Development of a fluorescent quantitative polymerase chain reaction technique for detection of grass carp reovirus HZ08 strain
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1. Pearl River Fishery Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China;2. College of Life Science and Technology, Dalian Ocean University, Dalian 116023, China

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    Abstract:

    Outbreaks of grass carp hemorrhage have caused widespread economic loss to the freshwater aquaculture industry in China. The disease is caused by grass carp reovirus (GCRV). Currently, strain HZ08 is responsible for the majority of outbreaks of grass carp hemorrhage. We developed a rapid, efficient, and specific method for detection of GCRV epidemic causing strains. We designed a pair of specific primers and TaqMan probes targeting the HZ08 strain S7 gene. The standard curve was established using the standard template plasmid PVAX1-S7, which exhibited a corresponding relationship between the Ct and the copies of plasmid. We then evaluated the specificity, sensitivity, and repeatability of this approach. The linear relationship of copies was excellent within the range 6.0×1010−6.0 (r=0.999 9). Using FQ-PCR we were able to detect at least 6.0 copies of the S7 gene in the plasmid suggesting this method has high sensitivity. The coefficients of variation were 0.82 and 0.41%−0.52% for intragroup and intergroup, respectively, indicating that this method had high repeatability. Furthermore, the method had high specificity, based on the lack of amplification of other aquatic viruses. We tested 32 suspected grass carp hemorrhage specimens and returned 28 positive samples using FQ-PCR but just 23 positive samples using conventional RT-PCR. In summary, we developed a FQ-PCR method for the detection of HZ08 strain GCRV that has high specificity, sensitivity, and repeatability. Our method may also be applied to GCRV rapid detection and preliminary quantification.

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刘宝芹,曾伟伟,王庆,张乐生,王英英,石存斌,吴淑勤. 草鱼呼肠孤病毒HZ08株FQ-PCR检测方法的建立及应用[J]. Jounal of Fishery Sciences of China, 2012,[volume_no](2):329-335

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  • Online: March 26,2012
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