Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR

Diseases of Aquatic Organisms
K F Tang, D V Lightner

Abstract

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infe...Continue Reading

Citations

Apr 16, 2010·Marine Biotechnology·T W Flegel, Kallaya Sritunyalucksana
Sep 3, 2013·Indian Journal of Virology : an Official Organ of Indian Virological Society·Praveen RaiIndrani Karunasagar
Mar 7, 2012·Transboundary and Emerging Diseases·S Vega-HerediaA Sánchez-Paz
Aug 1, 2002·Journal of Clinical Microbiology·John M BroylesDenise M Korniewicz
Mar 25, 2014·Archives of Virology·Santiago Ramos-CarreñoIvone Giffard-Mena
Jan 11, 2011·Journal of Invertebrate Pathology·D V Lightner
May 18, 2010·Journal of Virological Methods·Grit MrotzekHans P Saluz
Jul 23, 2009·Journal of Invertebrate Pathology·Dongchun YanDonald V Lightner
Apr 15, 2008·Molecular and Cellular Probes·Paisarn KhawsakWasana Sukhumsirichart
Aug 9, 2006·Journal of Virological Methods·K V RajendranPeter J Walker
May 31, 2019·Virusdisease·Arun K DharDarryl Jory
Jul 6, 2014·BMC Veterinary Research·Carlos A G LealHenrique C P Figueiredo

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