Establishment and Application of a SYBR Green I Real-Time Quantitative PCR for Detection of Micropterus salmoides Rhabdovirus
Establishment and Application of a SYBR Green I Real-Time Quantitative PCR for Detection of Micropterus salmoides Rhabdovirus
Ningyu Zhu, Runzhen He, Qianrong Liang, Xiaoye Zheng, Gaohua Yao, Wenjun Ma and Xueyan Ding*
ABSTRACT
In order to develop a rapid and convenient detection assay of Micropterus salmoides rhabdovirus (MSRV), the SYBR Green I real-time quantitative PCR(RT-qPCR) assay was established. A pair of primers was designed according to the G protein gene of MSRV and a recombinant plasmid containing the target gene was constructed as a standard control. The correlation coefficient of the standard curve was 0.998, which indicated a good linear relationship between initial templates and Ct values. The established SYBR Green I RT-qPCR assay had a detection limit of 2.78×101 copies/μL, which was 100 times more sensitive than the conventional PCR. Moreover, the coefficient of variations was less than 1% for both intra-assay and inter-assay, and no cross reaction was found in other aquatic viruses such as GCRV, KHV, ISKNV and NNV. 54 samples were detected positive from 104 clinical samples by the SYBR Green I RT-qPCR assay, while 22 samples were detected positive by the conventional PCR. The SYBR Green I RT-qPCR assay showed the characteristics of sensitivity and specificity. This method can provide reliable technical support for clinical diagnosis and epidemiological investigation of MSRV, which can effectively control the spread and epidemic of MSRV disease.
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