CN111500726A - Primer group for detecting pathogenic gene mutation of pheochromocytoma and paraganglioma and application method thereof - Google Patents
Primer group for detecting pathogenic gene mutation of pheochromocytoma and paraganglioma and application method thereof Download PDFInfo
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Abstract
The invention provides a primer group for detecting pheochromocytoma and paraganglioma pathogenic gene mutation and an application method thereof, wherein the primer group comprises the following steps: at least one primer pair of twenty primer pairs of SEQ ID No.1 to SEQ ID No.40 and a corresponding sequencing primer. The scheme can improve the detection flux of the pheochromocytoma and the paraganglioma pathogenic gene mutation.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer group for detecting pheochromocytoma and paraganglioma pathogenic gene mutation and an application method thereof.
Background
Pheochromocytoma and paraganglioma (PPG L) are tumors originating from adrenal medulla or extraadrenal sympathetic nerve chain, mainly synthesize and secrete a large amount of Catecholamines (CA), such as Norepinephrine (NE), epinephrine (E) and Dopamine (DA), causing a series of clinical symptoms such as high blood pressure rise of patients and causing serious complications such as heart, brain, kidney and the like.
Currently, a synthase Chain Reaction (PCR) detection test can be designed for exons of pheochromocytoma and paraganglioma pathogenic mutation sites in SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX genes, respectively, so as to detect the gene mutation condition.
However, since each PCR reaction can detect gene mutation only for one exon of the above-mentioned gene, the detection throughput of the pathogenic gene mutation of pheochromocytoma and paraganglioma is low.
Disclosure of Invention
The embodiment of the invention provides a primer group for detecting pathogenic gene mutation of pheochromocytoma and paraganglioma and an application method thereof, which can improve the detection flux of pathogenic gene mutation of pheochromocytoma and paraganglioma.
In order to achieve the purpose, the invention is realized by the following technical scheme:
PPG L is involved in germ-line mutation of disease-causing genes, and at present, PPG L disease-causing genes are classified into two types, the first type is that PPG L tumor shows disorder of citric acid circulation and hypoxia induction pathway, functional deletion gene mutation of SDHx gene, FH gene, EG L N1 gene or VH L gene is often found in the category, activation of hypoxia signal caused by HIF2A gene mutation is also found in the category, the second type is that PPG L tumor shows abnormal kinase activity caused by mutation of main regulatory factors (such as NF1 gene, MAX gene and TMEM127 gene) on a feedback loop, and gene mutation with improved RET gene function causes initiation of Ras/MEK and PI 3K/t kinase pathway, thus promoting proliferation and survival of tumor cells.
PPG L has large difference according to different gene types of patients, and patients with different gene mutations have obvious difference in PPG L tumor position, benign/malignant, CA secretion type and recurrence inclination, patients with SDHx gene mutation have head and neck and sympathetic PG L, wherein part of patients can combine kidney cancer, gastrointestinal stromal tumor and pituitary tumor, VH L, RET, NF1, TMEM127 or MAX gene mutation is commonly seen in PCC patients and mostly suffer bilateral adrenal gland involvement, RET gene mutation is also seen in multiple endocrine adenomatosis type II (MEN II), patients with SDHB and FH gene mutation are mostly suggested as malignant PG L, PCC with RET and NF1 gene mutation mainly secretes E, and tumors with VH L and SDHx gene mutation mainly have NE, therefore, the pathogenic site specific amplimers based on the exogenetic mutation of SDHB, SDHC, SDHD, VH L, RET, TMEM, MAX 127 gene and paraganglioma mutation are designed.
