30 results about "PIK3CA Gene Mutation" patented technology

The present invention discloses primers, probes, a detection system and a kit for one time detection of lung cancer multiple genes, wherein the primers, the probes, and the distribution way for detecting 25 EGFR gene mutations, 7 KRAS gene mutations, 6 BRAF gene mutations, 9 NRAS gene mutations, 5 HER2 gene mutations, 2 PIK3CA gene mutations, 5 fusion genes of ALK5, 13 fusion genes of ROS1, and 6 fusion genes of RET are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, and the corresponding fusion detection reagents and the internal control reagents are filled in the pipes 1-4 of the 12 linking PCR strip; and with the primers, the probes, the detection system and the kit, the one-time detection of the 24 fusions and the 54 mutations of the lung cancer can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on lung cancer patients can be provided for clinician.

The invention relates to a method and kit for detecting gene mutation, and specifically relates to a method and kit for detecting PIK3CA gene mutation. The invention is characterized in that the kit comprises two specific probes for performing genotyping on a 9 exon and a 20 exon of the PIK3CA gene, wherein the specific probe for the 9 exon contains a nucleotide sequence of E545K and E542K codons, and the specific probe for the 20 exon contains a nucleotide sequence of an H1047R codon. By using the conventional PCR (polymerase chain reaction) amplification in combination with the Cold-PCR amplification product enrichment technology and the high-resolution melting curve analysis technology, the kit provided by the invention can complete the judgment on the genotyping of a sample.

The invention discloses a liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation. The liquid phase chip mainly comprises (A) a wild-type and mutant-type allele specific primer extension (ASPE) primer pair designed for mutational sites of a PIK3CA gene respectively, microspheres which are respectively coated with specific anti-tag sequences and have different colors of codes and amplification primers for respectively amplifying the target sequences of the PIK3CA gene with the corresponding mutational sites of the Exon 9 and / or Exon 20, wherein the anti-tag sequences can correspondingly complement and form pair with the selected tag sequences. The liquid phase chip has the following advantages: the coincidence rate of the detection method provided by the invention and a sequencing method is as high as 100%; the prepared liquid phase chip has very good signal to noise ratio; cross reactions do not exist between the designed probes and the anti-tag sequences; and parallel detection of various mutant types and various mutational sites on the same site can be realized.

The invention relates to the field of molecular biology, in particular to a method for detecting gene mutations by adopting fluorescent quantitation PCR, as well as a fluorescent quantitation PCR detection kit and a detection method for PIK3CA (phosphatidylinositol3-kinase catalytic alpha) gene mutations. The method comprises the following steps: designing a specific primer and a probe; preparing a fluorescent quantitation PCR reaction system and designing reaction conditions; amplifying a mutant gene target sequence in a sample by utilizing the specific primer; detecting the fluorescence signal strengths of FAM, HEX and Cy5 in the reaction system, and taking the fluorescence signal strengths as judgement standards of results. The kit provided by the invention can detect 8 types of mutations of a PIK3CA gene simultaneously; the specificity and the selection capability are high; a PIK3CA mutated molecule, which is as low as 5 copy, can be detected in a clinical sample of which the mutation content is only 0.1 percent; the detection speed is high, that is, the detection can be fulfilled within 90 minutes.

The invention is applicable to biotechnology and the medical field and relates to a reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations. A first reagent, a second reagent, a third reagent, a fourth reagent and a fifth reagent are sequentially targeted to E542K, E545K, E545D, H1047R and H1047L mutations of a PIK3CA gene, and a sixth reagent is targeted to a conserved region of the PIK3CA gene and used for quality control. A testing method is carried out by the testing reagent kit by means of fluorescence quantitative PCR (polymerase chain reaction). The testing reagent kit is high in sensitivity and good in specificity and is a high-performance human PIK3CA gene mutation testing reagent kit. The fluorescence quantitative PCR method is adopted for performing specific amplification testing on sequences in PIK3CA gene mutation regions, and specific binding of the MGB (minor groove binder) blocked nucleic acid and a wild type DNA (deoxyribo nucleic acid) template in a sample to be tested is utilized to inhibit binding of the wild type DNA template and a primer probe. Forward primers are designed by methods of lowering the primer Tm value, tailing the 5' end and adding and inserting bases among the primers and the like, specificity of the reagent kit can be improved, then more than 45 cycle numbers are introduced, and accordingly effect of improving sensitivity of the regent kit is achieved.

