HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

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.

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 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.
AuthorsBenjaporn Panichareon, Paisarn Khawsak, Warin Deesukon, Wasana Sukhumsirichart
JournalJournal of virological methods (J Virol Methods) Vol. 178 Issue 1-2 Pg. 16-21 (Dec 2011) ISSN: 1879-0984 [Electronic] Netherlands
PMID21906627 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2011 Elsevier B.V. All rights reserved.
Chemical References
  • DNA Primers
Topics
  • Animals
  • DNA Primers (genetics)
  • Densovirus (classification, genetics, isolation & purification)
  • Electrophoresis, Agar Gel
  • Multiplex Polymerase Chain Reaction (methods)
  • Penaeidae (virology)
  • Real-Time Polymerase Chain Reaction (methods)
  • Roniviridae (classification, genetics, isolation & purification)
  • Sensitivity and Specificity
  • Transition Temperature
  • Virology (methods)
  • White spot syndrome virus 1 (classification, genetics, isolation & purification)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: