Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry

A. K. Dhar, M. M. Roux, K. R. Klimpel

Research output: Contribution to journalArticle

125 Scopus citations

Abstract

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the Gene-Amp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (CT) values over a range of 1 to 105 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp β-actin gene was amplified in parallel with the viral DNA. The CT values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard CT curves of these viruses. For each virus and its β-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

Original languageEnglish (US)
Pages (from-to)2835-2845
Number of pages11
JournalJournal of clinical microbiology
Volume39
Issue number8
DOIs
StatePublished - Aug 23 2001
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology (medical)

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