CN113637755A - Primer composition for detecting cancer susceptibility gene and application thereof - Google Patents
Primer composition for detecting cancer susceptibility gene and application thereof Download PDFInfo
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Abstract
The invention provides a primer composition for detecting cancer susceptibility genes, which comprises 226 pairs of primers, wherein each pair of primers comprises a forward primer and a reverse primer, and each pair of primers corresponds to an SNP locus. The primer composition can accurately and specifically identify 226 SNP loci related to 9 cancer susceptibility genes, and can detect the 226 SNP loci simultaneously. The primers in the primer composition have the advantages of small mutual interference among the primers, strong specificity and good amplification effect, and the primers can be connected with indexes of different sequencing platforms to meet the requirements of sequencing on the different sequencing platforms.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer composition for detecting cancer susceptibility genes and application thereof.
Background
Cancer is one of the major diseases and serious public health problems seriously threatening human survival and social development, and cancer control has become the key point of the health strategy of governments around the world. In 2020, the total number of the nine cancers of lung cancer, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, gastric cancer, esophageal cancer and cervical cancer accounts for 69.2% of the number of new cases all the year around, and if the cancer susceptibility gene can be detected, early monitoring, prevention, treatment guidance and reduction of the risk group can be achieved.
Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. The individual carries different gene Single Nucleotide Polymorphisms (SNPs), the risk of the disease is different, and the response to the drug is different. Therefore, the early detection and early warning of diseases can be realized by detecting individual gene Single Nucleotide Polymorphism (SNP), and doctors can be helped to screen out effective treatment schemes, make the most correct treatment decision for patients, and realize accurate early warning before and accurate medication after the diseases.
Most of the existing cancer detection primers are SNP sites related to chemotherapeutic drugs, so a primer group and a detection method which can simultaneously detect 9 cancer susceptibility genes are needed to guide healthy medication and provide help for avoiding high risk of diseases.
Disclosure of Invention
In view of the above, the present invention provides a primer set capable of simultaneously detecting 9 cancer susceptibility genes.
The technical scheme of the invention is realized as follows: the invention provides a primer composition for detecting cancer susceptibility genes, which comprises 226 pairs of primers, wherein each pair of primers comprises a forward primer and a reverse primer. On the basis of the technical scheme, preferably, the nucleotide sequence of the primer composition is shown as SEQ ID NO. 1-452.
On the basis of the above technical solution, preferably, the primer composition is a specific primer designed for 226 SNP sites, and the SNP sites are as follows: rs8032158, rs940187, rs1511412, rs873549, rs1229984, rs6435862, rs4712653, rs3768716, rs602633, rs7504990, rs975334, rs10936599, rs2272007, rs 17190, rs401681, rs6773854, rs10735810, rs6068816, rs3117582, rs 1051731730, rs2285947, rs 249494949494938, rs2736100, rs10023113, rs2395185, rs 752352352352352352352352352352358, rs11733008, rs36600, rs 17386, rs 1316296298, rs 7574865865865865865865865 865, rs 92105, rs 74779, rs 4354779, rs 64779, rs 427767779, rs 4354779, rs 427777779, rs 72779, rs 647777779, rs 4277779, rs 42777777779, rs 7277779, rs 72779, rs 7277779, rs 72779, rs 72793, rs 72779, rs 7279779, rs 72793, rs 72779, rs 72798, rs 7256300, rs 72798, rs 729, rs 72793, rs 72779, rs 729, rs 72779, rs 729, rs 72794354779, rs 72798, rs 729, rs 7294729472779, rs 729472779, rs 729, rs 72779, rs 729, rs 64049, rs 729, rs 64729, rs 729, rs 64049, rs 729, rs 72849, rs 729, rs 64049, rs 72849, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs10090154, rs9623117, rs2987983, rs1571801, rs1447295, rs 169901979, rs7017300, rs3760511, rs721048, rs1800896, rs2011077, rs 7652332331, rs339331, rs3218536, rs180177111, rs 3731231239, rs34330, rs28997576, rs 146747465, rs10895068, rs4986850, rs249954, rs144848, rs2420946, rs 28897757757757757757757757757756, rs11571833, rs180177122, rs12248560, rs 1801801804, rs1219648, rs 87117, rs 180701783, rs11200014, rs 17969, rs 1807124, rs 17817 817, rs 17878761, rs 29578 5757795779579, rs 1779577946, rs 1779577948, rs 177948, rs 17797948, rs 177948, rs 177957798, rs 1805747, rs 1807948, rs 180798, rs 180577948, rs 1805747, rs 1807946, rs 177146, rs 1746, rs 177146, rs 1746, rs 1747, rs 177946, rs 1747, rs 177946, rs 1747, rs 177947, rs 1747, rs 7948, rs 1747, rs 798, rs 177948, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17798, rs 177948, rs 177148, rs 17798, rs 177146, rs 177148, rs 17798, rs 177148, rs 177948, rs 17798, rs 177146 177948, rs 177146, rs 17798, rs 177948, rs 17798, rs 177148, rs 177948, rs 177148, rs 17717948, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17717948, rs 177148, rs 17798, rs 177148, rs 17798, rs 177948, rs 17798, rs 177148, rs 17717948, rs 177948, rs 177148, rs 17798, rs 177148, rs 177948.