The invention provides a primer group for detecting pheochromocytoma and paraganglioma pathogenic gene mutation, which comprises the following components: comprising at least one of the following twenty primer pairs:
the first primer pair is used for detecting the No.1 exon of the SDHB gene, the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the second primer pair is used for detecting the No.2 exon of the SDHB gene, the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4;
the third primer pair is used for detecting the No.3 exon of the SHDB gene, the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6;
the fourth primer pair is used for detecting the No.4 exon of the SDHB gene, the nucleotide sequence of the upstream primer of the fourth primer pair is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8;
the fifth primer pair is used for detecting exons 6 and 7 of the SDHB gene, the nucleotide sequence of the upstream primer of the fifth primer pair is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10;
the sixth primer pair is used for detecting the No.1 exon of the SDHC gene, the nucleotide sequence of the upstream primer of the sixth primer pair is shown as SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12;
the seventh primer pair is used for detecting the exon 2 of the SHDC gene, the nucleotide sequence of the upstream primer of the seventh primer pair is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 14;
the eighth primer pair is used for detecting the No.4 exon of the SDHC gene, the nucleotide sequence of the upstream primer of the eighth primer pair is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 16;
the ninth primer pair is used for detecting the No.5 exon of the SDHC gene, the nucleotide sequence of the upstream primer of the ninth primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 18;
the tenth primer pair is used for detecting exons 1, 2 and 3 of the SDHD gene, the nucleotide sequence of an upstream primer of the tenth primer pair is shown as SEQ ID NO.19, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.20, and the nucleotide sequence of a sequencing primer is shown as SEQ ID NO. 41;
the eleventh primer pair is used for detecting the No.4 exon of the SDHD gene, the nucleotide sequence of the upstream primer of the eleventh primer pair is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 22;
the twelfth primer pair is used for detecting the No.1 exon of the VH L gene, the nucleotide sequence of the upstream primer of the twelfth primer pair is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 24;
the thirteenth primer pair is used for detecting the No.2 exon of the VH L gene, the nucleotide sequence of the upstream primer of the thirteenth primer pair is shown as SEQ ID NO.25, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 26;
the fourteenth primer pair is used for detecting the No.3 exon of the VH L gene, the nucleotide sequence of the upstream primer of the fourteenth primer pair is shown as SEQ ID NO.27, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 28;
the fifteenth primer pair is used for detecting exons 10 and 11 of RET gene, the nucleotide sequence of the upstream primer of the fifteenth primer pair is shown as SEQ ID NO.29, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 30;
the sixteenth primer pair is used for detecting the 14 th and 16 th exons of the RET gene, the nucleotide sequence of the upstream primer of the sixteenth primer pair is shown as SEQ ID NO.31, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 32;
the seventeenth primer pair is used for detecting the No.1 exon of the TMEM127 gene, the nucleotide sequence of the upstream primer of the seventeenth primer pair is shown as SEQ ID NO.33, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 34;
the eighteenth primer pair is used for detecting exons 2 and 3 of the TMEM127 gene, the nucleotide sequence of the upstream primer of the eighteenth primer pair is shown as SEQ ID NO.35, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 36;
the nineteenth primer pair is used for detecting No.3 exon of the MAX gene, the nucleotide sequence of an upstream primer of the nineteenth primer pair is shown as SEQ ID NO.37, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 38;
the twentieth primer pair is used for detecting No.4 exon of the MAX gene, the nucleotide sequence of the upstream primer of the twentieth primer pair is shown as SEQ ID NO.39, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 40.
The primer group is used for PCR amplification reaction, so that the detection flux of the pheochromocytoma and paraganglioma pathogenic gene mutation can be improved.
It should be noted that at least one primer pair shown in SEQ ID Nos. 1 to 22 may be applied to a reagent or a kit for detecting exon 1 of SDHB gene, exon 2 of SDHB gene, exon 3 of SHDB gene, exon 4 of SDHB gene, exon 6 and 7 of SDHB gene, exon 1 of SDHC gene, exon 2 of SHDC gene, exon 4 of SDHC gene, exon 5 of SDHC gene, exon 1, 2 and 3 of SDHD gene, and exon 4 of SDHD gene, and at least one primer pair shown in SEQ ID Nos. 23 to 40 may be applied to a reagent or a kit for detecting exon 1 of VH L gene, exon 2 of VH L gene, exon 3 of VH L gene, exon 10 and 11 of RET gene, exon 14 and 16 of RET gene, exon 1 of TMEM gene, exon 127, exon 1, exon 2 of VH L gene, exon 3 of VH L gene, exon 3 of RET gene, and MAX gene.
