Development of a Duplex Real-time Fluorescence PCR for Simultaneous Detection of the Carp Edema Virus and Koi Herpesvirus

ZHANG Wen1 XU Lipu1 LYU Xiaonan1 WANG Xiaoliang1 CAO Huan1 WANG Shu1 WANG Jingbo1

(1.Beijing Fisheries Technical Extension Station, Beijing, China 100176)
【Novelty】For the first time to establish a highly efficient diagnostic method for simultaneous detection of the carp edema virus (CEV) and koi herpesvirus (KHV); One pair of specific primers and a fluorescein FAM-labeled probe designed by the CEFAS according to conserved sequences of the P4a gene of the CEV were optimized, and one pair of specific primers and a VIC-labeled probe were designed on the basis of conserved sequences of the ORF7 of KHV; The specificity of this PCR was high and there was no cross-reaction with other aquatic viruses.The limit of detection limit of this PCR was 15 copies/L if two pre-mixed plasmids were present.The coefficient of variation was <3% for intra-assay and inter-assay.

【Abstract】To establish a highly efficient diagnostic method for simultaneous detection of the carp edema virus (CEV) and koi herpesvirus (KHV), one pair of specific primers and a fluorescein FAM-labeled probe designed by the Center for Environment, Fisheries and Aquaculture Science according to conserved sequences of the P4a gene of the CEV were optimized, and one pair of specific primers and a VIC-labeled probe were designed on the basis of conserved sequences of the ORF7 of KHV. The duplex real-time polymerase chain reaction (PCR) was developed and optimized to detect the CEV and KHV in one reaction simultaneously. Results showed that the specificity of this PCR was high and there was no cross-reaction with other aquatic viruses. The limit of detection of this PCR was 15 copies/μL if two pre-mixed plasmids were present. The coefficient of variation was < 3% for intra-assay and inter-assay. It was shown that this duplex real-time PCR was a sensitive, specific and simple method for detection of the CEV and KHV which could be used as a rapid detection method.

【Keywords】 Carp edema virus (CEV) ; Koi herpesvirus (KHV) ; Duplex real-time fluorescence PCR;

【DOI】

【Funds】 Beijing Agricultural Bureau Science and Technology New Star Project (20180212), Subject: Establishment and application of duplex PCR of KHV and CEV Beijing Innovation Team (BAIC03-2018), Subject: Beijing Aquaculture Industry Innovation Team

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(Translated by BAI XD)

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This Article

ISSN:1000-8721

CN: 11-1865/R

Vol 34, No. 06, Pages 889-895

November 2018

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Article Outline

Novelty

Abstract

  • Materials and methods
  • Results
  • Discussion
  • References