| Part one Development of recombinase aided amplification assay to detect two SNPs.Objective:To establish a fast,efficient and economical method for accurate typing of two SNPs(rs6983267 and rs1447295)located on 8q24.Methods:A duplex probe-directed recombinase amplification(duplex-PDRA)detection method was established using recombinase-aided amplification(RAA)technology.Primers and probes for two SNPs were designed according to the principle of duplex-PDRA.The recombinant plasmid was constructed to evaluate the sensitivity and specificity of the method.From November 2018 to November 2019,a total of 179anticoagulated whole blood samples or blood clot samples were collected from prostate cancer patients at the Department of Laboratory Medicine,Fourth Hospital of Hebei Medical University.The samples were tested by duplex-PDRA method and sequencing,in order to evaluate the stability of the method.Results:The duplex-PDRA method established in this study can recognize four different alleles of two SNPs(rs6983267 and rs1447295)at39℃within 30 minutes.The sensitivities duplex-PDRA can reach 10~3～10~4copies/μL and no cross-reactivity was observed.The method has good stability for the detection of clinical samples,and can accurately type SNPs of 179anticoagulated whole blood samples or blood clot samples.The typing results are completely consistent with the sequencing method.Conclusions:The duplex-PDRA method established in this research is simple,fast,and cost-effective.It provides a feasible strategy for large-scale testing of SNPs and shows great potential advantages for widespread use in research and clinical settings.Part two Study on the correlation between two SNPs on 8q24 and prostate cancer.Objective:To investigate the relationship between two SNPs(rs6983267and rs1447295)on 8q24 and the susceptibility of prostate cancer.Methods:179 patients with prostate cancer who were treated at the Fourth Hospital of Hebei Medical University from November 2018 to November 2019 were used as the case group,and 179 local health checkups within the same age group were used as the control group.The genotypes of rs6983267 and rs1447295 were detected by a duplex-PDRA,and the chi-square test of SPSS21.0 software was used to analyze the relationship between the above two SNPs and the risk of prostate cancer.Results:In terms of genotype of rs1447295,compared with major allele homozygous genotype CC,CA genotype carriers have a 1.97-fold increased risk of prostate cancer(OR=1.97,95%CI 1.24～3.15,P<0.05),CA+AA genotype carriers have a 2.05-fold increased risk of prostate cancer(OR=2.05,95%CI 1.31～3.22,P<0.05).In terms of alleles,carriers of the A allele are 1.87times more likely to have prostate cancer than carriers of the C allele(OR=1.87,95%CI 1.27～2.77,P=0.001);In terms of genotype of rs6983267locus,compared with major allele homozygous genotype TT,GG genotype carriers suffer from prostate cancer risk increased by 2.43 times(OR=2.43,95%CI 1.29～4.58,P<0.05),and the risk of prostate cancer among GT+GG genotype carriers increased by 1.63 times(OR=1.63,95%CI 1.02～2.63,P<0.05).In terms of alleles,carriers of G allele were 1.48 times more likely to have prostate cancer than carriers of T allele(OR=1.48,95%CI 1.10～1.99,P<0.05).Conclusion:The two SNPs(rs1447295 and rs6983267)on 8q24 may be related to the susceptibility to prostate cancer.The A allele at rs1447295 and the G allele at rs6983267 may be risk alleles of prostate cancer. |
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