CN118932050A - Detection kit and detection method for multiple mutations of human PIK3CA gene - Google Patents

Abstract

The invention discloses a human PIK3CA gene mutation detection kit, which is characterized by comprising the following components: an internal reference detection reagent comprising an internal standard specific primer, an internal standard specific probe, and a dNTP solution; PIK3CA mutation detection reagent, which comprises PIK3CA gene mutation type specific primer, mutation type specific primer probe and Mg ²⁺ aqueous solution; taq DNA polymerase, PIK3CA negative and positive quality controls, which contain fragments of wild type and mutant and internal standard genes, respectively. The amplification product is detected by the Taq Man probe, so that the mutant gene is amplified but the wild type is not amplified basically, thereby achieving high detection specificity and high sensitivity.

Description

Detection kit and detection method for multiple mutations of human PIK3CA gene

Technical Field

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a detection kit and a detection method for multiple mutations of a human PIK3CA gene.

Background

The PIK3CA (Phosphatidylinositol-KINASE CATALYTICALPHA) gene is an oncogene detected by Volinia et al in 1994, a lipid kinase encoding gene, and a homolog of the retrovirus V-P3K oncogene in cells. PIK3CA is a 34kb long gene on chromosome 3q26.3, which consists of 20 exons encoding 1068 amino acids that produce a set of 124KD long proteins, p110 catalytic subunits encoding class i phosphatidylinositol-3-kinases (PI 3 Ks), PI3Kp110a.

PIK3CA is an important kinase molecule located on the cascade signaling pathway downstream of the Epidermal Growth Factor Receptor (EGFR), playing a role as a protooncogene in the development of tumors. It was found that about 80% of the PIK3CA gene mutations in human tumors are in the helical and kinase domains, i.e., exons 9 and 20. The PIK3CA gene mutation causes abnormal activation of p110a, in addition, ras protein combined with GTP is directly combined with p110a, and simultaneously, a growth factor receptor with a phosphorylated tyrosine residue, a connexin and a regulatory subunit p85 of PI3Ks are also involved in the abnormal activation of the PI3Ks together through a complex mechanism, the activated PI3Ks further activate downstream Akt and other signal molecules, the activated Akt activates or inhibits the downstream protein thereof through phosphorylation so as to regulate proliferation, differentiation, apoptosis, migration and the like of cells, and finally, the occurrence of tumors is caused. PIK3CA is used as a key component of PI3K-AKT-mTOR signaling pathway to regulate proliferation, differentiation, apoptosis, angiogenesis and the like of tumor cells. The PIK3CA gene is a common mutant gene of colon cancer, liver cancer and breast cancer except p53 mutation and HER-2 amplification, which is discovered in recent years, mutation sites are concentrated on exon 9 and exon 20, and the most common 5 mutation hot spots are: E542K, E545K, E545D and H1047R/L, wherein the E542K mutation frequency was 5.7%, the E545K mutation frequency was 8.5%, the E545D mutation frequency was 0.2% and the H1047R/L mutation frequency was 18.8%. Researches show that trastuzumab has poor curative effect on PIK3CA gene mutation population of PIK3-AKT (phosphatidylinositol-3 kinase/serine-threonine protein kinase B) pathway; additional studies have shown that colorectal cancer patients with mutations in the PIK3CA gene develop resistance to cetuximab; in addition, 2% of lung cancer patients are accompanied by PIK3CA mutations, and the mutated patients are associated with TKI-type drug resistance. In recent years, a new research target, namely PIK3CA gene, has emerged in colorectal cancer treatment. The research shows that the mutation rate of the colorectal cancer PIK3CA gene is obviously lower than that of related foreign results, and the analysis reasons are probably that the PIK3CA gene mutation is related to the environmental difference between the race and the region, and the mutation sites of the gene are different between different race regions. Although further research is needed about the influence of PIK3CA mutation on colorectal cancer, the existing research shows that the discovery of PIK3CA high-frequency mutation and mutation hot spot areas has certain clinical significance for colorectal cancer gene diagnosis and targeted therapy selection.

PI3Ks are a class of specifically phosphorylated inositol phospholipid 3-hydroxy kinases called PI3Ks family, class I PI3Ks are heterodimers consisting of one p110 catalytic subunit and one p85 regulatory subunit, activated by growth factor receptors and insulin receptors, etc., PI3Ks are normally expressed in the cytoplasm to produce inositol lipids possessing a second messenger role, playing a very important role in the regulation of cell morphology, viability, invasiveness, apoptosis and cell cycle. The PI3K-Akt signaling pathway is an important signaling pathway regulating cell function, and when PIK3CA gene is activated in the form of gene amplification or point mutation, its transcription and translation level is raised, akt is overactivated through PI3K/Akt pathway, growth and reproduction of cells are promoted without restriction, apoptosis of cells is inhibited, and finally tumor is caused. The mutation of the gene can occur in various cancers such as breast cancer, colorectal cancer, lung cancer and the like, wherein the mutation rate in the breast cancer is 26%, the mutation rate in the colorectal cancer is 25%, the mutation rate in the lung cancer is 2%, and the mutation state of the PIK3CA gene directly or indirectly influences the medication and prognosis of various cancers such as colorectal cancer, breast cancer, lung cancer and the like. The targeted therapeutic drugs for colorectal cancer are anti-EGFR monoclonal antibodies, such as cetuximab and panitumumab, which can block EGFR downstream signal pathway conduction, thereby playing an anti-tumor role.

