Establishment of Digital PCR Assay for Detection of Viral Haemorrhagic Septicemia Virus
ZHAO Huijun1, HU Qiang1, YU Ling1, WU Bin2, YI Jiaying3
1. Dalian Customs Technology Center, Dalian 116001, China; 2. China Certification and Inspection Group Liaoning Co., Ltd., Dalian 116003, China; 3. College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China
Abstract：In order to establish an accurate and rapid detection method for Viral hemorrhagic sepsis virus (VHSV), the VHSV digital PCR detection technology system was constructed with the specific primers and probes designed and synthesized for the specific conserved glycoprotein gene fragment of VHSV genome ,and the key reaction conditions such as annealing temperature, and sensitivity test, specificity test and reproducibility test were carried out. At the same time, it was compared with real-time fluorescence quantitative RT-PCR. The results showed that the optimum annealing temperature of VHSV digital PCR was 58.5 ℃. Sensitivity test showed that,with the continuous tenfold dilution of VHSV genomic RNA, when diluted to 105 times, the digital PCR system can still detect positive droplets. Therefore, it is determined that the RNA genomic nucleic acid concentration of 19 copies/μL is the detection lower limit of VHSV digital PCR. The specificity test revealed that this method had specific amplification only with the target virus VHSV, and had no amplification reaction with Infectious pancreatic necrosis virus (IPNV), Infectious hematopoietic necrosis virus (IHNV), Spring viraemia of carp virus (SVCV), Epizootic haematopietic necrosis virus (EHNV), Hirame rhabdovirus virus (HRV), Viral nervous necrosis virus(VNNV), Koi herpesvirus (KHV), Carp edema virus (CEV), Red sea bream iridovirus (RSIV) and Lymphocystis disease virus (LCDV). The repeatability test indicated that the coefficients of variation of VHSV digital PCR within and between batches were less than 1%. Using the digital PCR method, 1210 batches of domestic and imported fish samples were tested for VHSV. The results showed that the detection rate of digital PCR positive samples was 9.5%, and that of conventional real-time quantitative reverse transcription polymerase chain reaction positive samples varied from 7.69% to 7.77%. Clinical trials showed that the VHSV digital PCR method had higher detection sensitivity, can improve the detection rate of unqualified products and suitable for the accurate detection of aquatic animal diseases.
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2024, Vol. 43 
