CN114381518A - Primer and kit for rapidly detecting glioma mutation site and parting - Google Patents
Primer and kit for rapidly detecting glioma mutation site and parting Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to the technical field of gene mutation detection, and relates to a method, a primer combination and a kit for clinically and rapidly detecting human glioma mutation sites and parting. The method comprises the following steps: designing a primer group aiming at a mutation site to be detected, wherein the primer group consists of a plurality of forward primers and a reverse primer to form a primer pair for PCR reaction; obtaining DNA in a tissue sample or cell; performing a fluorescence PCR reaction by using the DNA as a template, and performing real-time signal detection on the PCR reaction by using a real-time fluorescence PCR instrument; and (4) judging the mutation information and the gene typing according to the accumulation of reaction products and a dissolution curve. The invention can determine the sequence information of three mutation sites at one time through the fluorescent PCR reaction, cover all mutation conditions and can also determine the corresponding genotyping information. The invention has low demand on sample tissues, simple operation, low cost and short time.
Description
Technical Field
The invention relates to the technical field of gene mutation detection, and relates to a primer and a kit for rapidly detecting glioma mutation sites and parting. In particular to a rapid detection method for IDH allelic gene mutation of glioma in operation based on allelic gene specific PCR technology.
Background
The prior art discloses glioma, abbreviated glioma, also known as glioblastoma, is the first tumor of the nervous system, which accounts for 40% -50% of craniocerebral tumors. The currently available WHO grades the glioma into four grades mainly according to the characteristics of atypical character, nuclear division index, endothelial cell proliferation and necrosis degree; wherein, the grade I is mainly benign hair cell type astrocytoma, accounts for about 5 percent of glioma, and can be generally cured; the II grade is general astrocytoma or mixed oligodendroastrocytoma, and accounts for about 30-40% of glioma; grade III is anaplastic astrocytoma, accounts for about 15-25% of glioma, and generally evolves from grade II; grade IV is glioblastoma, accounting for about 1/3 of glioma.
Research has shown that about 80% of glioma grades 2-3 has IDH mutation characteristics, most of which are IDH1 gene mutation, and the mutation is mainly concentrated on 132 th amino acid; the other part is IDH2 gene mutation, which is mainly concentrated on the 172 th amino acid, and the two gene mutations have various mutation combinations at the base level; therefore, the detection of IDH mutation in clinical practice becomes an important basis for glioma identification and molecular typing, and the molecular typing taking IDH mutation as a technology can make up the defects of the traditional pathological typing and provide a more detailed and specific reference basis for the formulation of treatment and surgical schemes.
It has been shown that, in addition to IDH mutation characteristics, there are some important molecular mutation characteristics in human glioma, such as TERT, TP53, ATRX, H3 gene locus, TERT region, etc., and different molecular mutation characteristics have different effects on tumor development, development and treatment, so that fine detection of different loci and different mutations is of great significance in clinical practice.
Usually, clinical diagnosis and classification of glioma are mainly based on histopathological methods, but depending on histopathology, it is sometimes difficult to provide very precise diagnosis and classification due to the characteristics of complexity, diversity and heterogeneity of tumor tissues. At present, clinical diagnosis and mutation analysis of glioma mainly focus on nucleic acid and protein molecule level, and the commonly used molecular biology techniques mainly include: (1) fluorescent PCR based on differences in PCR extension or product annealing; (2) hybridizing a gene chip array; (3) chromatography-mass spectrometry analysis alone or in combination; (4) sanger sequencing; (5) high throughput sequencing, etc.
The above detection techniques for gene mutation have advantages, but there are still various disadvantages for clinical sample detection, such as complex operation, long time period, high cost, single detection information, etc., especially for detecting glioma-like IDH mutation in the intraoperative process, the problems of small and precious tissue sample acquisition amount, acquisition of mutation information in as short a time as possible, and various mutation type combinations, etc., are faced, so that a novel detection technique and a corresponding kit with simple operation, low cost, short time consumption, low requirement for sample amount, and acquisition of more mutation site information at one time are still needed for clinical detection of IDH and other mutations.
Based on the foundation and the current situation of the prior art, the inventors of the present application intend to provide a primer and a kit for rapidly detecting glioma mutation sites and typing. In particular to a rapid detection method for IDH allelic gene mutation of glioma in operation based on allelic gene specific PCR technology.
Disclosure of Invention
The invention aims to provide a primer and a kit for rapidly detecting glioma mutation sites and typing based on the basis and the current situation of the prior art. In particular to a rapid detection method for IDH allelic gene mutation of glioma in operation based on allelic gene specific PCR technology.