Although the detection of colorectal cancer mutation is most direct by adopting a gene sequencing technology, for cost reasons, there are also some detection technologies based on PCR to detect mutation of colorectal cancer related mutant genes such as PIK3CA, for example, chinese patent No. CN201110344347 discloses a primer, a probe and a kit for human PIK3CA gene driven mutation, relating to the detection of gene mutation, chinese patent No. CN201210234133 discloses a kit for detecting colorectal cancer PIK3CA gene hotspot mutation sites, chinese patent No. CN201510213332 discloses a kit for detecting PIK3CA gene mutation and a detection method thereof, the kit contains an LNA lock nucleic acid modified specific probe aiming at PIK3CA gene mutation sites, chinese patent No. CN202010297730 discloses a kit for detecting PIK3CA gene mutation by dPCR, and Chinese patent No. CN202211089987 discloses a kit and a detection method thereof. The method adopted by the patent comprises the technology of driving mutation fluorescence PCR method, pyrosequencing technology, dPCR, sequencing analysis, enrichment analysis method and the like, and the technology may have the possibility that a primer for detecting mutation easily detects wild PIK3CA, and the influence on the interpretation of the result causes false positive. The most advanced technology for detecting PIK3CA gene mutation internationally adopts site-specific fluorescent quantitative PCR, and the method has the advantages of improving detection sensitivity and detection timeliness, but the real-time fluorescent quantitative detection technology has the problem of high false positive rate, namely poor specificity.

In view of the above, in order to solve the shortcomings and actual needs of the prior art, a kit for detecting multiple PIK3CA gene mutations with simple operation, strong specificity, and high speed and accuracy is needed.

Disclosure of Invention

The invention aims to solve the problems of low detection efficiency, easiness in sample pollution, long detection time, poor accuracy and the like of the conventional PIK3CA gene mutation detection method, and provides a multi-mutation detection kit and a detection method for human PIK3CA genes. The rapid and accurate detection of the unlimited sites is realized, the operation is simple, and the specificity is strong.

In order to achieve the above purpose, the present invention provides the following technical solutions: a human PIK3CA gene mutation detection kit comprising:

(1) An internal reference detection reagent comprising an internal standard specific primer, an internal standard specific probe, and a dNTP solution;

(2) PIK3CA mutation detection reagent, which comprises PIK3CA gene mutation type specific primer, mutation type specific primer probe and Mg ²⁺ aqueous solution;

(3) Taq DNA polymerase, PIK3CA negative quality control and positive quality control, which respectively contain wild type and mutant type and fragments of internal standard genes;

Wherein, the PIK3CA gene mutant in the PIK3CA mutation detection reagent is selected from the group consisting of:

Mutation name	Base mutant forms	Amino acid mutant forms	Exons
				B1(E542K)	GAA>AAA	Glu542Lys	9
B2(E545K)	GAG>AAG	Glu545Lys	9
				B3(E545D)	GAG>GAT	Glu545Asp	9
B4(H1047R)	CAT>CGT	His1047Arg	20
				B5(H1047L)	CAT>CTT	His1047Leu	20

The nucleotide sequences of the related primers and probes are as follows:

IC: internal standard specificity

Internal standard upstream primer IL: ATGGGAAACAGACGAATGC A

Internal standard downstream primer IR: ACAGCAAATAAAAGAAACTAAAACA internal standard probe IP: TCCTGAGACTTCCACACTGATGC B1: E542K mutations

Mutant upstream primer M1L: CTACACGAGATCCTCTCTGCA mutant downstream primer M1R: GAGATCAGCCAAATTCAGTTACT mutant probe M1P: ATGGAGTCACAGGTAAGTGCTAAAATGG B2: E545K mutant upstream primer M2L: CGAGATCCTCTCTCTGAAATTACTA mutant downstream primer M2R: GAGATCAGCCAAATTCAGTTAGT mutant probe M2P: ATGGAGTCACAGGTAAGTGCTAAAATGG B3: E545D mutations

Mutant upstream primer M3L: GATCCTCTCTCTGAAATCACTCGT mutant downstream primer M3R: GAGATCAGCCAAATTCAGTTACT mutant probe M3P: ATGGAGTCACAGGTAAGTGCTAAAATGG B4: H1047R mutation

Mutant upstream primer M4L: ATGAAACAAATGAATGATGCATG mutant downstream primer M4R: CAGAGTGAGCTTTCATTTTGT mutant probe M4P: TGGCTGGACAACAAAAATGGATTGG B5: H1047L mutation

Mutant upstream primer M5L: CATGAAACAAATGAATGATGCTAT A

Mutant downstream primer M5R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M5P: TGGCTGGACAACAAAAATGGATTGG B6: wild type

Wild type upstream primer WL: AGCTCAAAGCAATTTCTACACGT A

Wild type downstream primer WR: GAGATCAGCCAAATTCAGTTACT A

Wild type probe WP: ATGGAGTCACAGGTAAGTGCTAAAATGG.