Specifically, in order to avoid using interference between the different primer pairs during PCR amplification, the twenty primers can be divided into two groups (i.e., each group is a PCR tube during PCR reaction), which are specifically grouped as follows:
the first group includes: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair, and an eleventh primer pair;
the second group includes: the primer pairs of the twenty primer pairs other than the first group, i.e., the twelfth to twentieth primer pairs.
Specifically, when a primer set consisting of a primer pair shown in SEQ ID No. 1-22 is used for multiplex PCR amplification reaction, the No.1 exon of the SDHB gene, the No.2 exon of the SDHB gene, the No.3 exon of the SHDB gene, the No.4 exon of the SDHB gene, the No.6 and No.7 exons of the SDHB gene, the No.1 exon of the SDHC gene, the No.2 exon of the SHDC gene, the No.4 exon of the SDHC gene, the No.5 exon of the SDHC gene, the No.1, 2 and 3 exons of the SDHD gene and the No.4 exon of the SDHD gene can be amplified simultaneously in one amplification system, namely, the exons containing pathogenic mutation sites of pheochromocytoma and paraganglia in the SDHB, SDHC and SDHD genes can be amplified simultaneously.
Specifically, when the primer set of SEQ ID No. 23-40 is used for multiplex PCR amplification reaction, the 1 st exon of VH L gene, the 2 nd exon of VH L gene, the 3 rd exon of VH L gene, the 10 th and 11 th exons of RET gene, the 14 th and 16 th exons of RET gene, the 1 st exon of TMEM127 gene, the 2 nd and 3 rd exons of TMEM127 gene, the 3 rd exon of MAX gene and the 4 th exon of MAX gene are simultaneously amplified in another amplification system, namely, the exons containing pheochromocytoma and paraganglia pathogenic mutation sites in VH L, RET, TMEM127 and MAX genes are simultaneously amplified.
Wherein, the primer shown in SEQ ID NO.41 is a primer pair for sequencing.
The specific sequence includes that when the corresponding sequence includes the exon 1 of the SDHB gene by using the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, the length of the corresponding amplified fragment is 228bp, when the corresponding sequence includes the exon 2 of the SDHB gene by using the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.4, the length of the corresponding amplified fragment is 291bp, when the corresponding sequence includes the exon 3 of the SHDB gene by using the upstream primer SEQ ID NO.5 and the downstream primer SEQ ID NO.6, the length of the corresponding amplified fragment is 186bp, when the corresponding sequence includes the exon 4 of the SDHB gene by using the upstream primer SEQ ID NO.7 and the downstream primer SEQ ID NO.8, the length of the corresponding amplified fragment is 489bp, when the corresponding sequence includes the exon 6 of the SDHB gene, the exon 4, the length of the upstream primer SEQ ID NO.35 bp, when the corresponding sequence includes the exon 35 bp, the exon 35, the corresponding sequence includes the exon 35 bp, the SEQ ID NO.35, the corresponding sequence includes the SEQ ID NO.35 bp, when the corresponding sequence includes the SEQ ID NO.35 bp, the corresponding sequence includes the exon 35 bp, the SEQ ID NO.35 bp, the corresponding sequence includes the SEQ ID NO.35, the SEQ ID NO.35 bp, the SEQ ID NO.35, the SEQ ID NO.5, the SEQ ID NO.35, the SEQ ID NO.5, the SEQ ID NO.35, the SEQ ID NO.5, the SEQ ID NO.26, the SEQ ID NO.35, the SEQ ID NO.5, the SEQ ID NO.26, the SEQ ID NO.5, the SEQ ID NO.35, the SEQ ID NO.26, the SEQ ID NO. 5.
