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Open Access Methodology article

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

Carlos A G Leal123, Alex F Carvalho3, Rômulo C Leite23 and Henrique C P Figueiredo23*

Author Affiliations

1 Department of Veterinary Medicine, Federal University of Lavras, Lavras, Brazil

2 AQUAVET, Laboratory of Aquatic Animal Diseases, School of Veterinary Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil

3 AQUACEN, National Reference Laboratory for Aquatic Animal Diseases, Ministry of Fisheries and Aquaculture, Federal University of Minas Gerais, Belo Horizonte, Brazil

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BMC Veterinary Research 2014, 10:150  doi:10.1186/1746-6148-10-150

Published: 5 July 2014

Abstract

Background

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation.

Results

The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR.

Conclusion

The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Keywords:
Coinfection; Clinical sensitivity; Duplex qPCR; IHHNV; Litopenaeus vannamei; WSSV