A method for using a primer composition for cancer susceptibility gene detection, comprising the following steps:
s1, extracting sample genome DNA, then crushing the genome DNA to be below 500bp by using an ultrasonic disruptor, and carrying out Q-Sep detection on the crushed DNA;
s2, library construction, comprising two-step PCR amplification, PCR1 amplification: performing amplification by using the primer composition of claim 1 as a template, wherein the amplification procedure comprises: activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 60 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeating the procedures of denaturation-annealing-extension for 15 times; 5min at 72 ℃ and permanence at 4 ℃; after the PCR1 amplification is finished, purifying a PCR1 product by using magnetic beads; PCR2 amplification: using a purified product after PCR1 amplification as a template, adding an index tag sequence, and simultaneously amplifying, wherein the amplification procedure comprises the steps of activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 58 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeatedly cycling 6 times; quenching at 72 ℃ for 5min, and keeping the temperature at 4 ℃ for a long time; after the PCR2 amplification is finished, sorting and purifying a PCR2 product by using magnetic beads;
s4, high throughput sequencing: sequencing the obtained DNA library through a sequencing platform, and analyzing the sequencing data result.
On the basis of the above technical solution, preferably, the sample in step S1 is one of saliva, blood and serum.
Based on the above technical solution, preferably, the index label adding method in step S2 is: an index tag was added to the 5' -end of each of the above primers.
On the basis of the above technical solution, preferably, the index tag in step S2 is one of an Illumina MiSeq sequencing platform, a BGI-Seq sequencing platform, and a Life sequencing platform.
Based on the above technical solution, preferably, the sequencing platform in step S4 is consistent with the sequencing platform selected by the index tag.
The invention also provides a detection kit for detecting the cancer susceptibility gene, which comprises the primer composition.
The invention also provides application of the kit for detecting the cancer susceptibility genes in cancer detection.
The invention also provides application of the kit for detecting the cancer susceptibility genes in preparing anti-cancer drugs.
Compared with the prior art, the primer composition for detecting the cancer susceptibility gene and the application thereof have the following beneficial effects:
1. the primer composition can accurately and specifically identify SNP loci related to 9 cancer susceptibility genes, can simultaneously detect 226 SNP loci, can know the self physical condition by detecting the 226 SNP locus genes, achieves the early warning effect, and delays or prevents the occurrence of cancers as far as possible by regular detection.
2. The primer group can perform double-round multiple PCR at one time, simplifies the experimental operation process, and has the advantages of small mutual interference among the primers, strong specificity, good PCR amplification effect, high accuracy of the detection result and good repeatability.
3. The primer can be connected with indexes of different sequencing platforms, and meets the requirement of sequencing on different sequencing platforms. Such as Illumina MiSeq sequencing platform, BGI-Seq sequencing platform and Life sequencing platform.