Thus, after 2 kinds of multiplex PCR amplifications are carried out by using the primer group, DNA fragments with different lengths can be generated, so that the fragments with different lengths can be distinguished by electrophoresis subsequently. Furthermore, the DNA of different fragments can be cut and recovered, and the sequence can be measured.
Based on the above, the present invention also provides an application method of the primer set for detecting the mutations of the pathogenic genes of pheochromocytoma and paraganglioma, which comprises:
designing the primer set of claim 1;
extracting genome DNA from a sample to be detected as an amplification template;
preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;
performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;
and determining the gene mutation conditions of pathogenic mutation sites of pheochromocytoma and paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene and the MAX gene of the sample to be detected according to the PCR product.
In an embodiment of the present invention, the determining, according to the PCR product, the gene mutation conditions of the pathogenic mutation sites of pheochromocytoma and paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene, and the MAX gene of the test sample include:
detecting the PCR product through electrophoresis to obtain the amplified fragment size of the PCR product;
and when the amplified fragment of the PCR product is correct in size, carrying out nucleotide sequence determination on the PCR product to obtain the gene mutation conditions of the pathogenic mutation sites of the pheochromocytoma and the paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene and the MAX gene of the sample to be detected.
Specifically, since DNA fragments with different lengths can be generated after multiplex PCR amplification is performed using the primer set, it is determined whether the amplified PCR product is a desired DNA fragment based on the amplified fragment size of the PCR product, i.e., the fragment length of the PCR product, and when the amplified PCR product has the correct length, the PCR product can be sequenced.
Specifically, agarose gel electrophoresis or polyacrylamide gel electrophoresis can be used to resolve DNA fragments of different lengths.
The above application method is a method for non-diagnostic purposes.
In one embodiment of the present invention, the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTP and ultrapure water;
wherein the dosage of the DNA polymerase is 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-300 nM.
For the amount of DNA polymerase, 0.5-5U means any amount in the range of 0.5U to 5U, for example, 0.5U, 1U, 1.5U, 2U, 2.5U, 3U, 3.5U, 4U, 4.5U and 5U.
For the final concentration of each mixture of dNTPs, 50-500. mu.M refers to any concentration in the range of 50. mu.M to 500. mu.M, such as 50. mu.M, 80. mu.M, 100. mu.M, 120. mu.M, 150. mu.M, 180. mu.M, 200. mu.M, 230. mu.M, 250. mu.M, 280. mu.M, 300. mu.M, 320. mu.M, 350. mu.M, 380. mu.M, 400. mu.M, 425. mu.M, 450. mu.M, 475. mu.M and 500. mu.M.
For the final concentration of each primer, 20-300nM refers to any concentration in the range of 20nM to 300nM, e.g., 20nM, 50nM, 100nM, 150nM, 200nM, 250nM, and 300 nM.
The concentration degree of the PCR buffer solution can be 2 ×, 3 ×, 4 ×, 5 ×, 6 ×, 7 ×, 8 ×, 9 × or 10 ×.
In one embodiment of the present invention, the reaction conditions of the PCR reaction system are as shown in table 1:
TABLE 1
The storage condition of the amplified PCR product is 0-20 ℃.
For the temperature of thermal denaturation under PCR reaction conditions: 92-96 deg.C means any temperature in the range of 92 deg.C to 96 deg.C, such as 92 deg.C, 93 deg.C, 94 deg.C, 95 deg.C and 96 deg.C
For the time of thermal denaturation under the PCR reaction condition, 1-10 min refers to any time within 1min to 10min, such as 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min and 10 min.
For the temperature of the denaturation reaction under the PCR reaction conditions, 93-98 ℃ means any temperature in the range of 93 ℃ to 98 ℃, such as 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 and 98 ℃.
For the time of denaturation under PCR reaction conditions, 5 to 60s means any time within the range of 5s to 60s, for example, 5s, 8s, 10s, 14s, 17s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, and 60 s.
