[1]温嘉琦,马偲航,薛凯文,等.巢式PCR检测卵泡液HBVX区、C区基因扩增反应体系与条件的优化研究[J].现代检验医学杂志,2026,41(03):22-27.[doi:10.3969/j.issn.1671-7414.2026.03.004]
　WEN Jiaqi,MA Sihang,XUE Kaiwen,et al.Optimization of the Nested PCR Amplification Reaction System and Conditions for Detection of HBV X Region and C Region Genes in Follicular Fluid[J].Journal of Modern Laboratory Medicine,2026,41(03):22-27.[doi:10.3969/j.issn.1671-7414.2026.03.004]
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巢式PCR检测卵泡液HBVX区、C区基因扩增反应体系与条件的优化研究()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第41卷
期数:
2026年03期
页码:
22-27
栏目:
论著
出版日期:
2026-05-13

文章信息/Info

Title:
Optimization of the Nested PCR Amplification Reaction System and Conditions for Detection of HBV X Region and C Region Genes in Follicular Fluid
文章编号:
1671-7414（2026）03-022-06
作者:
温嘉琦1，马偲航1，薛凯文1，颜晓玥1，孔馨怡1，李友筑2，张 磊1
1.厦门大学公共卫生学院，福建厦门 361102;2.厦门大学附属第一医院生殖医学科，福建厦门 361003
Author(s):
WEN Jiaqi1，MA Sihang1，XUE Kaiwen1，YAN Xiaoyue1，KONG Xinyi1，LI Youzhu2，ZHANG Lei1
1.School of Public Health, Xiamen University, Fujian Xiamen 361102, China;2.Department of Reproductive Medicine, the First Affiliated Hospital of Xiamen University, Fujian Xiamen 361003, China
关键词:
巢式聚合酶链式反应; 乙型肝炎病毒; X区基因; C区基因; 卵泡液
分类号:
R373.21；R446.7
DOI:
10.3969/j.issn.1671-7414.2026.03.004
文献标志码:
A
摘要:
目的?为提高巢式聚合酶链式反应（PCR）检测乙型肝炎病毒（HBV）X区基因与C区基因的灵敏度，提高卵泡液HBV基因组的检测效率，探索HBVX区与C区基因巢式PCR扩增反应的最佳体系与最适条件。方法收集2024年9月~12月厦门大学附属第一医院8例乙肝表面抗原阳性、计划接受治疗的女性患者的卵泡液作为研究对象，经PCR-荧光探针法检测，得出样本HBVDNA滴度。利用软件Oligo7进行引物设计，分别从退火温度、模板量等进行优化。PCR扩增产物经1g/dl琼脂糖凝胶电泳以及测序分析后进行确认。结果不论HBVX区基因或C区基因，其最佳退火温度皆为53℃。X区基因巢式PCR反应检测中筛选出X3-X4引物组（XF3：ACTTATCGGGACTGACAACTCG、XR3：GGTGAACAGAC-CAATTTATGCCT；XF4：TCCTCTCTCGGAAATACACCT、XR4：AGCCTCCTAGTACAAAGACCT）可获得最佳扩增效果，8例样本检出率为87.5%；C区基因巢式PCR反应检测中筛选出C3-C4引物组（CF3：CCAGGGAATTAGTAGTCAGC、CR3：GTTTCCCACCTTATGAGTCCA；CF4：ATGTCAATGTTAATATGGGCCTA、CR4：TACTAACATTGAGATTCCCGAGA）可获得最佳扩增效果，8例样本检出率为100%。DNA模板量3μl与5μl皆可有效扩增目的基因。结论基于卵泡液建立了针对HBVX区与C区基因的最优巢式PCR体系，显著提升低滴度HBVDNA及阴性样本的HBV目的基因检出效能，为扩展乙肝病毒携带人员的相关基因组学研究提供检测方案。
Abstract:
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.

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备注/Memo

备注/Memo:
基金项目：国家自然科学基金项目（81102140、81472988）。
作者简介：温嘉琦（2002-），女，在读硕士，主要从事流行病学研究，E-mail：1374810249@qq.com。
通讯作者：张磊（1972-），女，博士研究生，硕导，主要从事流行病学研究，E-mail：DrZhanglei@xmu.edu.cn。
更新日期/Last Update: 2026-05-15