In particular, the method comprises the following steps of,
the technical problem to be solved by the invention is to provide a reagent for accurately detecting IDH gene mutation.
Another technical problem to be solved by the present invention is to precisely determine the genotype of IDH using the above-mentioned reagent.
In order to solve the above technical problems, the present invention provides a method for genotyping a mutation site of human glioma by using human glioma mutation as a research target and using a novel gene mutation and typing detection technology based on allele specific pcr (aspcr), wherein the method comprises the following steps:
the method comprises the following steps: designing a primer group aiming at a mutation site to be detected, wherein the primer group consists of a plurality of forward primers and a reverse primer to form a primer pair for PCR reaction;
step two: obtaining DNA in a tissue sample or cell;
step three: performing a fluorescence PCR reaction by using the DNA as a template, and performing real-time signal detection on the PCR reaction by using a real-time fluorescence PCR instrument;
step four: the mutation information and genotyping status were interpreted based on the accumulation of reaction products and the dissolution profile.
Preferably, the mutation sites are 3 continuous mutation sites, and the primer group consists of 12 forward primers and 1 reverse primer.
Preferably, the technical scheme adopted by the invention mainly comprises the following steps:
1) designing a group of primer combinations for single-site or continuous multi-site mutation detection and typing analysis aiming at the mutation sites to be detected;
2) rapidly cracking the collected clinical tissue sample to release the genomic DNA in the tissue sample;
3) and (3) performing fluorescence PCR reaction by using the lysis sample as a template. And carrying out real-time signal detection on the PCR reaction by a real-time fluorescent PCR instrument.
4) And determining mutation information and genotyping conditions according to the amplification curve and the dissolution curve of the reaction product.
More specifically, the method of the present invention comprises:
(1) cutting sample tissues with the size of rice grains, and putting the sample tissues into a small reaction tube suitable for PCR;
(2) adding lysis solution, incubating, adding lysis neutralizing solution, and placing the product on ice;
(3) absorbing the cleavage product as a PCR amplification template, and preparing a fluorescent quantitative PCR amplification system;
(4) putting the mixture into a fluorescent quantitative PCR instrument for amplification and real-time fluorescent detection;
(5) and comparing the amplification curve and the dissolution curve of the sample with a positive control and a negative control respectively, and judging the result.
Preferably, in the present invention, the human glioma mutation site comprises amino acid 132 of human glioma IDH1, H3K27M or TERT Promoter regions c228t and c250 t.
On the basis of software assistance, the invention designs a series of primers aiming at important mutation sites, and carries out screening and optimization of the primers according to the amplification result to obtain a group of primer combinations for detecting 132 th amino acid mutation sites and molecular typing of glioma IDH1, wherein the primer combinations comprise 12 upstream primers and a downstream primer, and the upstream primers and the downstream primer respectively are as follows:
IDH1-1：AAAACCTATCATCATAGGTA(SEQ ID NO 1)；
IDH1-2：AAAACCTATCATCATAGGTT(SEQ ID NO 2)；
IDH1-3：AAAACCTATCATCATAGGTC(SEQ ID NO 3)；
IDH1-4：AAAACCTATCATCATAGGTG(SEQ ID NO 4)；
IDH1-5：AAACCTATCATCATAGGTNA(SEQ ID NO 5)；
IDH1-6：AAACCTATCATCATAGGTNT(SEQ ID NO 6)；
IDH1-7：AAACCTATCATCATAGGTNC(SEQ ID NO 7)；
IDH1-8：AAACCTATCATCATAGGTNG(SEQ ID NO 8)；
IDH1-9：AACCTATCATCATAGGTNNA(SEQ ID NO 9)；
IDH1-10：AACCTATCATCATAGGTNNT(SEQ ID NO 10)；
IDH1-11：AACCTATCATCATAGGTNNC(SEQ ID NO 11)；
IDH1-12：AACCTATCATCATAGGTNNG(SEQ ID NO 12)；
IDH1-13：CCTTTAGCTAAATGTGTGTA(SEQ ID NO 13)。
the primer combination comprises a plurality of forward primers and a reverse primer aiming at mutation sites, so that the detection accuracy and sensitivity are improved, the cost is considered, and the probability among the primers is reduced.
Compared with the prior art, the invention can determine the sequence information of three mutation sites once through 12 groups of PCR reactions, cover all mutation conditions and also can determine corresponding genotyping information. The method has the advantages of low demand on sample tissues, simple operation, low cost and short time.
Based on the mode, the invention designs and optimizes the primers aiming at the important H3K27M and TERT Promoter region c228t and c250t mutation sites in glioma.