The temperature of the annealing reaction in the PCR reaction condition is 52-68 ℃ which is any temperature in the range of 52 ℃ to 68 ℃, for example, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃ and 68 ℃.
The time of the annealing reaction under the PCR reaction conditions is 10 to 60 seconds, for example, 10s, 15s, 20s, 25s, 30s, 32s, 38s, 40s, 45s, 50s, 53s, 56s, and 60 s.
For the temperature of the extension reaction and the temperature of the final extension reaction under the PCR reaction conditions, 68-72 ℃ means any temperature in the range of 68 ℃ to 72 ℃, for example, 68 ℃, 69 ℃, 70 ℃, 71 ℃ and 72 ℃.
For the time of the extension reaction under the PCR reaction condition, 1-8 min refers to any time within 1min to 8min, such as 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, and 8 min.
For the time of the final extension reaction under the PCR reaction condition, 0-20 min refers to any time within the range of 0min to 20min, such as 0min, 3min, 5min, 7min, 10min, 12min, 15min, 18min and 20 min.
Specifically, the method for extracting genomic DNA from a sample to be tested comprises the following steps: and (3) extracting by hand or using a kit, and extracting the extracted DNA to obtain the genome DNA.
Specifically, the sample to be tested is a sample of blood, cells, tissues or buccal swabs containing human genomic DNA.
Specifically, the invention also provides application of the primer group or any primer pair in preparation of a reagent or a kit for detecting gene mutation of pathogenic mutation sites of pheochromocytoma and paraganglioma in SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX genes.
Specifically, the invention also provides application of the primer group or any primer pair in gene mutation detection of pathogenic mutation sites of pheochromocytoma and paraganglioma in SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX genes.
Compared with the prior art, the invention at least has the following beneficial effects:
(1) the detection flux is improved, each reaction of the common PCR only aims at one nucleic acid fragment, the invention can simultaneously detect a plurality of nucleic acid fragments based on the multiplex PCR, and the gene mutation of the pathogenic mutation sites of pheochromocytoma and paraganglioma in the genes of SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX can be detected by one reaction.
(2) The cost is reduced: the invention can reduce the PCR reaction system from 20 systems/procedures to 2 systems/procedures, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and greatly reducing the detection cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the result of agarose gel electrophoresis;
FIG. 2 is another result of agarose gel electrophoresis according to an embodiment of the invention;
FIG. 3 is a partial nucleotide base sequence for a mutation site in the SDHB gene in the PCR product sequence determination results provided by one embodiment of the invention;
FIG. 4 shows a partial nucleotide base sequence of another mutation site in the SDHB gene in the PCR product sequence determination results provided by one embodiment of the invention;
FIG. 5 shows a partial nucleotide base sequence of a mutation site in VH L gene in the PCR product sequencing result provided by an embodiment of the present invention.
Detailed Description
Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1
Design and Synthesis of primer set
Step 1.1, designing specific upstream and downstream primers based on exons of pheochromocytoma and paraganglioma pathogenic mutation sites in SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX genes.
For designing the primers, Primer Quest and Primer Premier 5.0 are adopted to design the primers and analyze the mismatch of the dimer and the stem loop, the primers are designed at two ends containing mutation sites, and the annealing temperatures of 20 pairs of primers are basically kept consistent.
The primer sets provided by the embodiment cover pathogenic mutation sites of pheochromocytoma and paraganglioma in genes of SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX, because small sequence change can cause the primer amplification efficiency to be obviously reduced and the specificity to be deteriorated, multiple PCR primer sets are respectively designed aiming at different sites/exons, and after the screening of a pre-experiment, the primer sets with the best amplification effect are selected as shown in the following table 2 in order to avoid the interference of each primer pair during amplification and the influence on the amplification effect, twenty primer pairs in the table 2 can be divided into two groups, specifically, the primer pairs shown in SEQ ID NO.1 to SEQ ID NO.22 are a first group, and the primer pairs shown in the primer names SEQ ID NO.23 to SEQ ID NO.40 are a second group.