Further included are DNA polymerase and uracil DNA glycosylase having anti-contamination effect. A method for detecting human PIK3CA gene mutation comprises the following steps:

(1) Extracting nucleic acid in a sample to obtain a nucleic acid extracting solution;

(2) Preparing a fluorescent PCR amplification reaction system, and respectively mixing a nucleic acid extracting solution, DNA polymerase, dNTPs, primers and probes in 6 PCR reaction tubes, wherein the nucleotide sequences of the primers and probes contained in each reaction tube are respectively shown in the following table:

Tube numbering	Primer(s)	Probe with a probe tip
			1	IL、IR、M1L、M1R	M1P
2	IL、IR、M2L、M2R	M2P
			3	IL、IR、M3L、M3R	M3P
4	IL、IR、M4L、M4R	M4P
			5	IL、IR、M5L、M5R	M5P
6	IL、IR、WL、WR	WP

Wherein, the PIK3CA gene mutant in the PIK3CA mutation detection reagent is selected from the group consisting of:

The nucleotide sequences of the related primers and probes are as follows:

IC: internal standard specificity

Internal standard upstream primer IL: ATGGGAAACAGACGAATGC A

Internal standard downstream primer IR: ACAGCAAATAAAAGAAACTAAAACA internal standard probe IP: TCCTGAGACTTCCACACTGATGC B1: E542K mutations

Mutant upstream primer M1L: CTACACGAGATCCTCTCTGCA mutant downstream primer M1R: GAGATCAGCCAAATTCAGTTACT mutant probe M1P: ATGGAGTCACAGGTAAGTGCTAAAATGG B2: E545K mutant upstream primer M2L: CGAGATCCTCTCTCTGAAATTACTA mutant downstream primer M2R: GAGATCAGCCAAATTCAGTTAGT mutant probe M2P: ATGGAGTCACAGGTAAGTGCTAAAATGG B3: E545D mutations

Mutant upstream primer M3L: GATCCTCTCTCTGAAATCACTCGT mutant downstream primer M3R: GAGATCAGCCAAATTCAGTTACT mutant probe M3P: ATGGAGTCACAGGTAAGTGCTAAAATGG B4: H1047R mutation

Mutant upstream primer M4L: ATGAAACAAATGAATGATGCATG A

Mutant downstream primer M4R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M4P: TGGCTGGACAACAAAAATGGATTGG B5: H1047L mutation

Mutant upstream primer M5L: CATGAAACAAATGAATGATGCTAT A

Mutant downstream primer M5R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M5P: TGGCTGGACAACAAAAATGGATTGG B6: wild type

Wild type upstream primer WL: AGCTCAAAGCAATTTCTACACGT A

Wild type downstream primer WR: GAGATCAGCCAAATTCAGTTACT A

Wild type probe WP: ATGGAGTCACAGGTAAGTGCTAAAATGG A

(3) Respectively adding the nucleic acid extracting solution into a mutant reaction system and a wild reaction system, and performing fluorescent quantitative PCR reaction;

(4) And analyzing the detection result according to the difference value of the FAM signal Ct value of the mutant reaction system and the wild reaction system.

Wherein the nucleic acid extracting solution is extracted from a sample by a magnetic bead extraction method, and the extracting process comprises the following steps: adding nucleic acid extract into the sample, preserving heat, adding magnetic beads, uniformly mixing, applying a magnetic field, discarding the liquid, washing, and eluting.

Wherein each reaction tube further comprises an internal control nucleic acid, an internal control primer, and an internal control probe.

Wherein the PCR reaction conditions are as follows:

50 ℃ for 2 min 94 ℃ for 5min

94℃10 Seconds 55℃45 seconds 40 cycles

The fluorescent groups are FAM, the fluorescent groups of the internal standard primer probes are VIC/HEX, and all the probes are marked with the fluorescent groups and the quenching groups.

The reagents required by the magnetic bead extraction method comprise:

nucleic acid extract comprising guanidine isothiocyanate, sodium ethylenediamine tetraacetate, tween-20, sodium perchlorate, ethanol and pH buffer;

a washing liquid for the first washing comprising sodium perchlorate and ethanol;

A wash solution for the second wash comprising ethanol and a pH buffer.

Compared with the prior art, the invention has the following beneficial effects: the invention adopts real-time fluorescence PCR combined with ARMS-PCR, which is totally called an amplification impeding mutation system (amplification refractory mutation system, ARMS), also called allele characteristic PCR (allele-SPECIFIC PCR, AS-PCR) and the like, to detect the known mutant genes. The method comprises the steps of designing two 5 '-end primers, wherein one primer is complementary to normal DNA, the other primer is complementary to mutant DNA, and for homozygous mutation, two parallel PCR is carried out by respectively adding the two primers and the 3' -end primer, and only the primer which is completely complementary to the mutant DNA can be extended to obtain a PCR amplification product. If the mismatch is located at the 3' end of the primer, the PCR is rendered incapable of extension. ARMS-PCR is a commonly used method for detecting gene mutation in the laboratory at present. The ARMS-PCR method has high detection sensitivity, and can detect mutant genes with the mutation ratio of 1% or lower in tumor cells.

Compared with the existing detection kit, the PIK3CA mutant gene in the DNA sample is detected by a real-time fluorescent PCR method by combining a specific primer and a Taq Man probe technology, the specific primer is used for amplifying the mutant target sequence, meanwhile, the amplification of a wild type is blocked, and the amplification product is detected by the Taq Man probe, so that the mutant gene is amplified but the wild type is not amplified basically, and the high detection specificity and the high sensitivity are achieved.