The invention discloses a kit for detecting PIK3CA gene mutation and a detecting method thereof. The kit comprises a special probe which is modified by LNA locked nucleic acid and used for PIK3CA gene mutation sites; the special probe can be combined with wild type DNAs, a sample containing 0.01% PIK3CA gene mutation DNAs can be detected, and the minimum detectability ranges from 2 copies to 5 copies. The detecting method has the advantages that as for PIK3CA gene mutation detection, the specificity is high, the flexibility is high, pollution is small, operation is easy and rapid, and the safety performance is high; the detecting method is particularly suitable for detecting gene mutation from body fluid such as plasma, urine and saliva with low mutation content, and is suitable for early screening and diagnosis of colorectal cancers, and guidance is provided for personalized medicines.

The invention provides a PIK3CA gene mutation detection kit, comprising: (1) specific primers for amplifying two exons Exon 9 and 20 of a PIK3CA gene in samples, wherein primer sequences are SEQ ID Nos: 1 to 4 respectively; (2) sequencing primers for sequencing PCR, and conducting sequencing analysis on the two exons Exon 9 and 20 of the PIK3CA gene respectively, wherein primer sequences are SEQ ID Nos: 5 to 6 respectively; (3) two positive quality control tubes with PIK3CA wild type plasmids and mutant type plasmids mixed according to a mass ratio of 1 to 1 and a test tube with PCR reaction premix liquid. The kit has the advantage of detecting the main mutation site type of the PIK3CA gene in the samples through a Sanger sequencing method by directly utilizing paraffin embedding the samples containing some tissue parts of the human body.

The present invention relates to a PIK3CA gene mutation detection kit, which can be used for detection of PIK3CA mutations associated with cancer.

The invention relates to detection of a mutation site of a PIK3CA gene in the ctDNA in urine, and particularly provides a method for separating the ctDNA from a urine sample, a method for building a sequencing library based on the urine sample, a method for performing nucleic acid sequencing based on the urine sample according to the sequencing library, equipment for separating the ctDNA from theurine sample and a system for determining the sensitivity of an individual drug. The method for separating the ctDNA from the urine sample comprises the following steps: adding EDTA into the urine sample to obtain a first mixed solution; centrifugating the mixed solution, and collecting supernate; concentrating the supernate to obtain a concentrated solution; mixing the concentrated solution withanion exchange resin to obtain a second mixed solution; performing chromatographic separation treatment on the second mixed solution to obtain eluent; purifying the eluent to obtain the ctDNA. According to the detection disclosed by the invention, a large quantity of ctDNAs can be extracted quickly from a large quantity of urine samples, and can be subjected to nucleic acid sequencing, and the sensitivity of the individual drug can be determined, so that the application prospect of the ctDNA in the urine is enlarged.

Probes, liquid phase chips and methods for detecting PIK3CA gene mutations are provided, wherein the liquid chips for detecting PIK3CA gene mutations mainly comprise: microspheres coupled with probes; primers used for amplifying target sequences with exon 9 and / or exon 20. The liquid chips and methods for detecting PIK3CA gene mutations are useful for detecting the sites containing mutations of the PIK3CA gene with relatively high frequency simultaneously, and also are useful for detecting the exon 9 and exon 20 separately or simultaneously. The detection methods have identical reaction conditions of detection, good specificity of detection, high sensitivity, above 90% accuracy, and short time of detection.

The invention discloses a kit for detecting PIK3CA gene mutation, which comprises peptide nucleic acids and specific primers with the following sequences: (1) peptide nucleic acid and the specific primer aiming at the ninth exon of a PIK3CA gene: shown as SEQ ID NO.1-3; and (2) peptide nucleic acid and the specific primer aiming at the twentieth exon of a PIK3CA gene: shown as SEQ ID NO.4-6. A method for detecting the PIK3CA gene mutation by utilizing the kit comprises the following steps: (1) extracting the DNA of a to-be-detected sample; (2) by taking the extracted DNA as a template, carrying out quantitative PCR amplification by utilizing the kit for detecting the PIK3CA gene mutation, and detecting the value of deltaCt; and (3) making judgment, if deltaCt is greater than 8, judging that the genotype of the PIK3CA gene of the to-be-detected sample is the wild type, and if deltaCt is smaller than 8, judging that the genotype of the PIK3CA gene of the to-be-detected sample is the mutation type. The kit has the advantages that the operation is easy and convenient, the time consumed is short, the sensitivity is high, the detection result is accurate and reliable, etc.

The invention provides a PIK3CA gene mutation detection method and kit, and concretely discloses a primer and a probe for detecting PIK3CA gene mutation, and a kit comprising a primer and probe mixedsolution. 11 mutation types of the PIK3CA gene can be detected at the same time, and the method and the kit are suitable for dPCR detection, and have the advantages of multiple detectable mutation types, high sensitivity, small sample dependence and the like.