TABLE 2
The specific mutation sites of each gene are shown in table 3 below:
TABLE 3
Step 1.2: and (3) synthesizing the primer group designed in the step 1.1.
Example 2
Extraction of genomic DNA from a sample to be tested
Step 2.1: mouth shed cells or fresh peripheral blood samples were collected with mouth swabs.
In this embodiment, the sample to be tested is a mouth swab sample or a human fresh peripheral blood sample.
And 2.2, extracting the genomic DNA from the sample by adopting a Tiangen buccal swab genomic DNA extraction kit (DP322) or a blood/cell/tissue genomic DNA extraction kit (DP304), measuring the concentration and purity of the DNA by adopting NP80-touch (IMP L EN in Germany), and storing the genomic DNA.
Example 3
Preparation of PCR reaction System
Step 3.1: and (3) taking the genome DNA obtained in the step 2.2 as an amplification template, and adopting the primer group synthesized in the step 1.2 to prepare a multiple PCR reaction system.
In this example, a multiplex PCR amplification system was prepared by using DNA polymerase and buffer as basic raw materials in KOD FX enzyme system (cat. KFX-101) manufactured by Toyobo, Inc., and adjusting the primer concentration, dNTP concentration, buffer concentration and enzyme amount based on the amplification system in the enzyme system specification, and the specific composition of this reaction system is shown in Table 4 below.
TABLE 4
| Reagent composition | Volume of |
| 5×PCR buffer for FX | 26μl |
| 2mM dNTP | 5μl |
| Primer Mix (1. mu.M each Primer) | 5μl |
| KOD FX(1U/μl) | 1μl |
| Amplification template | 1μl |
| Ultrapure water | 12μl |
Mixing the primers in an equimolar way, wherein the total concentration of the primers is 10 micromoles; the amount of the DNA template can be adjusted, and 30ng of genomic DNA can be used in this example.
It is understood that the proportional scaling up/down of the reaction system is within the scope of the embodiments of the present invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.
Step 3.2: the procedure of the PCR instrument was set according to the multiplex PCR reaction conditions shown in Table 4 below, and a multiplex PCR amplification reaction was performed on the multiplex PCR reaction system prepared in step 3.1 to obtain a PCR product.
TABLE 4
It should be noted that the PCR product obtained in this example was stored at 4 ℃ until use.
Example 4
Electrophoretic detection
Step 4.1: and (3) detecting the PCR product obtained in the step 3.2 by agarose gel electrophoresis to obtain the size of the PCR product fragment.
One of the multiple PCR detection results is shown in fig. 1, wherein the marks of ZFY, ZHF, S L P and "empty" shown in fig. 1 are marks for distinguishing the electrophoresis results of the PCR products of different samples, the left-most column of fig. 1 shows a mark strip for characterizing the length, the right-most column of fig. 1 shows the electrophoresis results of the PCR products of the blank control group, and the middle columns show the electrophoresis results of the PCR products of different samples to be detected.
For example, 11 bands from bottom to top in the electrophoresis result of the sample indicated by ZFY, ZHF and S L P in FIG. 1 are PCR amplification products respectively corresponding to the SDHB-3 exon, SDHB-1 exon, SDHB-2 exon, SDHD-4 exon, SDHC-1 exon, SDHB-4 exon, SDHC-2 exon, SDHC-5 exon, SDHC-4 exon, SDHB 6-7 exon and SDHD 1-3 exon.
The other multiplex PCR detection result is shown in fig. 2, the labels of JYX, RJJ, S L P, ZFY, ZHF and "empty" shown in fig. 1 are labels for distinguishing the electrophoresis results of PCR products of different samples, the left-most column of fig. 2 shows a ruler bar, the right-most column shows the electrophoresis results of PCR products of a blank control group, and the middle columns show the electrophoresis results of PCR products of different samples.