The invention combines ARMS-PCR technology to make the primer designed by mutant base amplify only the corresponding mutant template, and makes the primer form mismatch with the template by introducing mismatched base at the 3' end of the primer to prevent false extension. The specificity level of the system is further improved, so that the detection of low-content mutant sequences under the high wild-type gene background is realized;

2. Compared with the prior art, the invention introduces ARMS-PCR technology, has simple operation, low detection cost and accurate detection result, and has good in-situ application value and market value;

3. The method can detect whether 5 human PIK3CA mutant genes exist in a sample by using a small amount of PCR tubes in one round of PCR, ensures the accuracy, the reliability, the specificity, the sensitivity and the repeatability, and particularly can avoid the generation of false positive results of erroneously amplifying wild PIK3CA by using mutant primers; and the conventional commercial real-time fluorescence PCR equipment is used, no transformation is needed, the operation is convenient, the cost is saved, and the internal control monitoring can be used for further controlling the false negative.

4. The invention establishes a real-time fluorescence PCR amplification reaction system to realize the rapid detection of PIK3CA gene mutation; and the operation is simple, and the result is easy to read. The Ct value of the VIC/HEX signal reflects the relevant information of a real-time fluorescence PCR amplification reaction system (whether the sample nucleic acid extraction is normal, whether the operation process is accurate, etc.);

5. The detection sensitivity reaches 0.02%, the detection process is closed-tube reaction, and the possibility of pollution is reduced;

6. the safety is good, the whole system does not contain toxic and harmful substances, and the system is harmless to experimental staff and the environment.

The present invention will be described in detail below with reference to specific drawings and examples for the purpose of facilitating understanding. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention refers to publications that are incorporated herein by reference in their entirety for the purpose of more clearly describing the invention as if repeated herein were set forth in their entirety.

Drawings

FIG. 1 shows a schematic diagram of qPCR results of a control for detecting wild-type nucleic acid of the PIK3CA gene, wherein lines 1 and 2 are internal control IC and line 3 is external control CtW. The resulting curves can be clearly distinguished, but have reached the limit.

FIG. 2 shows a schematic diagram of qPCR results positive for detecting any mutation site of 5 mutations in the PIK3CA gene, wherein the 1 st line is internal control IC, the 2 nd line is CtM, and the 3 rd line is external control CtW. The resulting curves can be clearly distinguished, but have reached the limit.

FIG. 3 shows a schematic diagram of qPCR results positive for detecting 5 mutation sites of PIK3CA gene, wherein the number 1 is internal control IC, the number 2 is CtM, and the number 3 is external control CtW. The resulting curves can be clearly distinguished, but have reached the limit.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Hereinafter, the invention will be described by means of specific examples.

EXAMPLE 1 extraction of nucleic acids

The test sample containing the wild type and mutant genes of human PIK3CA can be purchased from the national ministry of health temporary examination center and is a standard for extracting nucleic acid.

The extraction of nucleic acid is carried out according to a conventional magnetic bead extraction method, and in order to simultaneously adapt to the extraction of a large number of human PIK3CA gene mutant nucleic acids, the steps are as follows:

90uL of nucleic acid extract (formula and final concentration: guanidine isothiocyanate 1.5M, ethylenediamine tetraacetic acid sodium (pH8.0) 15mM, tween-20% 3% (W/W), sodium perchlorate 1M, ethanol 50% (V/V), tris-HCl (pH8.0) 10 mM) was added to 1uL of standard, and the mixture was incubated at 42℃for 10min,

Then 10uL was addedD-Beads DNA magnetic bead suspension (50 mg/mL, available products) is mixed uniformly by shaking, and then sleeved on a magnetic frame to apply a magnetic field, and the liquid is discarded;

Then 200uL of washing solution A (formula and final concentration: sodium perchlorate 1.5M and ethanol 50% (V/V)) was added, washing was carried out, washing solution A was discarded,

Then 200uL of washing liquid B (the formula and the final concentration are 70 percent (V/V) of ethanol) is added, washing liquid B is discarded after washing,

Finally, adding eluent (formula and final concentration: tris-HCl (pH8.0) 10 mM), preserving heat at 60 ℃ for 10min, and sucking out and retaining the liquid, thus obtaining the nucleic acid extract.

Example 2 human PIK3CA real-time PCR assay

1, Primer and probe sequences and fluorescent markers

The synthesis of the above-mentioned nucleotide sequence primers and probes was delegated.

Respectively entrusting synthesis of a fluorescence labeling FAM at the 5 'end of a probe of a wild type nucleotide sequence and a mutant nucleotide sequence such as M1P, M2P, M3P, M4P, M5P, WP, and marking a quenching group BHQ at the 3' end; the 5 'end of the probe with the internal standard nucleotide sequence shown as IP is entrusted with a synthesized marker fluorescence marker VIC, and the 3' end is marked with a quenching group BHQ; other nucleic acids or portions are not labeled.

2, Grouping of PCR reactions

A total of 6 PCR tubes were taken, containing the corresponding primers and probes, respectively, according to the following table:

3, PCR reaction conditions

The total volume of the PCR reaction system is 25 mu L, wherein the final concentration of each component is as follows: 15uL of nucleic acid extract (100-fold dilution); the concentration of the internal standard primer is 2.0 pmol/. Mu.L, and the concentration of the internal standard probe is 0.75 pmol/. Mu.L; the concentration of the mutation primer is 1.5 pmol/. Mu.L, and the concentration of the mutation probe is 1.0 pmol/. Mu.L; the control primer concentration was 2.0 pmol/. Mu.L and the mutation probe concentration was 0.75 pmol/. Mu.L; mg2+ (MgCl 2) concentration was 35mmol, tris-HCl (pH 8.0) concentration was 10.0mmol, dNTP concentrations were 1.0mmol each, UNG enzyme content was 0.2. Mu.m, taq DNA polymerase was 2.5. Mu.m, glycerol 15% (V/V), and the balance deionized water.