The second primer set for H3K27M in the present invention:
H3-1:GGCTACAAAAGCCGCTCGCA(SEQ ID NO 14)
H3-2:GGCTACAAAAGCCGCTCGCT(SEQ ID NO 15)
H3-3:GGCTACAAAAGCCGCTCGCC(SEQ ID NO 16)
H3-4:GGCTACAAAAGCCGCTCGCG(SEQ ID NO 17)
H3-5:GCTACAAAAGCCGCTCGCAA(SEQ ID NO 18)
H3-6:GCTACAAAAGCCGCTCGCAT(SEQ ID NO 19)
H3-7:GCTACAAAAGCCGCTCGCAC(SEQ ID NO 20)
H3-8:GCTACAAAAGCCGCTCGCAG(SEQ ID NO 21)
H3-9:CTACAAAAGCCGCTCGCAAA(SEQ ID NO 22)
H3-10:CTACAAAAGCCGCTCGCAAT(SEQ ID NO 23)
H3-11:CTACAAAAGCCGCTCGCAAC(SEQ ID NO 24)
H3-12:CTACAAAAGCCGCTCGCAAG(SEQ ID NO 25)
H3-13:CCTCCAGGTAAGATTATGGC(SEQ ID NO 26)。
the third primer set of the present invention for TERTPromoter regions c228t and c250 t:
TERTp228-1：CCGCCCCGTCCCGACCCCTC(SEQ ID NO 27)
TERTp228-2：CCGCCCCGTCCCGACCCCTT(SEQ ID NO 28)
TERTp250-1：GGGTCCCCGGCCCAGCCCCC(SEQ ID NO 29)
TERTp250-2：GGGTCCCCGGCCCAGCCCCT(SEQ ID NO 30)
TERTp-3：GGCCCCAGGCGCCGCACGAA(SEQ ID NO 31)
in the invention, the primer combination for rapidly detecting mutation sites such as glioma IDH1 and molecular typing has the specific sequence as described above; the second primer group and the third primer group are respectively used for different gene loci, so that the accuracy of genotyping can be further improved.
The invention also provides a kit for rapidly detecting mutation sites such as glioma IDH1 and the like and molecular typing, which comprises the primer combination.
The invention also provides a kit for rapidly detecting glioma mutation sites and molecular typing, which comprises the primer, a fluorescent PCR reagent, a negative quality control sample and a wild homozygous quality control sample; the kit also comprises a lysate for cracking clinical tissue samples and a PCR amplification system for realizing PCR reaction; preferably, the kit further comprises instructions, reaction vessels, simple means for grinding or disrupting tissue, and the like.
The invention provides a primer and a kit for clinically and rapidly detecting mutation sites and typing of human glioma, and simultaneously designs a set of primer combination for single-site or continuous multi-site mutation detection and typing analysis aiming at IDH1 allelic sites. The invention can determine the sequence information of three mutation sites at one time through the fluorescent PCR reaction, cover all mutation conditions and can also determine the corresponding genotyping information. The invention also has the advantages of low demand on sample tissues, simple operation, low cost, short time and the like.
Compared with the gene mutation clinical detection technology in the prior art, the gene mutation clinical detection method has the following advantages:
(1) the demand for sample tissues is low;
(2) the operation is simple, the cost is low, and the used time is short;
(3) in the case of unknown DNA mutations, three base sequences can be determined at a time, covering all DNA mutation cases;
(4) the molecular typing of the gene can be analyzed while determining the DNA mutation.
Drawings
Figure 1 is a flow diagram of an embodiment of the present invention,
as shown in the figure, the invention is sequentially divided into 4 steps:
1.1 designing a group of primer combinations for single-site or continuous multi-site mutation detection and typing analysis aiming at the mutation sites to be detected;
1.2 cracking the collected clinical tissue sample to release DNA in the cells of the tissue sample;
1.3, performing a fluorescence PCR reaction by using a cracking sample as a template, and performing real-time signal detection on the PCR reaction by using a real-time fluorescence PCR instrument;
1.4 according to the amplification curve and the dissolution curve of the reaction product, the mutation information and the genotyping situation are read.
FIG. 2 is a schematic diagram of the design of primer combinations according to the present invention.
FIG. 3 is a diagram showing the setup of the fluorescent PCR amplification procedure in the method of the present invention.
FIG. 4 is a graph showing the result of amplification curve of the application case of the present invention for rapidly detecting the IDH1 mutation site of glioma.
FIG. 5 is a graph showing the results of the dissolution curve of the application case of the present invention for rapidly detecting the IDH1 mutation site of glioma.
FIG. 6 shows the nucleotide sequence information and typing interpretation of the 132 th amino acid in sample 1.
