Publication:
Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

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Issued Date

2011

Resource Type

Article

File Type

application/pdf

ISSN

1660934

DOI

10.1016/j.jviromet.2011.07.010

Other identifier(s)

2-s2.0-80955142726

Rights Holder(s)

Scopus

Bibliographic Citation

Journal of Virological Methods. Vol 178, (2011), p.16-21

Suggested Citation

Panichareon B., Khawsak P., Deesukon W., Sukhumsirichart W. Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp. Journal of Virological Methods. Vol 178, (2011), p.16-21. doi:10.1016/j.jviromet.2011.07.010 Retrieved from: https://hdl.handle.net/20.500.14740/7211

Title

Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

Author(s)

Panichareon B.
 Khawsak P.
 Deesukon W.
 Sukhumsirichart W.

Abstract

A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40. fg, 50. fg, and 500. fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers. © 2011 Elsevier B.V.

Subject(s)

Agar gel electrophoresis
 Article
 Controlled study
 Densovirus
 Dissociation
 DNA synthesis
 DNA virus
 Gene amplification
 High resolution melting analysis
 Multiplex polymerase chain reaction
 Nonhuman
 Nucleic acid analysis
 Penaeidae
 Penaeus monodon
 Polyhedrosis virus
 Priority journal
 Real time polymerase chain reaction
 Sensitivity analysis
 Shrimp
 Virus detection
 White spot syndrome virus
 Yellow head virus
 Animals
 Densovirus
 DNA Primers
 Electrophoresis, Agar Gel
 Multiplex Polymerase Chain Reaction
 Penaeidae
 Real-Time Polymerase Chain Reaction
 Roniviridae
 Sensitivity and Specificity
 Transition Temperature
 Virology
 White spot syndrome virus 1
 Decapoda (Crustacea)
 Densovirus
 Litopenaeus stylirostris
 Nucleopolyhedrovirus
 Penaeidae
 Penaeus monodon
 Shrimp white spot syndrome virus
 Taura syndrome virus
 Yellow head virus

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URI

https://hdl.handle.net/20.500.14740/7211

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Scopus: Year 1983-2021

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