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.

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 GeneAmp 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 (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-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.

journal_name

J Clin Microbiol

authors

Dhar AK,Roux MM,Klimpel KR

doi

10.1128/JCM.39.8.2835-2845.2001

subject

Has Abstract

pub_date

2001-08-01 00:00:00

pages

2835-45

issue

8

eissn

0095-1137

issn

1098-660X

journal_volume

39

pub_type

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