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Establishment of a TaqMan RT-PCR assay for the detection of Madariaga virus |
YIN Qi-kai1,2, LIU Wen-jing1,2, WANG Guo-wei3, ZHANG Wei-jia1,2, FU Shi-hong1,2, HAN Kun4, XU Chong-xiao1,2, XU Song-tao1,2, LI Fan1,2, LIU Ya-ning1, HE Ying1,2, WANG Zhen-hai5, NIE Kai1,2, WANG Huan-yu1,2 |
1. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; 2. State Key Laboratory of Infectious Disease Prevention and Control, National Health Commission Key Laboratory of Biosafety, Beijing 102206, China; 3. Ningxia Medical University, Yinchuan 750004, China; 4. Ningxia Hui Autonomous Region Center for Disease Control and Prevention, Yinchuan 750004, China; 5. Department of Neurology, General Hospital of Ningxia Medical University, Engineering Research Center for Diagnosis and Treatment of Ningxia Nervous System Diseases, Yinchuan 750004, China |
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Abstract A rapid assay for the Madariaga virus (MADV) was established by using TaqMan reverse transcription-polymerase chain reaction (RT-PCR). The MADV gene sequences were downloaded from GenBank, and the highly conserved region of the NSP1 gene of MADV was selected to design specific primers and a probe according to the results of multiple sequence alignment. The sensitivity, specificity and stability of this assay were evaluated with an absolute quantitative analysis model of MADV by using in vitro transcribed RNA standards. The sensitivity of this assay was 1.0×102 copies/reaction, and no cross reaction with other arboviruses was observed. The coefficients of variation were all below 1.5%. A total of 81 serum samples from cases of encephalitis with unknown causes, and 161 batches of Culex samples collected from the Ningxia Hui Autonomous Region, Zhejiang and Yunnan Province, were tested with the established assay. The results were negative in all samples. Thus, a rapid TaqMan RT-PCR assay for MADV was established, which can be used for early detection of MADV in laboratory settings.
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Received: 04 November 2021
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Corresponding Authors:
Nie Kai, Email: niekai@ivdc.chinacdc.cn; Wang Huan-yu, Email: wanghy@ivdc.chinacdc.cn
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