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CN113913521A - Genotyping detection kit and application thereof - Google Patents

Genotyping detection kit and application thereof Download PDF

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CN113913521A
CN113913521A CN202111306837.5A CN202111306837A CN113913521A CN 113913521 A CN113913521 A CN 113913521A CN 202111306837 A CN202111306837 A CN 202111306837A CN 113913521 A CN113913521 A CN 113913521A
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吴俊杰
陈柏岑
张昊瑞
肖曼
邹子修
李玉涛
王一
郭士成
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Abstract

本发明提供了一种基因分型检测试剂盒及其应用，具有这样的特征，包括：用于对所述癌症患者的全血基因组进行PCR的引物组，该引物组包括第一上游引物和第一下游引物，其中，所述第一上游引物的核苷酸序列如序列表中SEQ NO:1所示，所述第一下游引物的核苷酸序列如序列表中SEQ NO:2所示。

Figure 202111306837

The present invention provides a genotyping detection kit and application thereof, and has the characteristics of comprising: a primer set for performing PCR on the whole blood genome of the cancer patient, the primer set comprising a first upstream primer and a first upstream primer A downstream primer, wherein the nucleotide sequence of the first upstream primer is shown in SEQ NO: 1 in the sequence listing, and the nucleotide sequence of the first downstream primer is shown in SEQ NO: 2 in the sequence listing.

