Establishment of TaqMan One-step Real-time Quantitative Polymerase Chain Reaction Assay to Detect Coxsackievirus B5

VernacularTitle:柯萨奇病毒B组5型TaqMan一步法RT-qPCR检测方法的建立
Author: Yuhan LIU ¹ ; Ming ZHANG ; Wei GUO
Author Information

1. 650118 昆明,中国医学科学院/北京协和医学院医学生物学研究所、云南省重大传染病疫苗研发重点实验室
Keywords: Coxsackievirus B5; Real-time quantitative polymerase chain reaction; TaqMan probe method
From: Journal of Medical Research 2024;53(7):84-88
CountryChina
Language:Chinese
Abstract: Objective To establish a TaqMan one-step real-time quantitative polymerase chain reaction(RT-qPCR)assay spe-cific for coxsackievirus B5(CV-B5).Methods Specific primers and TaqMan probes were designed based on the VP1 sequence of CV-B5.The target gene was inserted into the pMD18TM vector,amplified in DH5α receptor cells,and RNA standards were obtained from escherichia coli transcribed in vitro and a standard curve was established,and finally the reproducibility,sensitivity and specificity of the assay were evaluated.Results The method had good linearity(r2＞0.99)in the template range of 103-1011 copies/μl,amplifica-tion efficiency E=100.9％,sensitivity up to 1 × 103 copies/μl,and specific amplification curve for CV-B5 only,and good reproduc-ibility.Conclusion The TaqMan one-step RT-qPCR assay established in this experiment has high sensitivity,specificity,reproducibili-ty,good anti-interference performance,which can be used for clinical sample detection and absolute quantitative analysis of CV-B5.