Skip to content
Research Article Open access CC BY 4.0

KHV Detection Based on Thermal Lysis - Nested PCR (Polymerase Chain Reaction)

Ibnu Dwi Buwono

Asian Journal of Fisheries and Aquatic Research · pp. 49–57 · Published 19 Dec 2020

10.9734/ajfar/2020/v10i430189

Abstract

In the case of common carp (Cyprinus carpio) and goldfish (Carrasius auratus) that were lightly infected with Koi Herpes Virus (KHV), the use of the one-step PCR method was insensitive to detect the presence of KHV DNA. The nested PCR method has been developed for a more sensitive detection system for KHV infection. This study aims to test the sensitivity of one-step and nested PCR methods in detecting the KHV virus in lightly infected common carp to prevent early disease attacks. DNA extraction of the test samples used thermal lysis and PBS (Phosphate Buffered Saline). DNA isolate as a KHV template was used to amplify KHV sequences using KHV-TKf: 5'-GGGTTACCTGTACGAG-3 'and KHV-TKr: 5'-CACCCAGTAGATTATGC-3' primers in the one-step PCR method. The nested PCR method uses two pairs of primers, namely the KHV-TKf: 5'-GACACCACATCTGCAAGGAG-3' and KHV-TKr: 5'-GACACATGTTACAATGGTGGC-3') and the CEFAS primer pair (5'-CGTCGTGAGGAATACGACG-3' and 5'- ACCGTACAGCTCGTACTGG-3') for amplification of KHV sequences. The one-step PCR method was insensitive for detection of KHV in common carp and goldfish infected with viruses without clinical symptoms, and was sensitive enough to detect viruses with mild symptoms with a DNA fragment size of 409 bp using TK primers. The nested PCR method was more sensitive in detecting KHV DNA in fish without clinical symptoms with a DNA fragment size of 348 bp using CEFAS primers.

Sensitivity one step nested PCR koi herpes virus comon carp goldfish.

References (10)

  1. 1 Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally-infected Cyprinus carpio koi as assessed by real-time TaqMan PCR [DOI]
  2. 2 Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi [DOI]
  3. 3 Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae [DOI]
  4. 4 Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi [DOI]
  5. 5 Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA [DOI]
  6. 6 Identification of spring viraemia of carp virus (SVCV) by combined RT-PCR and nested PCR [DOI]
  7. 7 The Outbreak of Koi Herpesvirus (KHV) in Koi (Cyprinus carpio koi) from Chiang Mai Province, Thailand [DOI]
  8. 8 Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR [DOI]
  9. 9 Tests for the presence of koi herpesvirus (KHV) in common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) in the Czech Republic [DOI]
  10. 10 ISOLATION AND IDENTIFICATION OF CARP INTERSTITIAL NEPHRITIS AND GILL NECROSIS VIRUS (CNGV) IN FINGERLING COMMON CARP (CYPRINUS CARPIO L.)

Cited by 0

No indexed citations yet.

Related research

Article metrics

Real usage data collected on this platform.

30

Page views

0

PDF downloads

3

Outbound clicks

0

Citations

Views over time

Views by country

Approximate, from request IP at view time — not citizenship or institution. Countries with fewer than 5 views are grouped as "Other".

Traffic sources

Referring site, by host.

Views and downloads exclude known bots/crawlers. Citations combines this platform's own DOI-resolved index with each external source's own reported total — see Cited by above for individually listed citing works. Last refreshed 0 seconds ago.