| 摘要: |
| 目的 探究低载量HBV DNA S区基因扩增的可行性并对实验条件进行优化，为隐匿性HBV感染（OBI）患者HBV DNA S区基因突变检测提供依据。方法 采用传统巢式PCR和自建两轮PCR方法扩增6例低HBV DNA载量（100~200 IU/mL）和22例更低HBV DNA载量（20~99 IU/mL）的血清样本中HBV DNA S区基因，并对引物序列、引物量、PCR产物模板稀释倍数、退火温度、PCR反应循环数、PCR总反应体系等条件进行优化。PCR扩增产物经琼脂糖凝胶电泳后，切割目的条带凝胶进行克隆测序，然后对克隆测序结果进行核酸序列BLAST比对确认。结果 设计3对巢式PCR引物（P1~P3），扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化后，6例低HBV DNA载量的血清样本中仅2例经巢式PCR扩增出HBV DNA S区基因特异性靶序列，22例更低HBV DNA载量样本无一例扩增成功。自建两轮PCR法设计了P4~P15共12对引物，扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化并筛选出P13为最佳引物后，6例低HBV DNA载量的血清样本全部扩增出HBV DNA S区基因特异性靶序列；15例（15/22，68.18%）更低HBV DNA载量的样本扩增出HBV DNA S区基因特异性靶序列，经PCR产物克隆测序均证实为HBV DNA S区基因，该15例样本中HBV DNA载量最低为20.1 IU/mL。结论 基于引物P13自建的两轮PCR法更适用于低载量HBV DNA S区基因的扩增，扩增效率和特异性均优于传统巢式PCR；扩增产物可进一步应用于OBI者HBV DNA S区基因序列突变分析。 |
| 关键词: 乙型肝炎病毒 HBV DNA S区基因 聚合酶链反应 引物 测序 |
| DOI：10.16781/j.0258-879x.2021.10.1157 |
| 投稿时间：2021-03-30修订日期：2021-08-27 |
| 基金项目:浦东新区科技发展基金（PKJ2018-Y20），上海中医药大学附属第七人民医院人才培养计划（XX2020-13）. |
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| Amplification of low-load HBV DNA S-gene by self-built two-round PCR and optimization of experimental conditions |
| HE Cheng-shan,JIANG Xiu-di,XU Zheng,MA Chen-yun,YAO Xiao-yang,LU Zhi-cheng* |
(Department of Clinical Laboratory, Shanghai Seventh People's Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200137, China
*Corresponding author) |
| Abstract: |
| Objective To explore the feasibility of low-load hepatitis B virus (HBV) DNA S-gene amplification and optimize the experimental conditions, so as to provide a basis for the detection of HBV DNA S-gene mutation in occult HBV infection (OBI) patients. Methods HBV DNA S-gene in serum samples of 6 cases with low-load HBV DNA (100-200 IU/mL) and 22 cases with lower-load HBV DNA (20-99 IU/mL) was amplified by traditional nested polymerase chain reaction (PCR) and self-built 2-round PCR. The experimental conditions, such as primer sequences, primer amount, the dilution ratio of PCR product templates, annealing temperature, the number of PCR reaction cycles, and PCR reaction system, were optimized. The PCR amplification product was observed by agarose gel electrophoresis, the target band gel was cut for cloning and sequencing, and then the cloned sequencing result was confirmed by BLAST comparison of nucleic acid sequence. Results Three pairs of primers (P1-P3) for nested PCR were designed, and the amplified products theoretically contained the whole HBV DNA S-gene. After optimization of PCR amplification conditions, only 2 of the 6 serum samples with low-load HBV DNA were amplified by nested PCR, and 22 samples with low-load HBV DNA were failed to amplify any fragments. Twelve pairs of primers (P4-P15) were designed for the self-built 2-round PCR, and the amplified products theoretically contained the whole HBV DNA S-gene. After PCR amplification conditions were optimized and P13 was selected as the best primer, all 6 serum samples with low-load HBV DNA and 15 (15/22, 68.18%) samples with lower-load HBV DNA were amplified successfully. The PCR products were confirmed as the HBV DNA S-gene specific target sequences by cloning and sequencing. Among the 15 samples, the minimum HBV DNA load was 20.1 IU/mL. Conclusion The self-built 2-round PCR based on the primer P13 is more suitable for the amplification of low-load HBV DNA S-gene. The amplification efficiency and specificity are much better than traditional nested PCR. The amplified products can be further applied to analyze HBV DNA S-gene mutation in OBI patients. |
| Key words: hepatitis B virus HBV DNA S-gene polymerase chain reaction primers sequencing |
