Development Of Four Methods For Detection Of Cyprinid Herpesvirus2

Cyprinid herpesvirus2(CyHV-Ⅱ), also known as herpesviral hematopoieticnecrosis virus (HVHNV) and goldfish hematopoietic necrosis virus (GFHNV), is a veryimportant viral pathogen in cultured goldfish. It was first reported from ornamentalgoldfish in Japan, where it caused severe haematopoietic necrosis and high mortality(100%) in the spring of1992and1993. Subsequently, CyHV-Ⅱ was reported occurringin private ponds and aquaria or in small scale breeding facilities in the USA, Taiwan,Australia, southern England and New Zealand. These reports confirm that CyHV-Ⅱ iswidespread in world goldfish populations.In recent years, CyHV-Ⅱwhich made greateconomic loss to our cyprinidae aquaculture.To establish a rapid detection method, thisstudy according to CyHV-Ⅱ triplet protein gene sequence design specific primer incouldPCR, duplex PCR, SYBR GreenⅠreal-time fluorescent quantitative PCR and ringmediated isothermal amplification (LAMP) method.1. The PCR was applied to amplify the genes of triplex protein in Cyprinid. Thereaction conditions of the PCR were optimized, and electrophoresis bands were found intriplex protein gene amplification by PCR, with the amplified fragment of218bp. Thesensitivity of the duplex PCR showed that the two primers detected CyHV-Ⅱ at a level ofas few as2×104copies/uL, The detection results of samples collected from cities in themain Carassius auratus breeding area in China using the established PCR method revealedthat the positive rates of genotypes were87%. The PCR has good amplification efficiency,specificity, and sensitivity advantage and it provides a significant improvement in thedetection.2. The duplex PCR was applied to amplify the genes of triplex protein in Cyprinid.The reaction conditions of the duplex PCR were optimized, and two electrophoresis bandswere found in triplex protein gene amplification by duplex PCR, with the amplifiedfragment of218bp,369bp respectively. Furthermore, when ISKNV were used todetermine the specification of that rapid detection, no specific electrophoresis bands werefound in the duplex PCR amplification. The sensitivity of the duplex PCR showed that thetwo primers detected CyHV-Ⅱat a level of as few as3×102copies/uL, The detectionresults of samples collected from cities in the main Carassius auratus breeding area in China using the established duplex PCR method revealed that the positive rates ofgenotypes were87%.3.Accord to CyHV-Ⅱ triplet protein gene one premer sequence design foramplification140bp fragment, then cloning to pMD18-T carrier, the purification ofrecombinant plasmid in gradient diluted10times as a template, fluorescence quantitativePCR amplification for make standard curve, established CyHV-Ⅱ SYBR Green Ⅰreal-time fluorescent quantitative PCR detection method. And sensitivity, repeatability andspecificity test, The sensitivity of the SYBR Green Ⅰ real-time fluorescent quantitativePCR showed that the detected CyHV-Ⅱat a level of as few as30copies/ul, The detectionresults of samples collected from cities in the main Carassius auratus breeding area inChina using the established duplex PCR method revealed that the positive rates ofgenotypes were92.5%.4.This study established loop-mediated isothermal amplification (LAMP)method.Reaction time and temperature are optimized, the experimental results show thatwhen the reaction conditions of63℃for10min (loop primers) or32min (no loopprimers) when is the best reaction conditions.Detection limit of10copies/mL,100timeshigher than ordinary PCR detection limit.Use the LAMP for other pathogens DNAtemplate for testing, the results show that the results were negative for nucleic acidamplification of other pathogens, showed good specificity.And amplification products canbe through the use of SYBR Green Ⅰ visualization.In addition, about120suspectedillness carassius positive detection rate of92.5%.Four detection method has good amplification efficiency, specificity, and sensitivityadvantage and it provides a significant improvement in the detection, genotyping,molecular epidemiological study of CyHV-Ⅱ.