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 rapid and highly sensitive real-time PCR detection and quantification met hod for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a s ingle-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 chemist ry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon bin ding to dsDNA exhibits fluorescence enhancement. The enhancement of fluores cence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA an d cycle threshold (C-T) values over a range of 1 to 10(5) copies of the vir al genome. To control the variation in sampling and processing among sample s, 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 amplificatio n was monitored by using the dissociation curve of the amplified product. U sing 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 shrim p viruses and that it is amenable to high-throughout assay.