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

• Published in: Journal of virological methods 2011-12, Vol.178 (1-2), p.16-21
• Main Authors: Panichareon, Benjaporn, Khawsak, Paisarn, Deesukon, Warin, Sukhumsirichart, Wasana
• Format: Article
• Language: English
• Subjects: Animals; Densovirus; Densovirus - classification; Densovirus - genetics; Densovirus - isolation & purification; DNA Primers - genetics; Electrophoresis, Agar Gel; High-resolution melting analysis; Multiplex Polymerase Chain Reaction - methods; Multiplex real-time PCR; Nuclear polyhedrosis virus; Penaeidae - virology; Penaeus monodon; Penaeus monodon densovirus; Penaeus stylirostris; Real-Time Polymerase Chain Reaction - methods; Roniviridae - classification; Roniviridae - genetics; Roniviridae - isolation & purification; Sensitivity and Specificity; Shrimp viruses; Taura syndrome virus; Transition Temperature; Virology - methods; White spot syndrome virus; White spot syndrome virus 1 - classification; White spot syndrome virus 1 - genetics; White spot syndrome virus 1 - isolation & purification; Yellow-head virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.07.010

► The developed multiplex real-time PCR and HRM analysis for detection of PmDNV, WSSV, and YHV was highly specific and sensitive. ► Detection of the three viruses in forty-one shrimp samples demonstrated the applicability of this method to examine the viral infection in shrimp. ► The most attractive of this assay is its ability to simultaneously detect three of the major viruses, no requirement of probe and carcinogen dye, and capable to detect viral infection in cultured penaeid shrimp sample. 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 40fg, 50fg, and 500fg, 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.