CN115386632A - GPC5 rs2352028 gene-based genotyping detection kit and application thereof - Google Patents

GPC5 rs2352028 gene-based genotyping detection kit and application thereof Download PDF

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CN115386632A
CN115386632A CN202111578185.0A CN202111578185A CN115386632A CN 115386632 A CN115386632 A CN 115386632A CN 202111578185 A CN202111578185 A CN 202111578185A CN 115386632 A CN115386632 A CN 115386632A
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gpc5
gene
genotype
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lung cancer
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吴俊杰
王帆
邹子修
王一
郭士成
李玉涛
冯伟
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Fudan University
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Abstract

The invention provides a genotyping detection kit based on a GPC5 rs2352028 gene, which is used for detecting the genotype of a GPC5 rs2352028 locus gene of a cancer patient and has the characteristics of comprising the following components: a primer group for carrying out PCR on a whole blood genome of a cancer patient comprises 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 a sequence table, and the nucleotide sequence of the first downstream primer is shown as SEQ NO. 2 in the sequence table.

Description

GPC5 rs2352028 gene-based genotyping detection kit and application thereof
Technical Field
The invention belongs to the field of kits, and particularly relates to a genotyping detection kit based on a GPC5 rs2352028 gene 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 environmental factors, and the prediction difficulty is large. Therefore, finding effective predictive prognostic markers is crucial for predicting lung cancer patient prognosis, personalized treatment for lung cancer patients, and improving 5-year survival rate 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 is made to solve the above problems, and an object of the present invention is to provide a genotyping detection kit based on the GPC5 rs2352028 gene, and applications thereof. .
The invention provides a GPC5 rs2352028 gene-based genotyping detection kit, which is used for detecting the genotype of a GPC5 rs2352028 locus gene of a cancer patient and has the characteristics that: a primer group for carrying out PCR on a whole blood genome of a cancer patient comprises 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 a 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 based on the GPC5 rs2352028 gene provided by the invention can also have the following characteristics: wherein the genotype is any one of CC genotype, CT genotype and TT genotype.
The genotyping detection kit based on the GPC5 rs2352028 gene provided by the invention also has the following characteristics: a 5 'universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a 5 'specific ligation probe, and a 3' specific ligation probe.
The genotyping detection kit based on the GPC5 rs2352028 gene provided by the invention can also have the following characteristics: wherein the cancer is lung cancer.
The genotyping detection kit based on the GPC5 rs2352028 gene provided by the invention can also have the following characteristics: wherein the lung cancer is non-small cell lung cancer.
The invention provides application of a GPC5 rs2352028 gene-based genotyping detection kit in predicting prognosis survival of a lung cancer patient.
Action and effects of the invention
According to the GPC5 rs2352028 gene-based genotyping detection kit, the kit comprises a primer group, wherein the primer group comprises a first upstream primer and a first downstream primer, the primer group can be used for carrying out PCR amplification on the whole blood genome of a cancer patient, the genotype of the GPC5 rs2352028 locus gene of the cancer patient is judged according to the amplification result, and the prognosis survival of the cancer patient can be predicted according to the genotyping result of the genotype.
Drawings
FIG. 1 is a schematic diagram showing the procedure of genotyping detection in example 2 of the present invention.
Detailed Description
In order to make the technical means, creation characteristics, achievement purposes and effects of the invention easy to understand, the following embodiments are combined with the accompanying drawings to specifically describe the genotyping detection kit based on the GPC5 rs2352028 gene and the application thereof.
The Qiagen Blood DNA Extraction Kit (Qiagen, hilden, germany), cell Counting Kit-8, and Annexin V-FITC apoptosis detection kits used in the following examples are all commercially available kits.
< example 1>
The embodiment provides a genotyping detection kit based on a GPC5 rs2352028 gene, which comprises: a primer set for PCR on a cancer patient's whole blood genome, a 5' universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a 5' 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 10Mm.
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 biotech ltd, and the concentration and the use conditions were the same as those of the 2 × 48-Plex SNP scan TM kit from shanghai sky biotech ltd.
By using the GPC5 rs2352028 gene-based genotyping detection kit to perform PCR amplification on the whole blood genome of a cancer patient, whether the cancer patient has genetic mutation at the GPC5 rs2352028 site can be judged, and three genotyping detection results are obtained based on the genetic mutation at the GPC5 rs2352028 site: CC genotype, CT genotype, and TT genotype (where site T is mutant). That is, the genotype of the cancer patient at the GPC5 rs2352028 site can be detected as one of the three genotypes of CC, CT and TT.
< example 2>
Embodiment 2 provides an application of the GPC5 rs2352028 gene-based genotyping detection kit of embodiment 1 in predicting prognostic survival of lung cancer patients. 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 research is approved by the ethical committee of the institute of life science of the university of reinserture, and the subjects are informed to approve the collection of epidemiological investigation data and blood samples.
2.1 detection of the patient's GPC5 rs2352028 genotype Using the GPC5 rs2352028 Gene-based genotyping assay kit of example 1
2.1.1 blood sample Collection
All patients gave informed consent and 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 GPC5 rs2352028 Gene-based genotyping assay kit of example 1
FIG. 1 is a schematic diagram showing the procedure of genotyping detection in example 2 of the present invention.
As shown in figure 1, the high specificity of ligase ligation reaction is adopted to realize the identification of SNP locus alleles, then non-specific sequences with different lengths are introduced into the tail section of a ligation probe, ligation products with different lengths corresponding to the loci are obtained through ligase ligation reaction, and the PCR amplification is carried out on the ligation products by using a fluorescence-labeled universal primer.
The PCR amplification reaction system comprises: 2 XEs Taq MasterMix (Beijing Baiolaibobo technologies, ltd.), primer sets (each 0.5 mM), and 100ng of human whole blood genomic DNA.
The PCR cycling conditions were: each cycle was 94 ℃ 30s,60 ℃ 30s,72 ℃ 30s for 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 GPC5 rs2352028 site, and determining which of three genotypes (CC genotype, CT genotype and TT genotype (wherein, the site T 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 Chi 2 test. And respectively adopting an allele model, a genotype model, a dominant model and a recessive model to carry out cox regression analysis and a Kaplan-Meier method to draw a survival curve, calculating a Hazard Ratio (HR) and a 95% Confidence Interval (CI), and correcting by adopting age and gender. 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 tumor subtypes, 367 cases (43.7%), 282 cases of squamous carcinoma (33.6%), SCLC 72 cases (8.6%), and 118 cases of other types (14.1%). Including adenosquamous carcinoma (ASC), large Cell Carcinoma (LCC), carcinosarcoma (CS), and Mucous Epidermoid 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 general characteristics Table of Chinese Lung cancer patients (n = 839)
Figure BDA0003426057450000071
# Other cancers include adenosquamous carcinoma, large cell carcinoma, carcinosarcoma, and mucoepidermoid carcinoma
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 months, 33.20 months; P = 0.003). Median survival time was significantly higher for patients without smoking than for patients 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 relationship between GPC5 rs2302028 and prognosis of Chinese Lung cancer patients
Figure BDA0003426057450000081
a Adjusting according to age and sex
As shown in table 2, in the total patients, the CT genotype patients had a lower risk of pre-death compared to the CC genotype (adjusted HR = 0.84% ci.
TABLE 3 relationship between GPC5 rs2352028 allelic patterns of Chinese lung cancer patients and prognosis
Figure BDA0003426057450000082
Figure BDA0003426057450000091
a Adjusting according to age and sex
TABLE 4 relationship between GPC5 rs2352028 and prognosis in genotype model of Chinese lung cancer patients
Figure BDA0003426057450000092
a Adjusting according to age and sex
TABLE 5 relationship between GPC5 rs2352028 in dominant model of Chinese Lung cancer patients and prognosis
Figure BDA0003426057450000093
Figure BDA0003426057450000101
a Adjusting according to age and sex
TABLE 6 relationship between GPC5 rs2302028 and prognosis in recessive models of Chinese lung cancer patients
Figure BDA0003426057450000102
a Adjusting according to age and sex
As shown in tables 3 to 6, in patients of age > =60, patients with CT genotype have lower risk of pre-death compared to CC genotype (adjusted HR =0.80, 95% ci; patients with dominant model CT + TT had a lower risk of prognosis for mortality compared to CC (adjusted HR =0.81, 95% ci from 0.66-0.98, p = 0.032); in smoking patients, the risk of mortality prognosis for CT patients is lower compared to CC genotype (adjusted HR =0.82, 95% ci, 0.68-1.00, P =0.046 log-rank P = 0.021); in patients with a family history of malignancy, the risk of mortality is lower in the prognosis of CT patients compared to CC genotype, (adjusted HR =0.74, 95% ci; in early stage lung cancer patients (stage I + II), CT patients have a lower risk of prognosis of death compared to CC genotype (adjusted HR =0.56, 95% ci from 0.33 to 0.93, p = 0.024); patients with dominant model CT + TT had a lower risk of prognosis of death compared to CC (adjusted HR =0.56, 95% ci.
Overall, the log-rank test analysis results indicated that GPC5 rs2352028 mutant genotype CT had a protective effect on the prognostic survival of lung cancer patients aged > =60 (P = 0.030), with a history of smoking (P = 0.046), with a family history of malignancy (P = 0.034), early in lung cancer (P = 0.024). The SNP of GPC5 site is closely related to environment, and the SNP of the site can be used as a functional survival prognosis prediction marker for predicting the risk and prognosis of cancer patients.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Sequence listing
<110> university of Compound Dan
<120> genotyping detection kit based on GPC5 rs2352028 gene and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcatgtggtt gcagttgtgt 20
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acatgtagct gtttgaatgt tacct 25

Claims (6)

1. A genotyping detection kit based on GPC5 rs2352028 gene, which is used for detecting the genotype of GPC5 rs2352028 site gene of a cancer patient, and comprises:
a primer set for PCR of the whole blood genome of the 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.
2. The GPC5 rs2352028 gene-based genotyping detection kit according to claim 1, wherein:
wherein the genotype is any one of CC genotype, CT genotype and TT genotype.
3. The GPC5 rs2352028 gene-based genotyping detection kit according to claim 1, further comprising:
a 5 'universal probe, a 3' universal probe, a site recognition sequence, a qPCR universal primer, a 5 'specific ligation probe, and a 3' specific ligation probe.
4. The GPC5 rs2352028 gene-based genotyping detection kit according to claim 1, wherein:
wherein the cancer is lung cancer.
5. The GPC5 rs2352028 gene-based genotyping detection kit according to claim 4, wherein the kit comprises:
wherein the lung cancer is non-small cell lung cancer.
6. Use of the GPC5 rs2352028 gene-based genotyping detection kit of any one of claims 1 to 5 for predicting prognostic survival of a lung cancer patient.
CN202111578185.0A 2021-12-22 2021-12-22 GPC5 rs2352028 gene-based genotyping detection kit and application thereof Pending CN115386632A (en)

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