For example, in the electrophoresis result of each sample in FIG. 2, the bottom-up 9 bands are usually PCR amplification products corresponding to the Max-3 exon, Max-4 exon, VH L-1 exon, VH L-2 exon, VH L-3 exon, TMEM127-1 exon, RET exons 10-11, TMEM127 exons 2-3 exon, RET exons 14-16 exon, respectively.
Referring to fig. 1 and 2, it can be known from the electrophoresis results of the blank set that the environmental factors have no adverse effect on the electrophoresis detection results of the sample to be detected. According to the electrophoresis result of each sample to be detected, the number of the bright bands of the PCR amplification product corresponding to the existing bright bands is consistent with the theory; the bright bands are clear and have obvious intervals, different bright bands are not overlapped and have no smear, and the bright band effect is good. Thus, it can be shown that when two multiplex PCR amplifications are performed using the PCR amplification primer set designed in step 1.1, only the expected target product is generated, but no other irrelevant product is generated, and the primer set is reasonably designed.
Step 4.2: after the size of the PCR product fragment is verified to be correct, the sequence of the PCR product can be determined.
Example 5
Sequence determination
Step 5.1: after determining that the size of the PCR product fragment obtained in step 4.1 is correct, the PCR product obtained in step 3.2 is sent to a sequencing company for sequence determination, and a sequencing result in the format of.ab 1 is obtained.
And 5.2, analyzing the sequencing result obtained in the step 5.1 by using Chromas sequence analysis software to obtain the gene mutation conditions of the pathogenic mutation sites of pheochromocytoma and paraganglioma in the genes of SDHB, SDHC, SDHD, VH L, RET, TMEM127 and MAX.
The partial sequencing results are shown in FIGS. 3 to 5.
Referring to FIG. 3, FIG. 3 shows the nucleotide base sequences at and upstream and downstream of the c.343C > T (p.Arg115Ter) mutation site of the gene of SDHB. Referring to the frame line part in FIG. 3, it can be seen that the mutation of the gene from C to T does not occur at position 343, i.e., the mutation of the gene does not occur at the site of the mutation. The gene mutation condition is consistent with the premise that the sample source is a normal human body.
Referring to FIG. 4, FIG. 4 shows nucleotide base sequences at and upstream and downstream of the mutation site c.574T > C (p.Cys192Arg) of the SDHB gene. Referring to the frame line part in FIG. 4, it can be seen that no gene mutation from T to C occurred at position 574, i.e., no gene mutation occurred at the site of the gene mutation. The gene mutation condition is consistent with the premise that the sample source is a normal human body.
Referring to FIG. 5, FIG. 5 shows nucleotide base sequences at C.445G > T (p.Ala149Ser) and upstream and downstream of the gene mutation site of VH L. referring to the box line part in FIG. 5, it can be seen that no gene mutation from G to T occurs at position 445, i.e., no gene mutation occurs at the gene mutation site.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110> Beijing and Hei medical diagnostic technology GmbH
<120> primer set for detecting pathogenic gene mutation of pheochromocytoma and paraganglioma and application method thereof
<160>41
<170>PatentIn version 3.3
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ggaccgagaa agtgcacgac ttagaaa 27
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ccttgggaca ctgccctgga aatatg 26
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ttgctatcct ccaccggact gtca 24
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gggaaggttt ggagaagatg gtcagg 26
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tttcccacct taccctctcg tttcc 25
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Claims (6)
1. The primer group for detecting the pheochromocytoma and paraganglioma pathogenic gene mutation is characterized by comprising at least one primer pair of the following twenty primer pairs:
the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4;
the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6;
the nucleotide sequence of the upstream primer of the fourth primer pair is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8;
the nucleotide sequence of the upstream primer of the fifth primer pair is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10;
the nucleotide sequence of the upstream primer of the sixth primer pair is shown as SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12;
the nucleotide sequence of the upstream primer of the seventh primer pair is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 14;
