleotide sequence shown by SEQ ID NO: 3. 4, 13, 17; and/or

The kit comprises a fifth container, wherein a fifth primer probe mixed solution is contained in the fifth container, and the fifth primer probe mixed solution contains a nucleotide sequence shown by SEQ ID NO: 3. 4, 12, 17; and/or

The kit comprises a sixth container, wherein a sixth primer probe mixed solution is contained in the sixth container, and the sixth primer probe mixed solution contains a nucleotide sequence shown by SEQ ID NO: 3. 4, 11, 17.

9. A method for detecting a mutation in PIK3CA gene, comprising the steps of:

(1) providing a DNA sample of an object to be detected;

(2) preparing a dPCR reaction system and carrying out dPCR detection:

wherein, the dPCR reaction system comprises: the DNA sample provided in step (1), the primer set of claim 1, and the probe set of claim 4.

10. Use of the primer pair set of claim 1 and/or the probe set of claim 4 for preparing a PCR detection kit for detecting PIK3CA gene mutation.