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