1.Establishment of TaqMan RT-PCR assay for Wuxiang virus
Danhe HU ; Xiaohui YAO ; Shihong FU ; Fan LI ; Tianmeng GU ; Junjie CHEN ; Ying HE ; Jiayu YIN ; Songtao XU ; Xiangdong LI ; Kai NIE ; Huanyu WANG ; Guodong LIANG
Chinese Journal of Experimental and Clinical Virology 2022;36(4):460-464
Objective:To establish a real-time fluorescent quantitative TaqMan reverse transcription polymerase chain reaction (Real-time RT-PCR) detection method for Wuxiang virus (WUXV).Methods:All gene sequences of WUXV were downloaded from GenBank, and multi-sequence alignment analysis was performed using Mega-X. Primers and probes designed for the highly conservative region of S-segment genes were selected to evaluate the specificity, sensitivity and stability of detection reactions.Results:The established method can specifically detect WUXV and does not cross-react with various arboviruses. The lowest detection limit was 1 pfu/ml. The inter-batch variation coefficients of repeated detection cycle threshold ( Ct) of the same sample were all less than 1.00%. TaqMan RT-PCR was used to detect 30 batches of sandflies nucleic acid samples, and 7 of them showed positive amplification curve of WUXV. Conclusions:TaqMan RT-PCR with high sensitivity, specificity and repeatability has been successfully established, which can be used to screen large quantities of samples of WUXV.
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