The reaction thermal cycle conditions were as follows:

FAM and VIC detection wavelengths were collected by fluorescence using an ABI 7500 real-time fluorescence PCR instrument.

4, Judging the result

The baseline and threshold settings were the ABI 7500 fluorometer default auto settings. According to the determination method shown in the third aspect, the detection result of the sample to be detected is determined.

Exemplary test results are shown in fig. 1-3. The above test was repeated 100 times with samples containing different mutations and wild-type PIK3CA, and the results were all correct, indicating that the invention has good accuracy and reliability. The method can detect 1% multi-site mutation in 2 ng/. Mu.L human genome nucleic acid, has no mutual interference in detection and false negative result, especially has good control on wild type in mutation detection, has no false positive result of wild type PIK3CA, and achieves hypersensitive detection sensitivity.

Specifically, in a first aspect, the present invention provides for simultaneous detection of 5 PIK3CA gene mutations using 5 mutant reaction solutions, while providing 1 wild-type reaction solution, and analyzing the detection results based on the difference in Ct values of mutant reaction systems and wild-type reaction systems, i.e., external control FAM signals.

The kit for detecting the colorectal cancer PIK3CA gene hotspot mutation site comprises PCR amplification reaction liquid and yin-yang quality control products, and is characterized in that: the kit comprises an upstream primer and a downstream primer and a probe for detecting a No. 9 exon E542K, E545K, E545D mutant gene, an upstream primer and a downstream primer and a probe for detecting a No. 20 exon H1047R, H1047L mutant gene, an internal standard, an upstream primer and a downstream primer and a probe for a wild type gene, and the sequences are as follows:

IC: internal standard upstream primer IL: ATGGGAAACAGACGAATGC A

Internal standard downstream primer IR: ACAGCAAATAAAAGAAACTAAAACA internal standard probe IP: TCCTGAGACTTCCACACTGATGC B1: E542K mutations

Mutant upstream primer M1L: CTACACGAGATCCTCTCTGCA mutant downstream primer M1R: GAGATCAGCCAAATTCAGTTACT mutant probe M1P: ATGGAGTCACAGGTAAGTGCTAAAATGG B2: E545K mutant upstream primer M2L: CGAGATCCTCTCTCTGAAATTACTA mutant downstream primer M2R: GAGATCAGCCAAATTCAGTTAGT mutant probe M2P: ATGGAGTCACAGGTAAGTGCTAAAATGG B3: E545D mutations

Mutant upstream primer M3L: GATCCTCTCTCTGAAATCACTCGT mutant downstream primer M3R: GAGATCAGCCAAATTCAGTTACT mutant probe M3P: ATGGAGTCACAGGTAAGTGCTAAAATGG B4: H1047R mutation

Mutant upstream primer M4L: ATGAAACAAATGAATGATGCATG mutant downstream primer M4R: CAGAGTGAGCTTTCATTTTGT mutant probe M4P: TGGCTGGACAACAAAAATGGATTGG B5: H1047L mutation

Mutant upstream primer M5L: CATGAAACAAATGAATGATGCTAT mutant downstream primer M5R: CAGAGTGAGCTTTCATTTTGT mutant probe M5P: TGGCTGGACAACAAAAATGGATTGG B6: wild type

Wild type upstream primer WL: AGCTCAAAGCAATTTCTACACGT A

Wild type downstream primer WR: GAGATCAGCCAAATTCAGTTACT A

Wild type probe WP: ATGGAGTCACAGGTAAGTGCTAAAATGG A

The primers in the above technical scheme are reasonably designed so as to amplify only the part containing the codons E542K, E K/D and H1047R/L of the PIK3CA gene. The specific primer of the present invention is one of the inventions of the present invention, and the primer can be prepared by a method for preparing the primer in a usual manner by a person skilled in the art. When preparing the primers according to the invention, a suitable assay sample can be obtained which is an amplified DNA fragment and has a specific length.

Preferably, the PCR reaction solution comprises DNA polymerase, dNTPs, PCR reaction buffer solution and water.

Preferably, the DNA polymerase is selected from Taq DNA polymerase.

In a second aspect, as a preferred embodiment, the method for detecting PIK3CA gene mutation for the purpose of non-disease diagnosis and/or treatment comprises:

(1) Genomic DNA to be analyzed is conventionally collected and the test sample is paraffin-embedded slice (preferably, extracted by a magnetic bead method).

(2) Preparing a real-time fluorescence PCR amplification system, and respectively mixing a nucleic acid extracting solution, DNA polymerase, dNTPs, primers and probes in 6 PCR reaction tubes, wherein the nucleotide sequences of the primers and probes contained in each reaction tube are respectively shown in the following table:

In addition to these 6 PCR tubes, other PCR tubes may be included, for example as negative control tubes, in which the nucleic acid extract is replaced with water; as a further example, a positive control tube, wherein the nucleic acid extract is replaced with a sample known to be present with mutant or wild-type PIK3 CA.

In this document, the term "number" is used to distinguish between multiple objects of the same class (i.e., to denote that the objects assigned to different numbers are not identical) and may be used to refer to and reference, rather than to a specific number that is deterministic. Specific numbers may be remapped in a one-to-one correspondence with other numbers.