The invention discloses a PIK3CA gene g.1792224821G>A mutation and application thereof to auxiliary diagnosis of breast cancer and belongs to the field of medical and biological technology. Compared in sequence with PIK3CA normal genes, the PIK3CA gene g.1792224821G>A mutation has a g.1792224821G>A site mutation. The invention also provides a breast cancer pathogenesis new mutation site, a specific primer for detecting the breast cancer pathogenesis new mutation site and a kit containing the specific primer. The PIK3CA gene g.1792224821G>A mutation can be applied to early diagnosis of breast cancer.

The invention relates to a primer for detecting mutation of a human PIK3CA gene, a probe and a kit. The screened specific ARMS primer and the probe, and an optimized reaction system can obtain the kitfor detecting mutation of human PIK3CA gene with excellent detection performance. The detection of the kit is fast and detection can be completed in 90 minutes, the sensitivity is high, and the kit can detect PIK3CA gene mutation with the content as low as 1% in a 10ng DNA sample; the specificity is high, no amplification is generated with wild-type samples; the kit is easy to operate, has low cost, is conducive to large scale and marketization, and can be effectively applied to clinical precision guidance medication.

The invention discloses a primer combination for detecting mutation of a PIK3CA gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.9 exon mutation and a primer group for detecting No.20 exon mutation. According to a method, a trace amount of DNA is pre-amplified to obtain a high-concentration DNA template, and the mutation condition of the PIK3CA gene in a sample is detected by combining a direct sequencing method; by applying the primer combination to preparation of a detection reagent for detecting mutation of the PIK3CA gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.9 exon and a No.20 exon of the PIK3CA gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for PIK3CA gene mutation detection on a clinical tumor patient and has the very good clinical application value.

The invention discloses a PCR-RFLP method for detecting H1047R-site mutation of a PIK3CA gene. According to the method, a pair of specific primers are designed for the H1047R mutation site of the twentieth codon of the PIK3CA gene; and an Fst I digestion site is introduced to establish the PCR-RFLP method. The PCR-RFLP method mainly comprises the following steps: (1) extracting the genome DNA of a sample to be detected; (2) carrying out PCR amplification on a PIK3CA gene segment which contains the H1047R mutation site; (3) taking the PIK3CA gene segment as a template to carry out Fst I restriction enzyme reaction; (4) carrying out gel electrophoresis to obtain a length polymorphism atlas of restriction segments; and (5) determining the PIK3CA gene mutation state of the sample to be detected according to the length and quantity of the restriction fragments. The method has the advantages of being simple to operate, strong in specificity, high in sensitivity and low in cost, and can be used for effectively detecting the PIK3CA gene mutation state.

The invention discloses a kit utilizing stem loop primers to detect PIK3CA gene mutation sites. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites comprises a set of stemloop primers, and two MGC fluorescent probes; sequence of upstream primers of the set of the stem loop primers is shown as SEQ ID NO. 1 while sequence of downstream primers of the set of the stem loop primers is shown as SEQ ID NO. 8; and the two MGC fluorescent probes are shown as SEQ ID NO. 9-10. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites is capable of overcoming defect of low throughput of existing first-generation sequencing technologies, long time consumption of existing second-generation sequencing technologies, limited detection capacity of fluorescent quantitative PCR and the like; moreover, the kit has the advantages of being high in sensitivity, good in specificity, rapid, high in throughput and so on.

The invention discloses a primer combination for detecting mutation of a PIK3CA gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.9 exon mutation and a primer group for detecting No.20 exon mutation. According to a method, a trace amount of DNA is pre-amplified to obtain a high-concentration DNA template, and the mutation condition of the PIK3CA gene in a sample is detected by combining a direct sequencing method; by applying the primer combination to preparation of a detection reagent for detecting mutation of the PIK3CA gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.9 exon and a No.20 exon of the PIK3CA gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for PIK3CA gene mutation detection on a clinical tumor patient and has the very good clinical application value.

The invention discloses a breast cancer PIK3CA mutation gene and its application. The mutation gene has a c.1658_1659GT>C mutation site, and its nucleotide sequence is shown in SEQ ID NO: 1; PIK3CA gene mutations were detected in 88 breast cancer tissues, 19 breast cancer adjacent tissues, and 22 normal human blood samples. For the first time, it was found that the PIK3CA gene S553fs*7 mutation was as high as 44.3% in breast cancer tissues, and 44.3% in breast cancer adjacent tumors. There is no mutation in tissue and normal human blood samples; it is suggested that this mutation can be used to assist the early diagnosis of breast cancer; therefore, the present invention is of great significance for clinical prevention and treatment of the occurrence and development of breast cancer.