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 described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the Q-Sep identification result of the fragmented genomic DNA of a saliva sample of subject 1;
FIG. 2 is a diagram showing the Q-Sep identification result of the fragmented genomic DNA of the saliva sample of subject 2;
FIG. 3 is a diagram showing the Q-Sep identification result of the fragmented genomic DNA of the saliva sample of the subject 3;
FIG. 4 is a diagram showing the results of Q-Sep library identification of saliva samples from subject 1;
FIG. 5 is a diagram showing the results of Q-Sep assay of the library from saliva sample of subject 2;
FIG. 6 is a diagram showing the results of Q-Sep assay of the library from saliva sample of subject 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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 one primer design and dilution
Designing a primer: the invention designs 226 pairs of primers aiming at 9 kinds of cancers, covers 226 SNP loci, and has the primer sequences as shown in the following table:
TABLE 1 SNP sites and primer set sequences
Dilution of the cancer primer set mixture: diluting the dry powder of each primer to 100 mu M according to requirements; the primers were mixed into a primer mixture, with a final concentration of 0.5. mu.M for each primer.
EXAMPLE II extraction of biological samples to be tested
S1, sampling: 3 subjects are recruited to prepare to collect fresh saliva, rinse the mouth with clear water 30min before sampling, during which no water or food is available, and collect the saliva 30min later, specifically, the oral mucosa is scraped lightly with teeth, 2mL of fresh saliva is collected for a few times, and the saliva is uniformly mixed with 2mL of saliva preservation solution and then stored at normal temperature for standby.
S2, extracting sample DNA: taking 200 mu l of each biological sample, and extracting human genome DNA in the sample by using a QIAGEN kit; storing at-20 deg.C for use.
S3, disruption of genomic DNA: the genome DNA of 3 subjects is broken to be below 500bp by an ultrasonic breaking instrument under the conditions that: 20 sON; 10 sOFF; 8 cycles.
S4, quality control detection: the broken DNA was subjected to Q-Sep detection, and the results of DNA quality detection are shown in FIGS. 1-3 and Table 2.
TABLE 2 DNA quality inspection data
| Parameter(s) | cancer-1 | cancer-2 | cancer-3 |
| SNP site coverage | 100％ | 100％ | 100％ |
| Length range of building warehouse | 23-901bp | 23-900bp | 24-499bp |
| Average length | 225.5bp | 230.2bp | 238.2bp |
FIGS. 1-3 show: 98% of the DNA fragments are within 20-500bp, and the library building requirement is met. Shown in Table 2: the SNP site coverage rates of the subjects 1-3 are all 100%, namely 226 SNP sites can be detected, which shows that the primer group has good amplification effect and wide coverage range.
Example three Gene library construction
S1, first round PCR reaction: the PCR1 reaction was performed using the primer set described above, and the amplification procedure was: activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 60 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeating the procedures of denaturation-annealing-extension for 15 times; 72 ℃ for 5min, 4 ℃ permanent, as shown in Table 3.
TABLE 3 PCR1 reaction System and amplification conditions
S2, magnetic bead purification: (1) cooling the first round PCR reaction product to 25 ℃, adding 1.5X Buffer YC magnetic beads, uniformly mixing, and standing for 5 min; (2) centrifuging the reaction solution for a short time, placing on a magnetic frame, and discarding the supernatant after 5 min; (3) adding 200 μ l 80% ethanol, rinsing the magnetic beads, and removing ethanol after 30 s; (4) step S3 is repeated once; (5) the PCR tube was placed at 25 ℃ and 10.5. mu.l ddH was added after the beads were dried2And O. Mixing, and standing for 5 min; (6) after the PCR tube was centrifuged briefly, it was placed on a magnetic stand and after 5min, 9.5. mu.l of the supernatant was pipetted into a new PCR tube.
S3, second round PCR: using a purified product amplified by the PCR1 as a template, adding an index tag sequence, adding an index to the 5' end of each primer, and simultaneously amplifying, wherein the amplification procedure comprises the steps of activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 58 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeatedly cycling the denaturation-annealing-extension procedure for 6 times; quench at 72 ℃ for 5min, 4 ℃ permanently. As shown in table 4.