The nucleic acid extract may be directly detected, but it is preferable that the sample is treated to obtain a nucleic acid extract and then detected. Therefore, the nucleic acid extract is preferably an extract obtained by extracting nucleic acids in a sample, for example, by a magnetic bead extraction method. The nonspecific magnetic bead extraction method adopts paramagnetic particles with nonspecific nucleic acid adsorption substances coated on the surfaces, nucleic acid can be adsorbed on the surfaces of the paramagnetic particles under the conditions of low pH (e.g. pH value of 5-7) and high salt concentration, and after magnetic separation and full washing, the nucleic acid can be eluted under the conditions of high pH (e.g. pH value of 8-9) and low salt concentration, so that a sample enriched with nucleic acid (e.g. target nucleic acid) can be used for PCR test; magnetic bead extraction methods with specific adsorption (e.g., hybridization adsorption and immunoadsorption) may also be used. Such procedures are well known to those skilled in the art and see also Zheng Xiufen et al, template DNA magnetic bead extraction, journal of Chinese legal medicine, 18 (3): 107-108; chinese patents 200610030229.5, 200710118802.2, 201110105181.0, etc.

Preferably, the magnetic bead extraction method is further optimized, is more reliable for extracting nucleic acid in human tissue samples, and is convenient in steps.

The process of extracting nucleic acid from the test sample is as follows: nucleic acid extract (preferably nucleic acid extract comprising guanidine isothiocyanate, sodium ethylenediamine tetraacetate, tween-20, sodium perchlorate, ethanol and pH buffer (e.g. Tris-HCl)) is added to the sample, magnetic beads are added after incubation, after mixing well, the liquid is discarded after applying a magnetic field, followed by washing (preferably washing twice, more preferably wherein the washing liquid used for the first washing comprises sodium perchlorate and ethanol, and even more preferably wherein the washing liquid used for the second washing comprises ethanol), and eluting (preferably the eluting liquid used for the elution comprises pH buffer (e.g. Tris-HCl)).

Thus, in the method of the first aspect of the invention, the nucleic acid extraction solution is extracted from the sample, for example, wherein the extraction is by magnetic bead extraction. That is, it is preferable that the method of the first aspect of the present invention includes a step of extracting nucleic acids in a sample to obtain a nucleic acid extract.

(3) Respectively adding the genome DNA into each detection reaction system and each quality control reaction system to perform fluorescent quantitative PCR reaction; in addition, a negative-positive control corresponding to each mutation is arranged, the same operation is carried out with the genome DNA of the sample to be detected, and whether the sample to be detected has the mutation is judged according to the Ct value analysis detection result. The real-time fluorescent quantitative PCR instrument of the invention collects fluorescent signals simultaneously in the PCR amplification process according to the set requirements. The multichannel fluorometer performs multichannel fluorescence acquisition according to preset, and the acquired fluorescence channels are FAM and VIC/HEX. The kit is provided with an internal standard system in the amplification detection system, and is used for monitoring the whole process from nucleic acid extraction to amplification detection.

In the real-time fluorescent PCR reaction, the Ct value represents the number of cycles that each PCR reaction tube experiences when the fluorescent signal reaches the threshold value set by default in the real-time fluorescent PCR instrument, which has good reproducibility, and thus can be used as an excellent indicator of the interpretation result. In the method of the first aspect of the present invention, for step (3), wherein the Ct value calculated as a result of fluorescence detection of one target nucleic acid is <40, then such target nucleic acid is present in the sample; if the Ct value calculated as a result of fluorescence detection of a target nucleic acid is >40, such target nucleic acid is not present in the sample. This is the experience we have found through a large number of long-term experiments, and is excellent in reliability and repeatability. Thus in the method of the first aspect of the invention, an internal control is also preferably employed. The internal control comprises an internal control nucleic acid, an internal control primer, and an internal control probe.

(4) The amplification system in the above step was 25. Mu.L, and it included 10. Mu.L of the amplification reaction solution and 15. Mu.L of the genomic DNA of the sample to be tested. The detection sensitivity of the method of the first aspect of the present invention can be further improved by increasing the concentration of the probe. Therefore, it is preferred that in the method of the first aspect of the present invention, the concentration of each target nucleic acid probe in the single PCR reaction tube is greater than 5pmol/ml, preferably 6 to 12pmol/ml, more preferably 7 to 10pmol/ml, and most preferably 8pmol/ml.

In addition, other reagents required for the PCR reaction, such as salts, are further mixed in the single PCR reaction tube in step (1). Glycerol concentration is also preferred to extend the durability of enzyme activity under PCR conditions. In a specific embodiment of the invention, the salt is preferably a Mg salt. Other pH buffers (e.g., phosphate buffers, etc.) can be readily selected by those skilled in the art to adjust to the appropriate pH, and other soluble salts (e.g., KCl, etc.) can be readily selected to adjust the ionic strength. In addition, antioxidants (reducing agents), protein (enzyme) protective agents (e.g., bovine Serum Albumin (BSA), human Serum Albumin (HSA), etc.) may be further mixed. The choice of these ingredients is well known to those skilled in the art.

