Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.