Designment and evaluation of the primers for cyprinid herpesvirus 2 and spring viremia of carp virus duplex RT-PCR detection

To develop the rapid method for detecting of Cyprinid herpesvirus 2(CyHV2) and Spring viremia of carp virus (SVCV), 2 pairs of specific primers for duplex reverse transcriptase polymerase chain reaction (RT-PCR) and 5 overlapping oligo primers were designed according to the nucleotide sequence information of CyHV2 and SVCV published in GenBank, and a DNA fragment about 228 bp of the SVCV G gene was synthesized in vitro by overlap extension PCR to construct the recombinant plasmid pMD19-T-SVCVG. Then, the 2 pairs of specific primers were evaluated via a serial of tests, including reaction temperature optimization test, sensitivity and specificity tests. The results showed that the 2 pairs of designed specific primers are suitable for CyHV2 and SVCV duplex RT-PCR detection which is a rapid method with high specificity and sensitivity, the detection limits for CyHV2 and SVCV detection were both approximately 88 copies of the cloned viral genomic fragments, as well as resulted in no cross-reaction for GCRV, Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus isolated from fish, and the DNA (cDNA) of the normal Carassius auratus gibelio samples