One skilled in the art can design PCR reaction conditions based on the primers and the corresponding nucleic acid to be PCR amplified. In order to balance the amplification conditions of different mutants in the detection of the present invention, the present inventors have long sought out that the conditions are:

And analyzing the detection result according to the Ct value difference of the mutant reaction system and the wild reaction system, namely the external control FAM signal. Firstly, the negative control FAM signal is not hatched or the Ct value is more than 40, the internal standard, namely the VIC/HEX signal, meets the Ct value less than or equal to 30, the Ct value of the positive control FAM signal is less than or equal to 30, and the internal standard, namely the VIC/HEX signal, is not hatched or is not hatched. For judging whether a sample to be tested is mutant, firstly, the Ct value of internal standard (VIC/HEX) signals is required to be less than or equal to 30, the Ct value of FAM signals obtained by a mutant reaction system is set to CtM, the Ct value of FAM signals obtained by a wild reaction liquid system (external control FAM) is set to CtW, and then, the judgment is carried out according to the difference delta Ct= CtM-CtW between the Ct values of FAM signals of the mutant reaction system and the wild reaction system:

contains PIK3CA: the difference value of FAM signal Ct value in the detection reaction system of the primer and the probe of E542K (c.1624G > A) mutation and the external control reaction system is more than 10 and is less than or equal to 10 and is E542K (c.1624G > A) mutation type;

Contains PIK3CA: the difference between the detection reaction system of the E545K (c.1633G > A) mutant primer and probe and the FAM signal Ct value in the external control reaction system is more than 10 and less than or equal to 10 is the wild type, and is E545K (c.1633G > A) mutant;

Contains PIK3CA: the difference value of FAM signal Ct value in the detection reaction system of the primer and the probe of E545D (c.1635G > T) mutation and the external control reaction system is more than 10 and less than or equal to 10 is the wild type, and is E545D (c.1635G > T) mutation type;

Contains PIK3CA: the difference between the detection reaction system of the primers and the probes of the H1047R (c.3140A > G) mutation and the FAM signal Ct value in the external control reaction system is more than 10 and less than or equal to 10, is a wild type, and is H1047R (c.3140A > G) mutation type;

Contains PIK3CA: the difference between the detection reaction system of the H1047L (c.3140A > T) mutant primer and probe and the FAM signal Ct value in the external control reaction system is more than 10 and less than or equal to 10 is the wild type, and is E545D H1047R (c.3140G > A) mutant.

The kit of the second aspect of the invention may further comprise reagents required for a magnetic bead extraction method, preferably wherein the reagents required for a magnetic bead extraction method comprise:

nucleic acid extracts, preferably nucleic acid extracts comprising guanidine isothiocyanate, sodium ethylenediamine tetraacetate, tween-20, sodium perchlorate, ethanol and pH buffer (e.g. Tris-HCl);

The washing liquid used for the first washing preferably comprises sodium perchlorate and ethanol;

The washing liquid used for the second washing preferably comprises ethanol; and the eluent used for elution, preferably it contains a pH buffer (e.g., tris-HCl).

It is also preferable that the kit of the second aspect of the present invention further comprises a reagent vessel for detecting the nucleic acid extract, and more preferably a reagent vessel for performing real-time PCR detection.

In a third aspect, the invention provides the use of a kit according to the second aspect of the invention for the preparation of a detection reagent product for use in a method of detecting human PIK3CA gene mutations. In this context, the detection reagent product may be the detection kit itself, or may be a larger packaged product incorporating a plurality of detection kits. From the foregoing, one skilled in the art will readily understand the components of the assay kit and the process flow therein.

Claims (8)

1. A kit for detecting mutation in the human PIK3CA gene, comprising:

(1) An internal reference detection reagent comprising an internal standard specific primer, an internal standard specific probe, and a dNTP solution;

(2) PIK3CA mutation detection reagent, which comprises PIK3CA gene mutation type specific primer, mutation type specific primer probe and Mg ²⁺ aqueous solution;

(3) Taq DNA polymerase, PIK3CA negative quality control and positive quality control, which respectively contain wild type and mutant type and fragments of internal standard genes;

Wherein, the PIK3CA gene mutant in the PIK3CA mutation detection reagent is selected from the group consisting of:

Mutation name Base mutant forms Amino acid mutant forms Exons B1(E542K) GAA>AAA Glu542Lys 9 B2(E545K) GAG>AAG Glu545Lys 9 B3(E545D) GAG>GAT Glu545Asp 9 B4(H1047R) CAT>CGT His1047Arg 20 B5(H1047L) CAT>CTT His1047Leu 20

The nucleotide sequences of the related primers and probes are as follows:

IC: internal standard specificity

Internal standard upstream primer IL: ATGGGAAACAGACGAATGC A

Internal standard downstream primer IR: ACAGCAAATAAAAGAAACTAAAACA A

Internal standard probe IP: TCCTGAGACTTCCACACTGATGC A

B1: E542K mutations

Mutant upstream primer M1L: CTACACGAGATCCTCTCTGCA A

Mutant downstream primer M1R: GAGATCAGCCAAATTCAGTTACT A

Mutant probe M1P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B2: E545K mutations

Mutant upstream primer M2L: CGAGATCCTCTCTCTGAAATTACTA A

Mutant downstream primer M2R: GAGATCAGCCAAATTCAGTTAGT A

Mutant probe M2P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B3: E545D mutations

Mutant upstream primer M3L: GATCCTCTCTCTGAAATCACTCGT A

Mutant downstream primer M3R: GAGATCAGCCAAATTCAGTTACT A

Mutant probe M3P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B4: H1047R mutation

Mutant upstream primer M4L: ATGAAACAAATGAATGATGCATG A

Mutant downstream primer M4R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M4P: TGGCTGGACAACAAAAATGGATTGG A

B5: H1047L mutation

Mutant upstream primer M5L: CATGAAACAAATGAATGATGCTAT A

Mutant downstream primer M5R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M5P: TGGCTGGACAACAAAAATGGATTGG A

B6: wild type

Wild type upstream primer WL: AGCTCAAAGCAATTTCTACACGT A

Wild type downstream primer WR: GAGATCAGCCAAATTCAGTTACT A

Wild type probe WP: ATGGAGTCACAGGTAAGTGCTAAAATGG.

