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摘要
Covert mortality nodavirus (CMNV), an emerging RNA virus, is the pathogen of viral covert mortality disease (VCMD), which has emerged as a cause of serious losses in shrimp aquaculture in China. To improve VCMD diagnosis, a one-step, real-time TaqMan probe-based reverse transcription quantitative PCR (RT-qPCR) was developed in this study. The TaqMan RT-qPCR was optimized firstly, whereby the best results were obtained with 0.2 mu M of each primer, 0.2 mu M probe, and 0.5 mu L Enzyme Mix II. The optimal reaction program was determined as 15 min at 51 degrees C for reverse transcription and 5 min at 95 degrees C, followed by 40 cycles of denaturation at 94 degrees C for 10 s, and annealing and extension at 52.7 degrees C for 30 s. The optimized assay detected as little as 9.6 pg total RNA from CMNV-infected shrimp and 5.7 copies of the target plasmid. The RT-qPCR assay for CMNV with a high correlation coefficient (r(2) = 0.996) was developed basing on the standard curve generated by plotting the threshold cycle values (y) against the common logarithmic copies (log(10)n(c) as x; n(c) is copy number) of pMD20-CMNV. The diagnostic sensitivity and specificity of this assay versus the previously reported RT-qPCR was 96.2% and 98.0%, respectively. This method is highly specific to CMNV, as it showed no cross-reactivity with other common shrimp viruses. It is anticipated that the newly developed and optimized RT-qPCR assay will be instrumental for the rapid diagnosis and quantitation of CMNV.
- 出版日期2018-12
- 单位上海海洋大学; 中国水产科学研究院