Figure 202111306837

Description

Genotyping detection kit and application thereof
Technical Field
The invention belongs to the field of kits, and particularly relates to a genotyping detection kit and application thereof.
Background
Lung cancer is the most common cancer in the world and the leading cause of cancer death. With the remarkable achievement of surgical treatment, immunotherapy, chemoradiotherapy and molecular targeted therapy in the aspect of lung cancer treatment, the lung cancer treatment is greatly improved, but the 5-year survival rate of lung cancer patients in China is still less than 20%. The prognosis of lung cancer patients is interfered by various factors, and the prediction difficulty is large. Therefore, finding effective biomarkers is crucial for predicting lung cancer patient prognosis, personalized treatment for lung cancer patients, and improving 5-year survival of lung cancer patients. A great deal of research evidence indicates that the occurrence and development of lung cancer are influenced by various factors such as environment and individual heredity. Genetic polymorphisms are the most common genetic variations in humans and have been shown to affect gene expression and predict prognosis in a variety of cancer patients, including lung cancer.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a genotyping detection kit and its use.
The invention provides a genotyping detection kit, which is characterized by comprising: a primer set for PCR of a whole blood genome of a cancer patient, the primer set comprising a first upstream primer and a first downstream primer,
wherein, the nucleotide sequence of the first upstream primer is shown as SEQ NO. 1 in the sequence table, and the nucleotide sequence of the first downstream primer is shown as SEQ NO. 2 in the sequence table.
The genotyping detection kit provided by the present invention may further have the following characteristics: the genotyping detection is used for detecting the genotype of a gene at a locus CAMKK1rs7214723, and the genotyping detection result comprises the following steps: TT genotype, TC genotype, and CC genotype.
The genotyping detection kit provided by the present invention may further include: a5 'universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a5 'specific ligation probe, and a 3' specific ligation probe.
The genotyping detection kit provided by the present invention may further have the following characteristics: wherein the cancer is lung cancer.
The genotyping detection kit provided by the present invention may further have the following characteristics: wherein the lung cancer is non-small cell lung cancer.
The invention provides application of a genotyping detection kit in predicting prognosis survival of a lung cancer patient.
Action and Effect of the invention
According to the genotyping detection kit, the kit comprises a primer group, the primer group comprises a first upstream primer and a first downstream primer, the whole blood genome of a cancer patient can be subjected to PCR amplification through the primer group, the genotype of the CAMKK1rs7214723 locus gene of the cancer patient can be judged according to the amplification result, and the prognostic survival of the cancer patient can be predicted according to the genotyping result of the genotype.
Drawings
FIG. 1 is a graph showing the results of a CCK8 cell proliferation experiment in example 2 of the present invention, in which FIG. 1(a) is A549 and FIG. 1(b) is NCI-H358;
FIG. 2 is a graph showing the results of A549 cell scratching experiments in example 2 of the present invention, in which FIGS. 2(a) - (d) are respectively A549 wild type 0h, A549 wild type 24h, A549CAMKK1rs7214723 site mutant 0h, and A549CAMKK1rs7214723 site mutant 24 h;
FIG. 3 is a graph showing the results of the NCI-H358 cell scratch test in example 2 of the present invention, wherein FIGS. 3(a) to (d) are NCI-H358 wild-type 0H, NCI-H358 wild-type 24H, NCI-H358CAMKK 1rs7214723 site mutant 0H, and NCI-H358CAMKK 1rs7214723 site mutant 24H, respectively;
FIG. 4 is a graph showing the results of apoptosis experiments in example 2 of the present invention, wherein FIG. 4(a) is A549 wild type, FIG. 4(b) is A549CAMKK1rs7214723 site mutant (A549 CAMKK1-W), FIG. 4(c) is NCI-H358 wild type, and FIG. 4(b) is NCI-H358CAMKK 1rs7214723 site mutant (NCI-H358CAMKK 1-W); and
FIG. 5 is a schematic view of the procedure of genotyping assay in example 3 of the present invention.
Detailed Description
In order to make the technical means, creation features, achievement purposes and effects of the invention easy to understand, the following embodiments are specifically described with reference to the accompanying drawings.
The Qiagen Blood DNA Extraction Kit (Qiagen, Hilden, Germany), Cell Counting Kit-8, Annexin V-FITC apoptosis detection kits used in the following examples are all commercially available kits.
< example 1>
This embodiment provides a genotyping detection kit, which comprises: a primer set for PCR on a cancer patient's whole blood genome, a5 ' universal probe, a 3 ' universal probe, a site recognition sequence, a qPCR universal primer, a5 ' specific ligation probe, and a 3 ' specific ligation probe.
Wherein the primer group comprises a first upstream primer and a first downstream primer. The nucleotide sequence of the first upstream primer is shown as SEQ NO. 1 in the sequence table, and the nucleotide sequence of the first downstream primer is shown as SEQ NO. 2 in the sequence table.
The concentration of the first upstream primer and the first downstream primer is 10 Mm.
The 5 'universal probe, the 3' universal probe, the site recognition sequence, the qPCR universal primer, the 5 'specific ligation probe and the 3' specific ligation probe were purchased from shanghai sky biotechnology limited, and the concentration and the use conditions were the same as those of the 2 × 48-Plex SNP scan tm kit from shanghai sky biotechnology limited.
The whole blood genome of the cancer patient is subjected to PCR amplification by using the genotyping detection kit, so that whether the cancer patient has gene mutation at the CAMKK1rs7214723 site or not can be judged, and three genotyping detection results are obtained based on the gene mutation condition of the CAMKK1rs7214723 site: TT genotype, TC genotype, and CC genotype (where site C is mutant).
< example 2>
Example 2 provides a test for verifying the influence of genetic mutation at the CAMKK1rs7214723 site on cells at the cellular level, which utilizes CRISPR-Cas9 gene editing technology to perform point mutation at the CAMKK1rs7214723 site, and the specific process is as follows:
1.1 nucleotide sequences designed for CRISPR-Cas9 Gene editing
1.1.1 select sgRNA sequences.
The sequence of sgRNA is shown as SEQ NO. 3 in the sequence table.
1.1.2 design ssDNA sequences containing the CAMKK1rs7214723 mutation site.
The designed ssDNA sequence containing CAMKK1rs7214723 mutation site is shown as SEQ NO. 4 in the sequence table.
1.1.3 design of oligo primers
The oligo primers include a second forward primer and a second reverse primer. Wherein, the sequence of the second upstream primer is shown as SEQ NO. 5 in the sequence table, and the sequence of the second downstream primer is shown as SEQ NO. 6 in the sequence table.
1.1.4 annealing of ssDNA containing the mutation site of CAMKK1rs7214723 to double stranded DNA using oligo primers.
1.2 expression vector construction
The used vector is plasmid pSpCas9(BB) -2A-Puro (PX459) V2.0, is cut by Bbs1 endonuclease, is recovered after the enzyme cutting, is connected with double-stranded DNA containing CAMKK1rs7214723 mutation sites by T4 DNA Ligase, and is transformed and extracted to obtain the plasmid.
1.3 Liposomal transfection
The successfully constructed expression vector, sgRNA and ssDNA containing CAMKK1rs7214723 mutation sites are jointly transferred into A549 cells and NCI-H358 cells, after 24 hours, Puromycin Dihydroxychloride (Biyun, ST551) is used for screening, positive clones are selected for PCR, and the PCR product is sequenced and identified.
The primers used for PCR identification comprise a third upstream primer, a third downstream primer and a sequencing primer. The sequence of the third upstream primer is shown as SEQ NO. 7 in the sequence table, the sequence of the third downstream primer is shown as SEQ NO. 8 in the sequence table, and the sequence of the sequencing primer is shown as SEQ NO. 9 in the sequence table.
1.4 cell expansion culture
The correctly identified A549-CAMKK1-W, NCI-H358-CAMKK1-W cells were expanded and the wild type corresponding thereto was used as a control for subsequent cell experiments.
1.5 cell assay
1.5.1 CCK8 cell proliferation assay
According to the experimental method (Cell Counting Kit-8, CK04), cells were inoculated into a 96-well plate, after 24 hours, Cell proliferation was detected with a full-wavelength microplate reader (biotek full-wavelength microplate reader Epoch) at an absorbance of 450nm, and control wells were set for each set of experiments and repeated three times.
1.5.2 cell scratch test
The experiment is used for detecting the migration capacity of cells, and specifically comprises the following steps: adding about 5X 10 to 6-well plate5The cells are separated, the tip is used for comparing with the ruler on the next day, the transverse line scratch on the back is made as perpendicular as possible, the cells are washed for 3 times by PBS, the scratched cells are removed, a serum-free culture medium is added, and 5% CO at 37 ℃ is added2Culturing in an incubator. Samples were taken at 24 hours and photographed, and control wells were set for each set of experiments and repeated three times.
1.5.3 apoptosis assay
Adding 5 × 10 of AnnexinV-FITC apoptosis detection kit (Beyotime, C1062S) into a 6-well plate5Individual cells were sampled for 24 hours and the apoptosis rate was measured using flow cytometry. Control wells were set for each set of experiments and repeated three times.
1.6 results of the experiment
1.6.1 cell proliferation
FIG. 1 is a graph showing the results of the cell proliferation experiment of CCK8 in example 2 of the present invention, wherein FIG. 1(a) is A549 and FIG. 1(b) is NCI-H358.
As shown in figure 1, the cell proliferation test of CCK8 shows that the mutant strain of A549CAMKK1rs7214723 site and the mutant strain of NCI-H358CAMKK 1rs7214723 site have 11.6 percent and 13.3 percent of cell proliferation respectively compared with the wild type by the average value of three biological repetitions. Therefore, the CAMKK1rs7214723 site mutant strain has influence on the proliferation capacities of the A549 cell strain and the NCI-H358 cell strain, and both the proliferation capacities are reduced.
1.6.2 cell migration
FIG. 2 is a graph showing the results of A549 cell scratching experiments in example 2 of the present invention, in which FIGS. 2(a) - (d) are respectively A549 wild type 0h, A549 wild type 24h, A549CAMKK1rs7214723 site mutant 0h, and A549CAMKK1rs7214723 site mutant 24 h. FIG. 3 is a graph showing the results of the NCI-H358 cell scratch test in example 2 of the present invention, wherein FIGS. 3(a) to (d) are NCI-H358 wild-type 0H, NCI-H358 wild-type 24H, NCI-H358CAMKK 1rs7214723 site mutant 0H, and NCI-H358CAMKK 1rs7214723 site mutant 24H, respectively.
As shown in FIG. 2 and FIG. 3, it was found by cell scratch test that the cell migration of the A549 wild type and the NCI-H358 wild type was stronger than that of the A549CAMKK1rs7214723 site mutant and that of the NCI-H358CAMKK 1rs7214723 site mutant after normal culture for 24 hours. The effect of CAMKK1rs7214723 site mutation on the migration ability of A549 and NCI-H358 cells can be judged.
1.6.3 cell migration
FIG. 4 is a graph showing the results of apoptosis experiments in example 2 of the present invention, in which FIG. 4(a) is A549 wild type, FIG. 4(b) is A549CAMKK1rs7214723 site mutant (A549 CAMKK1-W), FIG. 4(c) is NCI-H358 wild type, and FIG. 4(b) is NCI-H358CAMKK 1rs7214723 site mutant (NCI-H358CAMKK 1-W).
As shown in FIG. 4, the apoptosis rate of the mutant strain of A549CAMKK1rs7214723 site (A549 CAMKK1-W) is higher than that of the wild type A549 by 5.63 percent through the detection of apoptosis experiments. The apoptosis rate of NCI-H358CAMKK 1rs7214723 site mutation (NCI-H358CAMKK1-W) is 3.98 percent higher than that of NCI-H358 wild type. Therefore, the CAMKK1rs7214723 site mutation can promote the apoptosis of A549 and NCI-H358.
The above experimental results of example 2 show that mutation at the CAMKK1rs7214723 site has an effect on proliferation, migration and apoptosis of a549 cells and NCI-H358 cells, which indicates that CAMKK1rs7214723 can be used as a biomarker for further research on non-small cell lung cancer cells.
< example 3>
Example 3 provides the use of the genotyping detection kit of example 1 to predict prognostic survival in a patient with lung cancer. The specific process is as follows:
536 cases of major hospitals affiliated to navy military medical university (second military medical university) and 352 cases of 888 primary lung cancer patients in Thai health science research institute of Compound denier university are collected during 1-11 months in 2009. Inclusion criteria were: the new lung cancer patients confirmed by the pathological histological examination have no history of malignant tumors of other organs and no age and sex limitation. Clinical data are from medical records, follow-up data are from telephone interviews, and all patients are Chinese. The study was approved by the ethical committee of the institute of Life sciences of the university of Compound Dan, and informed consent of the study subjects was obtained for epidemiological survey data collection and blood sample collection.
2.1 detection of the patient's CAMKK1rs7214723 genotype Using the genotyping assay kit of example 1
2.1.1 blood sample Collection
All patients donated 5mL of blood.
2.1.2 DNA extraction
Genomic DNA in Blood was extracted using Qiagen Blood DNA Extraction Kit (Qiagen, Hilden, Germany).
2.1.3 detection of genomic DNA Using the genotyping assay kit of example 1 FIG. 5 is a schematic representation of the procedure for the genotyping assay of example 3 of the invention.
As shown in FIG. 5, the SNP locus allele is identified by the high specificity of the ligase ligation reaction, and then different lengths of ligation products corresponding to the locus are obtained by introducing non-specific sequences with different lengths at the tail end of the ligation probe and the ligase ligation reaction, and the ligation products are subjected to PCR amplification by using a fluorescence-labeled universal primer.
The PCR amplification reaction system comprises: 2 XEs Taq MasterMix (Beijing Baiolai Boke technology Co., Ltd.), primer sets (each 0.5mM), and 100ng of human whole blood genomic DNA.
The PCR cycling conditions were: each cycle was carried out at 94 ℃ for 30s, at 60 ℃ for 30s and at 72 ℃ for 30s, for a total of 25 cycles.
And (3) carrying out electrophoretic separation on the amplified product by fluorescence capillary electrophoresis, finally analyzing the GeneMapper software to obtain the genotype of each SNP site, judging the gene mutation condition of the CAMKK1rs7214723 site, and determining which of three genotypes (TT genotype, TC genotype and CC genotype (wherein, the site C is a mutant)) the amplified product belongs to.
2.2 statistics of patient clinical data
Basic clinical data of the patient including sex, age, smoking history, family history of malignant tumor, pathological type of lung cancer and TNM stage are collected.
And 2.3, inferring the prognostic survival of the lung cancer patient according to the data statistical conclusion, and predicting the prognostic death risk of the lung cancer patient according to the statistical analysis conclusion of the lung cancer patient based on the genotyping detection result and the clinical information of the lung cancer patient.
2.4 Statistical analysis
Statistical processing was performed using rv3.6.2 software. Demographic variability, population smoking status, family cancer history, allelic frequency differences between control and case groups, and Hardy-Weinberg equilibrium (HWE) test were analyzed using the χ 2 test. Cox regression analysis was performed using an allele model, a genotype model, a dominant model, and a recessive model, respectively, and the risk ratio (HR) and 95% Confidence Interval (CI) were calculated, and age and gender were used for correction. Further, the models were analyzed according to age, sex, smoking condition, family history, histological type of lung cancer, and the like, respectively, to evaluate the relationship between the genetic polymorphism and the prognosis of lung cancer. P < 0.05 indicates that the difference is statistically significant.
2.5 results of the experiment
2.5.1 demographic and clinical characteristics of patients
The follow-up time started in 2009 1 to 2019, 11 and 15. After excluding 49 patients, 839 patients were analyzed for incomplete clinical data. Study samples were han-nationality, 668 cases (79.6%) died, 103 cases (12.3%) survived for more than 5 years, and 68 cases (8.1%) were missed. 610 male (72.7%), 524 aged 60 or more (62.5%), 582 smoking history (69.4%), 302 malignant tumor family history (36%). From the tumor subtypes, 367 cases of adenocarcinoma (43.7%), 282 cases of squamous carcinoma (33.6%), 72 cases of SCLC (8.6%), and 118 cases of other types (14.1%). Including adenosquamous carcinoma (ASC), Large Cell Carcinoma (LCC), Carcinosarcoma (CS), and mucoepidermoid carcinoma (MEC). 154 (18.4%) patients were diagnosed with stage I and II disease, 625 (74.5%) patients were diagnosed with stage III and IV disease (Table 1).
TABLE 1 Chinese Lung cancer patient characteristic distribution and prognosis analysis table
Figure BDA0003340589580000111
Figure BDA0003340589580000121
Other cancers include adenosquamous carcinoma (ASC), Large Cell Carcinoma (LCC), Carcinosarcoma (CS), mucoepidermoid carcinoma (MEC);#MST, median lifetime.
2.5.2 correlation of patient characteristics with prognosis of Lung cancer
As shown in table 1, median survival time was significantly lower for male patients than for female patients (male, 34.27 months; female, 40.17 months; P ═ 0.01). Median survival times were significantly higher for patients < 60 years of age than for patients > 60 years of age (40.87, 33.20 months; P0.003). Median survival time was significantly higher for patients without smoking than for those with smoking (41.03 months, 33.90 months; P < 0.001)). Furthermore, median survival time was significantly reduced in patients with advanced tumors compared to patients with early tumors (29.4 months, 113.93 months; P < 0). There were no significant statistical differences in the correlation of different hospital patient characteristics with lung cancer prognosis.
TABLE 2 CAMKK1rs7214723 genotyping (Gene polymorphism) and Chinese Lung cancer patient prognosis relation Table
Figure BDA0003340589580000122
Figure BDA0003340589580000131
*CI, confidence interval; HR, risk ratio; ref, ref.aAdjusted according to age, sex and hospital.
As shown in Table 2, there are 341 TT, 400 TC and 91 CC genotypes at rs7214723 site of CAMKK1 gene, and the detection rate of the genotypes is 99.17%. The genotype frequency of rs7214723 site T/C of the CAMKK1 gene is consistent with that of HWE (P is 0.102), which indicates that the surveyed population is in genetic balance (i.e. the data of the population survey is credible). Furthermore, 1082T alleles and 582C alleles were detected at locus rs7214723 of the CAMKK1 gene. In the group of patients with death, the frequency of the T and C alleles was 65.16% (864/1326) and 34.84% (462/1326), respectively. Individuals with recessive CC genotype have a lower risk of death compared to TT + TC genotype (CC adjusted HR 0.78; 95% CI 0.61-1.00, P0.049).
Sequence listing
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accgccacct tcatatct 18

