巢式PCR检测卵泡液HBV X区、C区基因扩增反应体系与条件的优化研究OA

Objective To enhance the sensitivity of nested polymerase chain reaction(PCR)for detecting hepatitis B virus(HBV)X and C region genes,improve the detection efficiency of HBV genomic DNA in follicular fluid,and explore the optimal system and conditions for nested PCR amplification of HBV X and C region genes.Methods Follicular fluid samples were collected from eight hepatitis B surface antigen-positive pregnant patients scheduled for treatment at the First Affiliated Hospital of Xia-men University between September and December 2024.HBV DNA titers in the samples were determined using PCR-fluorescent probe methods.Primers were designed using the Oligo 7 software and optimization was performed based on factors such as an-nealing temperature and template concentration.The amplified products were confirmed via 1g/dl agarose gel electrophoresis and subsequent sequencing analysis.Results The optimal annealing temperature was 53℃for both the HBV X region and C region genes.In the nested PCR assay for the X region gene,the X3-X4 primer set(XF3:ACTTATCGGGACTGACAACTCG,XR3:GGTGAACAGACCAATTTATGCCT;XF4:TCCTCTCTCGGAAATACACCT,XR4:AGCCTCCTAGTACAAAGACCT)yield-ed the optimal amplification results,achieving a detection rate of 87.5%across 8 samples.In the nested PCR assay for the C re-gion gene,the C3-C4 primer set(CF3:CCAGGGAATTAGTAGTCAGC,CR3:GTTTCCCACCTTATGAGTCCA;CF4:ATGT-CAATGTTAATATGGGCCTA,CR4:TACTAACATTGAGATTCCCGAGA)yielded the optimal amplification results,achieving a detection rate of 100%for the 8 samples.Both 3 μl and 5 μl of DNA template volume were effective for amplifying the target gene.Conclusions A highly sensitive and specific nested PCR system for HBV X region and C region genes was established using follicular fluid,which significantly improved the detection efficiency of HBV target genes in low-titer HBV DNA and negative samples,and provided a detection protocol for the relevant genomic studies in hepatitis B surface antigen carriers.