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Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection

文献类型： 外文期刊

作者： Wang, Wei 1 ; Liu, Shuang 2 ; Yao, Liang 1 ; Xia, Jitao 1 ; Xu, Tingting 2 ; Wang, Chong 2 ; Li, Chen 2 ; Zhang, Qingli 1 ;

作者机构： 1.Shanghai Ocean Univ, Coll Fisheries & Life Sci, Shanghai 201306, Peoples R China

2.Chinese Acad Fishery Sci, Funct Lab Marine Fisheries Sci & Food Prod Proc, Key Lab Maricultural Organism Dis Control,Minist, Qingdao Natl Lab Marine Sci & Technol,Yellow Sea, Qingdao 266071, Peoples R China

关键词： TaqMan probe based reverse transcription quantitative PCR (TaqMan RT-qPCR); covert mortality nodavirus (CMNV); optimized method; rapid molecular detection; national proficiency test

期刊名称：VIRUSES-BASEL （ 影响因子：5.818； 五年影响因子：5.811 ）

ISSN：

年卷期： 2022 年 14 卷 7 期

页码：

收录情况： SCI

摘要： Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 degrees C for 15 min and denaturation at 95 degrees C for 1 min, followed by 40 cycles including denaturation at 95 degrees C for 10 s, and annealing and extension at 54.9 degrees C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R-2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

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