The invention provides a primer group for detecting PIK3CA gene mutation and an application method thereof. The primer group comprises at least one primer pair of the following three primer pairs: a primer pair for detecting the No.1 exon of a PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2; a primer pair for detecting the No.9 exon of the PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primeris shown as SEQ ID NO.4; and a primer pair for detecting the No.20 exon of the PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6. The gene detection flux is improved.

The invention discloses a PIK3CA gene mutation detection system and a kit thereof. The PIK3CA gene mutation detection system comprises primers, a probe and an amplification blocking primer, wherein the primers include a general forward primer, detecting reverse primers and a quality-control reverse primer. The detecting reverse primers include the detecting reverse primer for the E542K mutation site of a PIK3CA gene, the detecting reverse primer for the E545K mutation site of the PIK3CA gene, the detecting reverse primer for the H1047R mutation site of the PIK3CA gene and the detecting reverse primer for the H1047L mutation site of the PIK3CA gene. The 5' end of a probe sequence is provided with a fluorescent marker, and the 3' end of the nucleotide sequence of the probe is provided with another fluorescent marker. The 3' end of the nucleotide sequence of the amplification blocking primer is provided with a phosphorylated modifier. The PIK3CA gene mutation detection system and the kit of the system can detect the E542K, E545K, H1047R or H1047L mutation site of the PIK3CA gene and are high in sensitivity, strong in specificity and fast and convenient to use.

The invention provides a digital PCR detection method for human PIK3CA gene mutation and application. Specifically, the invention provides a preferred primer pair aiming at sequences of PIK3CA E545, E542 and PIK3CA H1047, an amplification method, a nucleic acid probe and a detection system, and further provides a kit for detecting PIK3CA gene mutation. By optimizing the primer pair, the probe and corresponding reaction conditions, the PIK3CA gene mutation can be detected in a high-sensitivity and strong-specificity manner for different samples.

The invention discloses a kit utilizing stem loop primers to detect PIK3CA gene mutation sites. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites comprises a set of stemloop primers, and two MGC fluorescent probes; sequence of upstream primers of the set of the stem loop primers is shown as SEQ ID NO. 1 while sequence of downstream primers of the set of the stem loop primers is shown as SEQ ID NO. 8; and the two MGC fluorescent probes are shown as SEQ ID NO. 9-10. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites is capable of overcoming defect of low throughput of existing first-generation sequencing technologies, long time consumption of existing second-generation sequencing technologies, limited detection capacity of fluorescent quantitative PCR and the like; moreover, the kit has the advantages of being high in sensitivity, good in specificity, rapid, high in throughput and so on.

The invention discloses primers and probes and a kit for detecting five mutation types of a human PIK3CA gene. According to each group of the five groups (PIM1-PIM5) of the primers and probes, the mutation primer can be combined with a conserved sequence of the PIK3CA gene, a mutation ARMS primer can be specially combined with a corresponding mutation sequence of the mutation ARMS primer to amplify the mutation sequence, the detection probe can be combined with an amplified fragment, and the blocking probe can be specially combined with a wild type sequence corresponding to a mutation locus to inhibit wild type nonspecific amplification. Accordingly, by adopting a specific mutation primer and probe blocking technology, the five mutation types of the PIK3CA gene can be accurately detected; through the established kit of a real-time fluorescence PCR amplification reaction system, rapid detection on the PIK3CA gene mutation is facilitated, operation is easy, and a result is easy to read; the detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, no nonspecific amplification is generated in 20-ng wild type genome DNA, and the detectability reaches up to 1%.

The present invention discloses primers, probes, a detection system and a kit for one time detection of lung cancer multiple genes, wherein the primers, the probes, and the distribution way for detecting 25 EGFR gene mutations, 7 KRAS gene mutations, 6 BRAF gene mutations, 9 NRAS gene mutations, 5 HER2 gene mutations, 2 PIK3CA gene mutations, 5 fusion genes of ALK5, 13 fusion genes of ROS1, and 6 fusion genes of RET are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, and the corresponding fusion detection reagents and the internal control reagents are filled in the pipes 1-4 of the 12 linking PCR strip; and with the primers, the probes, the detection system and the kit, the one-time detection of the 24 fusions and the 54 mutations of the lung cancer can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on lung cancer patients can be provided for clinician.