Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp.

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.