Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

Abstract

BackgroundThe White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry.MethodsIn this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations.ResultsA universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method.ConclusionThe designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.

Presence of Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV) in Native Shrimps from Southern Mexico

“…And, in Figure 1). Although results from this study are similar to those reported in other regions of Mexico, the presence of IHHNV and WSSV has not been reported before this work in the Yucatan Peninsula [19]. Likewise, this is the first report of IHHNV in P. notialis or P. brasiliensis.…”

Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

“…In order to evaluate the RPA assay, we also established this published qPCR assay in this study, which showed a slightly higher sensitivity of 10 copies per reaction compared to that of [28].…”

Prevalence and distribution of W hite S pot S yndrome V irus in cultured shrimp

“…), Multiplex PCR (Jeeva et al . ) and qPCR(Mendoza‐Cano and Sánchez‐Paz ), in situ hybridization method(Chang et al . ) and recently developed loop‐mediated isothermal method (Chang et al .…”