the nucleotide sequence of the upstream primer of the eighth primer pair is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 16;
the nucleotide sequence of the upstream primer of the ninth primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 18;
the nucleotide sequence of the upstream primer of the tenth primer pair is shown as SEQ ID NO.19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.20, and the nucleotide sequence of the sequencing primer is shown as SEQ ID NO. 41;
the nucleotide sequence of the upstream primer of the eleventh primer pair is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 22;
the nucleotide sequence of the upstream primer of the twelfth primer pair is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 24;
the nucleotide sequence of the upstream primer of the thirteenth primer pair is shown as SEQ ID NO.25, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 26;
the nucleotide sequence of the upstream primer of the fourteenth primer pair is shown as SEQ ID NO.27, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 28;
the nucleotide sequence of the upstream primer of the fifteenth primer pair is shown as SEQ ID NO.29, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 30;
the nucleotide sequence of the upstream primer of the sixteenth primer pair is shown as SEQ ID NO.31, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 32;
the nucleotide sequence of the upstream primer of the seventeenth primer pair is shown as SEQ ID NO.33, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 34;
the nucleotide sequence of the upstream primer of the eighteenth primer pair is shown as SEQ ID NO.35, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 36;
the nucleotide sequence of the upstream primer of the nineteenth primer pair is shown as SEQ ID NO.37, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 38;
the nucleotide sequence of the upstream primer of the twentieth primer pair is shown as SEQ ID No.39, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 40.
2. The method for using the primer set for detecting the pathogenic gene mutation of pheochromocytoma and paraganglioma according to claim 1, which comprises:
designing the primer set of claim 1;
extracting genome DNA from a sample to be detected as an amplification template;
preparing a multiple Polymerase Chain Reaction (PCR) reaction system containing the primer group and the amplification template;
performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;
and determining the gene mutation conditions of pathogenic mutation sites of pheochromocytoma and paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene and the MAX gene of the sample to be detected according to the PCR product.
3. The method of claim 2,
the method for determining the gene mutation conditions of the pathogenic mutation sites of the pheochromocytoma and the paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene and the MAX gene of the sample to be detected according to the PCR product comprises the following steps:
detecting the PCR product through electrophoresis to obtain the amplified fragment size of the PCR product;
and when the amplified fragment of the PCR product is correct in size, carrying out nucleotide sequence determination on the PCR product to obtain the gene mutation conditions of the pathogenic mutation sites of the pheochromocytoma and the paraganglioma in the SDHB gene, the SDHC gene, the SDHD gene, the VH L gene, the RET gene, the TMEM127 gene and the MAX gene of the sample to be detected.
4. The method according to claim 2 or 3,
the multiplex PCR reaction system further comprises: DNA polymerase, PCR buffer solution corresponding to the DNA polymerase, a mixture of 4 kinds of deoxyribonucleoside triphosphate dNTP and ultrapure water;
wherein the dosage of the DNA polymerase is 0.5-5U, the final concentration of each dNTP is 50-500 mu M, and the final concentration of each primer in the primer group is 20-300 nM.
5. The method of claim 4,
the DNA polymerase comprises: taq polymerase and/or KOD FX polymerase.
6. The method of claim 4,
the reaction conditions of the PCR reaction system comprise: performing thermal denaturation at the temperature of 92-96 ℃ for 1-10 min; denaturation at 93-98 ℃ for 5-60 s; annealing at 52-68 ℃ for 10-60 s; extending for 1-8 min at 68-72 ℃; 5-60 s of denaturation at the temperature of 93-98 ℃, 10-60 s of annealing at the temperature of 52-68 ℃ and 1-8 min of extension at the temperature of 68-72 ℃ are circulated for 25-40 times, and 68-72 ℃ is finally extended for 0-30 min.
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| CN116516009A (en) * | 2023-05-06 | 2023-08-01 | 中国医学科学院北京协和医院 | Amplification primer group for detecting pheochromocytoma and paraganglioma pathogenic genes and application thereof |
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