TABLE 4 PCR2 reaction reagents
S4, Buffer YC magnetic bead double-round fragment screening: (1) add the second round PCR reaction product to ddH2Supplementing O to 50 μ l, adding 0.65X Buffer YC magnetic bead, mixing, and standing at room temperature for 5 min; (2) placing the reaction solution on a magnetic frame, and sucking the reaction solution into a fresh PCR tube after 5 min; (3) adding 0.2X Buffer YC magnetic beads into a new PCR tube, mixing with the reaction solution uniformly, and standing for 5min at 25 ℃; (4) centrifuging the reaction solution for a short time, placing the reaction solution on a magnetic frame, and discarding the supernatant after 5 min; (5) adding 200 mul of ethanol with volume fraction of 80% to rinse the magnetic beads, and discarding the ethanol after 30 s; (6) step 5 is repeated once; (7) the PCR tube was placed at 25 ℃ and 11. mu.l ddH was added after the beads were dried2O, standing for 5min after uniformly mixing; (8) the PCR tube was centrifuged briefly and placed on a magnetic stand, and after 5min, the supernatant was pipetted into a new PCR tube.
Example four quality control assays
The results of the detection by the Qubit fluorescence quantifier are measured by the Qubit 3.0, and are shown in the following table:
TABLE 5 fluorescent quantitation appearance test results
| Sample numbering | Concentration (ng/. mu.l) | Volume (μ l) | Total amount (ng) |
| cancer-1 | 4.40 | 10 | 44.00 |
| cancer-2 | 5.56 | 10 | 55.60 |
| cancer-3 | 5.85 | 10 | 58.50 |
Q-sep quality inspection: q-sep quality inspection is carried out on the constructed sample library, and the library result is shown in figures 4-6 and table 6.
TABLE 6 library quality test data
| Parameter(s) | cancer-1 | cancer-2 | cancer-3 |
| SNP site coverage | 100％ | 100％ | 100％ |
| Length range of building warehouse | 24-878bp | 24-894bp | 24-923bp |
| Average length | 248.5bp | 251.0bp | 253.4bp |
FIGS. 4-6 show: the quality inspection results show that target fragments are amplified uniformly, and the library meets the sequencing requirement. Shown in Table 6: the SNP site coverage rates of the subjects 1-3 are all 100%, namely 226 SNP sites can be detected, which shows that the primer group has good amplification effect and wide coverage range.
Example five sequencing
Quantitatively mixing the library of the sample for carrying out the illumina sequencing, and distinguishing sequencing data through an Index tag sequence added at the 5' end, so that the detection requirement can be met and the cost can be saved. And obtaining related data, carrying out genotyping, and giving an individual gene detection interpretation report according to a gene detection result. Through a gene detection report, the health condition of the user is known, so that the objective environment and the dietary structure can be improved in a targeted manner by better utilizing modern medicine, and the occurrence of diseases is effectively delayed or prevented.
The invention also provides a detection kit for cancer risk early warning, which comprises the 226 pair primer group, and the 226 pair primers are detected to detect the cancer susceptibility gene, so that early monitoring, prevention, guidance and treatment can be achieved, and the morbidity risk of risk groups is reduced. If the mutation of the SNP site corresponding to the cancer is detected, a medicine aiming at the SNP site can be developed to inhibit the spread of cancer cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A primer composition for detecting cancer susceptibility genes, which is characterized in that: comprises 226 pairs of primers, each pair of primers comprises a forward primer and a reverse primer, and the nucleotide sequence of the primer composition is shown as SEQ ID NO. 1-452.
