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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
Authors: Panichareon B.
Khawsak P.
Deesukon W.
Sukhumsirichart W.
Keywords: agar gel electrophoresis
controlled study
DNA synthesis
DNA virus
gene amplification
high resolution melting analysis
multiplex polymerase chain reaction
nucleic acid analysis
Penaeus monodon
Polyhedrosis virus
priority journal
real time polymerase chain reaction
sensitivity analysis
virus detection
White spot syndrome virus
Yellow head virus
DNA Primers
Electrophoresis, Agar Gel
Multiplex Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Transition Temperature
White spot syndrome virus 1
Decapoda (Crustacea)
Litopenaeus stylirostris
Penaeus monodon
Shrimp white spot syndrome virus
Taura syndrome virus
Yellow head virus
Issue Date: 2011
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.
ISSN: 1660934
Appears in Collections:Scopus 1983-2021

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