Claims (6)

1.一种基因分型检测试剂盒，用于对癌症患者进行基因分型检测，其特征在于，包括：1. a genotyping detection kit for carrying out genotyping detection to cancer patients, is characterized in that, comprising: 用于对所述癌症患者的全血基因组进行PCR的引物组，该引物组包括第一上游引物和第一下游引物，a primer set for performing PCR on the whole blood genome of the cancer patient, the primer set comprising a first upstream primer and a first downstream primer, 其中，所述第一上游引物的核苷酸序列如序列表中SEQ NO:1所示，所述第一下游引物的核苷酸序列如序列表中SEQ NO:2所示。Wherein, the nucleotide sequence of the first upstream primer is shown in SEQ NO: 1 in the sequence listing, and the nucleotide sequence of the first downstream primer is shown in SEQ NO: 2 in the sequence listing. 2.根据权利要求1所述的基因分型检测试剂盒，其特征在于：2. genotyping detection kit according to claim 1, is characterized in that: 其中，所述基因分型检测为检测CAMKK1 rs7214723位点基因的基因型，Wherein, the genotyping detection is to detect the genotype of the CAMKK1 rs7214723 locus gene, 所述基因分型检测的结果包括：TT基因型、TC基因型以及CC基因型。The results of the genotyping test include: TT genotype, TC genotype and CC genotype. 3.根据权利要求1所述的基因分型检测试剂盒，其特征在于，还包括：3. genotyping detection kit according to claim 1, is characterized in that, also comprises: 5’通用探针、3’通用探针、位点识别序列、qPCR通用引物、5’特异连接探针以及3’特异连接探针。5' universal probe, 3' universal probe, site recognition sequence, qPCR universal primer, 5' specific ligation probe, and 3' specific ligation probe. 4.根据权利要求1所述的基因分型检测试剂盒，其特征在于：4. genotyping detection kit according to claim 1, is characterized in that: 其中，所述癌症为肺癌。Wherein, the cancer is lung cancer. 5.根据权利要求4所述的基因分型检测试剂盒，其特征在于：5. genotyping detection kit according to claim 4, is characterized in that: 其中，所述肺癌为非小细胞肺癌。Wherein, the lung cancer is non-small cell lung cancer. 6.一种如权利要求1-5中任意一项所述的基因分型检测试剂盒在预测肺癌患者预后生存中的应用。6. The application of the genotyping detection kit according to any one of claims 1-5 in predicting the prognosis and survival of lung cancer patients.
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