2. The primer composition for cancer susceptibility gene detection according to claim 1, wherein: the primer composition is a specific primer designed aiming at 226 SNP sites, and the SNP sites are as follows: rs8032158, rs940187, rs1511412, rs873549, rs1229984, rs6435862, rs4712653, rs3768716, rs602633, rs7504990, rs975334, rs10936599, rs2272007, rs 17190, rs401681, rs6773854, rs10735810, rs6068816, rs3117582, rs 1051731730, rs2285947, rs 249494949494938, rs2736100, rs10023113, rs2395185, rs 752352352352352352352352352352358, rs11733008, rs36600, rs 17386, rs 1316296298, rs 7574865865865865865865865 865, rs 92105, rs 74779, rs 4354779, rs 64779, rs 427767779, rs 4354779, rs 427777779, rs 72779, rs 647777779, rs 4277779, rs 42777777779, rs 7277779, rs 72779, rs 7277779, rs 72779, rs 72793, rs 72779, rs 7279779, rs 72793, rs 72779, rs 72798, rs 7256300, rs 72798, rs 729, rs 72793, rs 72779, rs 729, rs 72779, rs 729, rs 72794354779, rs 72798, rs 729, rs 7294729472779, rs 729472779, rs 729, rs 72779, rs 729, rs 64049, rs 729, rs 64729, rs 729, rs 64049, rs 729, rs 72849, rs 729, rs 64049, rs 72849, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs 729, rs 64729, rs 729, rs 64729, rs 729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 4354779, rs 729, rs 4354779, rs 64729, rs 729, rs 4354779, rs10090154, rs9623117, rs2987983, rs1571801, rs1447295, rs 169901979, rs7017300, rs3760511, rs721048, rs1800896, rs2011077, rs 7652332331, rs339331, rs3218536, rs180177111, rs 3731231239, rs34330, rs28997576, rs 146747465, rs10895068, rs4986850, rs249954, rs144848, rs2420946, rs 28897757757757757757757757757756, rs11571833, rs180177122, rs12248560, rs 1801801804, rs1219648, rs 87117, rs 180701783, rs11200014, rs 17969, rs 1807124, rs 17817 817, rs 17878761, rs 29578 5757795779579, rs 1779577946, rs 1779577948, rs 177948, rs 17797948, rs 177948, rs 177957798, rs 1805747, rs 1807948, rs 180798, rs 180577948, rs 1805747, rs 1807946, rs 177146, rs 1746, rs 177146, rs 1746, rs 1747, rs 177946, rs 1747, rs 177946, rs 1747, rs 177947, rs 1747, rs 7948, rs 1747, rs 798, rs 177948, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17798, rs 177948, rs 177148, rs 17798, rs 177146, rs 177148, rs 17798, rs 177148, rs 177948, rs 17798, rs 177146 177948, rs 177146, rs 17798, rs 177948, rs 17798, rs 177148, rs 177948, rs 177148, rs 17717948, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17798, rs 177948, rs 17798, rs 177948, rs 177148, rs 17717948, rs 177148, rs 17798, rs 177148, rs 17798, rs 177948, rs 17798, rs 177148, rs 17717948, rs 177948, rs 177148, rs 17798, rs 177148, rs 177948.
3. A method for using a primer composition for cancer susceptibility gene detection, which is characterized by comprising the following steps: the method comprises the following steps:
s1, extracting sample genome DNA, then crushing the genome DNA to be below 500bp by using an ultrasonic disruptor, and carrying out Q-Sep detection on the crushed DNA;
s2, library construction, comprising two-step PCR amplification, PCR1 amplification: performing amplification by using the primer composition of claim 1 as a template, wherein the amplification procedure comprises: activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 60 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeating the procedures of denaturation-annealing-extension for 15 times; 5min at 72 ℃ and permanence at 4 ℃; after the PCR1 amplification is finished, purifying a PCR1 product by using magnetic beads; PCR2 amplification: using a purified product after PCR1 amplification as a template, adding an index tag sequence, and simultaneously amplifying, wherein the amplification procedure comprises the steps of activating at 94 ℃ for 3min, denaturing at 94 ℃ for 15sec, annealing at 58 ℃ for 20sec, extending at 72 ℃ for 10sec, and repeatedly cycling 6 times; quenching at 72 ℃ for 5min, and keeping the temperature at 4 ℃ for a long time; after the PCR2 amplification is finished, sorting and purifying a PCR2 product by using magnetic beads;
s4, high throughput sequencing: sequencing the obtained DNA library through a sequencing platform, and analyzing the sequencing data result.
4. The method of claim 3, wherein the primer composition for detecting a cancer susceptibility gene comprises: the sample of step S1 is one of saliva, blood and serum.
5. The method of claim 3, wherein the primer composition for detecting a cancer susceptibility gene comprises: the index label adding method in step S2 includes: an index tag is added to the 5' end of each primer of claim 1.
6. The method of claim 3, wherein the primer composition for detecting a cancer susceptibility gene comprises: the index label in the step S2 is one of an illumina Miseq sequencing platform, a BGI-Seq sequencing platform and a Life sequencing platform, and the sequencing platform in the step S4 is consistent with the sequencing platform selected by the index label.
7. A kit for detecting a cancer susceptibility gene, comprising the primer composition of claim 1.
8. The use of the kit of claim 7 for the detection of a cancer susceptibility gene in the detection of cancer.
9. The use of the kit according to claim 7 for detecting susceptibility genes to cancer in the preparation of anti-cancer drugs.
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