Journal of Pure and Applied MicrobiologyVol. 8 No. May 2014 Special Edition

A PCR Method for the Detection of Cyprinid Herpesvirus 2

Yang Zexiao1*, Li Guili1, Xu Qiumei2, Wang Yin1,Yao Xueping1, Zhu Ling1, Xu Zhiwen1, Chen Defang3 and Wang Kaiyu1*

1College of Veterinary Medicine, Sichuan Agricultural University, Yaan Sichuan - 625014, China. 2College of Basic Sciences, Sichuan Agricultural University, Yaan Sichuan - 625014, China. 3College of Animal Science and Technology, Sichuan Agricultural University, Yaan Sichuan - 625014, China.

Received on 12 April 2014 and accepted on 09 May 2014

 

ABSTRACT

Cyprinid herpesvirus 2(CyHV2) is a fatal pathogen for some species of the Cyprininae fish and, furthermore, is responsible for negatively impacting the goldfish industry worldwide. In order to develop a polymerase chain reaction (PCR) capable of detecting CyHV2, 2 specific primers and 9 overlapping oligo primers were designed based upon the nucleotide sequence information of CyHV2 published in GenBank (accession no:HM014349), and a 413 bp DNA fragment of the CyHV2 DNA-dependent DNA polymerase gene was synthesized in vitro by using an overlap extension PCR to construct the recombinant plasmid,pMD19-T-CyHV2. Then, a primary PCR method was established after set of serial tests, including reaction conditions optimization test, sensitivity test, and specificity test. The final results indicate that this developed PCR assay is a rapid method that maintains both a strong specificity and a high sensitivity for detecting CyHV2. The PCR detection limit could reach approximately 62 copies of the cloned viral genomic fragments (pMD19-T-CyHV2) as well as resulted in no amplifications for Aeromonas veronii, Aeromonas hydrophila, Pseudomonas fluorescens and Streptococcus, which are common pathogens isolated from fish by this detection approach.

Keywords : Cyprinid herpesvirus 2, PCR, overlap extension PCR.