FIG. 7 shows the nucleotide sequence information and typing interpretation of the 132 th amino acid in sample 2.
Detailed Description
The invention provides a primer and a kit for rapidly detecting glioma mutation sites and typing, which can rapidly confirm glioma IDH1 allelic point mutation information, cover all base mutation combinations and genotyping under the condition of unknown mutation types, and have very important practical significance for clinical treatment and post-cure decision schemes. The invention is described in detail below with reference to the drawings and the detailed description.
In order to make the technical solution and implementation of the present invention more clearly explained and illustrated, several preferred embodiments for implementing the technical solution of the present invention are described below. It should be understood that the specific embodiments described below are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The detection method mainly comprises the following steps, and the flow of the method is shown in figure 1;
the method comprises the following steps: respectively designing a primer group consisting of a group of four forward primers and simultaneously designing a universal reverse primer aiming at each of three continuous mutation sites to be detected to form a primer pair for PCR reaction;
as shown in FIG. 2, taking the detection of the 132 th amino acid mutation site of IDH1 as an example, the sequences of three sets of forward primers were designed as described above;
step two: cutting and cracking a small amount of tissues of the collected clinical tissue sample to release DNA in cells of the tissue sample;
step three: performing a fluorescent PCR reaction by using the cleavage product as a template, and performing real-time signal detection on the PCR reaction by using a real-time fluorescent PCR instrument;
step four: the mutation information and genotyping status were interpreted based on the accumulation of reaction products (i.e., amplification curve) and the melting curve.
Example 2
The application of the invention is specifically illustrated by taking clinical samples of glioma IDH1 as analysis objects.
Glioma, the first tumor of the nervous system, is a major intractable disease due to its characteristics of rapid progression and short survival. Determination of mutation sites and molecular typing is an important basis in the therapeutic process. In addition, the tissue sample for detection is complex to obtain and has a small sample amount, and the mutation types are many, mainly R132H.
The part is used as a detection pre-experiment, two glioma clinical tissue samples are used as reaction starting materials, one part of the two glioma clinical tissue samples is negative in mutation through preliminary generation sequencing verification, and the other part of the glioma clinical tissue samples is heterozygous for R132H (G-A) mutation.
(1) Cutting sample tissues with the size of rice grains, and putting the sample tissues into a 200-microliter PCR tube;
(2) adding 30 μ L of the first lysis solution, incubating at 50 deg.C for 10min, and optionally beating and mixing once;
(3) incubating at 95 deg.C for 5min, adding 20 μ L lysis neutralizing solution, and temporarily storing the product at 4 deg.C for further use;
(4) 2 mu L of lysate is taken as a PCR amplification template;
(5) the PCR amplification system was prepared as follows:
(6) putting the mixture into a Bio-Rad fluorescence quantitative PCR instrument for amplification and real-time fluorescence detection, wherein the setting procedure is shown in figure 3;
(7) and (4) carrying out result interpretation according to the amplification curve and the dissolution curve.
As a result, as shown in fig. 4 and 5, the first-rising set of curves (7) in fig. 4 was a positive test group, and the second-rising set of curves was a negative test group. The positive groups in FIG. 4 all showed an initial rise at around 20 cycles and corresponded to the consistent dissolution curves shown in FIG. 5; the negative groups in FIG. 4 all appeared to have risen after 30 cycles and corresponded to the chaotic dissolution profile shown in FIG. 5, with non-specific amplification.
The two samples are read and typed for IDH1 locus sequence as shown in figure 6 and figure 7, the reading result is consistent with the prior verification result, the sample 1 is unmutated and homozygous, and the sample 2 is G-A mutation and heterozygous.
In addition, in the embodiment, the double-stranded fluorescent dye is used for signal detection, and the Taqman fluorescent probe can also be used for signal detection, so that the sensitivity and the accuracy of a detection result can be further improved.
The present invention is not limited to the above-mentioned preferred embodiments, and any structural changes made under the teaching of the present invention shall fall within the scope of the present invention, which is similar or similar to the technical solutions of the present invention.
Sequence listing
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Claims (10)
1. A method for genotyping a human glioma mutation site, said method comprising the steps of:
the method comprises the following steps: designing a primer group aiming at a mutation site to be detected, wherein the primer group consists of a plurality of forward primers and a reverse primer to form a primer pair for PCR reaction;
step two: obtaining DNA in a tissue sample or cell;
step three: performing a fluorescence PCR reaction by using the DNA as a template, and performing real-time signal detection on the PCR reaction by using a real-time fluorescence PCR instrument;
step four: the mutation information and genotyping status were interpreted based on the accumulation of reaction products and the dissolution profile.