2. The kit for detecting mutation in human PIK3CA gene according to claim 1, further comprising a DNA polymerase and uracil DNA glycosylase having an anti-contamination effect.

3. The human PIK3CA gene mutation detection method is characterized by comprising the following steps of:

(1) Extracting nucleic acid in a sample to obtain a nucleic acid extracting solution;

(2) Preparing a fluorescent PCR amplification reaction system, and respectively mixing a nucleic acid extracting solution, DNA polymerase, dNTPs, primers and probes in 6 PCR reaction tubes, wherein the nucleotide sequences of the primers and probes contained in each reaction tube are respectively shown in the following table:

Tube numbering Primer(s) Probe with a probe tip 1 IL、IR、M1L、M1R M1P 2 IL、IR、M2L、M2R M2P 3 IL、IR、M3L、M3R M3P 4 IL、IR、M4L、M4R M4P 5 IL、IR、M5L、M5R M5P 6 IL、IR、WL、WR WP

Wherein, the PIK3CA gene mutant in the PIK3CA mutation detection reagent is selected from the group consisting of:

Mutation name Base mutant forms Amino acid mutant forms Exons B1(E542K) GAA>AAA Glu542Lys 9 B2(E545K) GAG>AAG Glu545Lys 9 B3(E545D) GAG>GAT Glu545Asp 9 B4(H1047R) CAT>CGT His1047Arg 20 B5(H1047L) CAT>CTT His1047Leu 20

The nucleotide sequences of the related primers and probes are as follows:

IC: internal standard specificity

Internal standard upstream primer IL: ATGGGAAACAGACGAATGC A

Internal standard downstream primer IR: ACAGCAAATAAAAGAAACTAAAACA internal standard probe IP: TCCTGAGACTTCCACACTGATGC A

B1: E542K mutations

Mutant upstream primer M1L: CTACACGAGATCCTCTCTGCA A

Mutant downstream primer M1R: GAGATCAGCCAAATTCAGTTACT mutant probe M1P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B2: E545K mutations

Mutant upstream primer M2L: CGAGATCCTCTCTCTGAAATTACTA mutant downstream primer M2R: GAGATCAGCCAAATTCAGTTAGT mutant probe M2P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B3: E545D mutations

Mutant upstream primer M3L: GATCCTCTCTCTGAAATCACTCGT mutant downstream primer M3R: GAGATCAGCCAAATTCAGTTACT mutant probe M3P: ATGGAGTCACAGGTAAGTGCTAAAATGG A

B4: H1047R mutation

Mutant upstream primer M4L: ATGAAACAAATGAATGATGCATG mutant downstream primer M4R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M4P: TGGCTGGACAACAAAAATGGATTGG A

B5: H1047L mutation

Mutant upstream primer M5L: CATGAAACAAATGAATGATGCTAT mutant downstream primer M5R: CAGAGTGAGCTTTCATTTTGT A

Mutant probe M5P: TGGCTGGACAACAAAAATGGATTGG A

B6: wild type

Wild type upstream primer WL: AGCTCAAAGCAATTTCTACACGT A

Wild type downstream primer WR: GAGATCAGCCAAATTCAGTTACT A

Wild type probe WP: ATGGAGTCACAGGTAAGTGCTAAAATGG A

(3) Respectively adding the nucleic acid extracting solution into a mutant reaction system and a wild reaction system, and performing fluorescent quantitative PCR reaction;

(4) And analyzing the detection result according to the difference value of the FAM signal Ct value of the mutant reaction system and the wild reaction system.

4. The method for detecting human PIK3CA gene mutation according to claim 3, wherein the nucleic acid extracting solution is extracted from the sample by a magnetic bead extraction method, and the extracting process is as follows: adding nucleic acid extract into the sample, preserving heat, adding magnetic beads, uniformly mixing, applying a magnetic field, discarding the liquid, washing, and eluting.

5. The method of claim 4, wherein each reaction tube further comprises an internal control nucleic acid, an internal control primer, and an internal control probe.

6. The method for detecting human PIK3CA gene mutation according to claim 5, wherein the PCR reaction conditions are:

50 ℃ for 2 min 94 ℃ for 5min

94℃For 10 seconds 55℃for 45 seconds X40 cycles.

7. The primer and probe for detecting 5 mutations in human PIK3CA gene according to claim 1, wherein the fluorescent moiety is FAM, the primer probe fluorescent moiety of the internal standard is VIC/HEX, and all the probes are labeled with a fluorescent moiety and a quencher.

8. The method for detecting human PIK3CA gene mutation according to claim 4, wherein the reagents required for the magnetic bead extraction method comprise:

nucleic acid extract comprising guanidine isothiocyanate, sodium ethylenediamine tetraacetate, tween-20, sodium perchlorate, ethanol and pH buffer;

a washing liquid for the first washing comprising sodium perchlorate and ethanol;

A wash solution for the second wash comprising ethanol and a pH buffer.