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

Fernando Mendoza-Cano,Arturo Sánchez-Paz

doi:10.1186/1743-422x-10-186

Fernando Mendoza-Cano, Arturo Sánchez-Paz

Open Access

https://doi.org/10.1186/1743-422x-10-186

Copy DOI

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.

Highlights

The 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
Primer design and PCR amplification Based on a multiple sequence alignment the PCR primers evaluated in this study were designed based on a highly conserved region of the WSSV VP28 gene
VP28, the most abundant exposed protein in the WSSV envelope, is encoded by the open reading frame (ORF) 421 [47], and the resulting protein contains 204 amino acid residues with a theoretical molecular mass of 22 kDa [48]

Summary

Introduction

The 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. 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. The White Spot Syndrome Virus (WSSV) is an extremely lethal and contagious shrimp pathogen It has emerged globally as the major threat for shrimp farming during the last decades as outbreaks of WSSV lead to cumulative mortalities of 100% within 3–10 days after the onset of clinical signs [1,2]. There is no known therapeutic treatment available to prevent or reduce the adverse effects of WSSV, and conducting a routine and intensive surveillance program in both brood-stock and larvae, based on appropriate diagnostic tests, as an effective strategy to prevent the occurrence of outbreaks in shrimp farming facilities, has been recommended [5,6]

Methods

Results

Conclusion