2. The method of claim 1, wherein the mutation sites are 3 consecutive mutation sites, and the primer set comprises 12 forward primers and 1 reverse primer.
3. The method of claim 1, wherein the method comprises:
(1) cutting sample tissues with the size of rice grains, and putting the sample tissues into a small reaction tube suitable for PCR;
(2) adding lysis solution, incubating, adding lysis neutralizing solution, and placing the product on ice;
(3) absorbing the cleavage product as a PCR amplification template, and preparing a fluorescent quantitative PCR amplification system;
(4) putting the mixture into a fluorescent quantitative PCR instrument for amplification and real-time fluorescent detection;
(5) and comparing the amplification curve and the dissolution curve of the sample with a positive control and a negative control respectively, and judging the result.
4. The method of claim 1, wherein the mutation site of human glioma IDH1 comprises amino acid 132 of human glioma IDH1, H3K27M, or TERT Promoter regions c228t and c250 t.
5. A primer combination for determining the genotyping of a human glioma mutation site, wherein the sequence of the primer combination is as follows:
IDH1-1：AAAACCTATCATCATAGGTA，SEQ ID NO 1；
IDH1-2：AAAACCTATCATCATAGGTT，SEQ ID NO 2；
IDH1-3：AAAACCTATCATCATAGGTC，SEQ ID NO 3；
IDH1-4：AAAACCTATCATCATAGGTG，SEQ ID NO 4；
IDH1-5：AAACCTATCATCATAGGTNA，SEQ ID NO 5；
IDH1-6：AAACCTATCATCATAGGTNT，SEQ ID NO 6；
IDH1-7：AAACCTATCATCATAGGTNC，SEQ ID NO 7；
IDH1-8：AAACCTATCATCATAGGTNG，SEQ ID NO 8；
IDH1-9：AACCTATCATCATAGGTNNA，SEQ ID NO 9；
IDH1-10：AACCTATCATCATAGGTNNT，SEQ ID NO 10；
IDH1-11：AACCTATCATCATAGGTNNC，SEQ ID NO 11；
IDH1-12：AACCTATCATCATAGGTNNG，SEQ ID NO 12；
IDH1-13：CCTTTAGCTAAATGTGTGTA，SEQ ID NO 13。
6. the primer combination of claim 5, wherein the primer combination further comprises a second primer set;
the second primer group is directed to H3K27M and comprises:
H3-1:GGCTACAAAAGCCGCTCGCA，SEQ ID NO 14；
H3-2:GGCTACAAAAGCCGCTCGCT，SEQ ID NO 15；
H3-3:GGCTACAAAAGCCGCTCGCC，SEQ ID NO 16；
H3-4:GGCTACAAAAGCCGCTCGCG，SEQ ID NO 17；
H3-5:GCTACAAAAGCCGCTCGCAA，SEQ ID NO 18；
H3-6:GCTACAAAAGCCGCTCGCAT，SEQ ID NO 19；
H3-7:GCTACAAAAGCCGCTCGCAC，SEQ ID NO 20；
H3-8:GCTACAAAAGCCGCTCGCAG，SEQ ID NO 21；
H3-9:CTACAAAAGCCGCTCGCAAA，SEQ ID NO 22；
H3-10:CTACAAAAGCCGCTCGCAAT，SEQ ID NO 23；
H3-11:CTACAAAAGCCGCTCGCAAC，SEQ ID NO 24；
H3-12:CTACAAAAGCCGCTCGCAAG，SEQ ID NO 25；
H3-13:CCTCCAGGTAAGATTATGGC，SEQ ID NO 26。
7. the primer combination of claim 5, wherein the primer combination further comprises a third primer set;
the sequences of the third primer set are directed to TERT Promoter regions c228t and c250t, comprising:
TERTp228-1：CCGCCCCGTCCCGACCCCTC；
TERTp228-2：CCGCCCCGTCCCGACCCCTT；
TERTp250-1：GGGTCCCCGGCCCAGCCCCC；
TERTp250-2：GGGTCCCCGGCCCAGCCCCT；
TERTp-3：GGCCCCAGGCGCCGCACGAA。
8. kit for determining genotyping of the IDH1 mutation site of human glioma, characterized in that it comprises the primer combination according to any one of claims 5 to 7.
9. The kit of claim 8, wherein the kit further comprises a fluorescent PCR reagent, a tissue lysate, a negative quality control, or a wild homozygous quality control.
10. Use of the primer combination of any one of claims 5-7 for preparing a genotyping kit for determining the site of IDH1 mutation in human glioma.
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