Development of a highly sensitive single-tube nested PCR protocol directed toward the sequence of virion envelope proteins for detection of white spot syndrome virus infection: Improvement of PCR methods for detection of WSSV

•No step for opening tube in the middle of PCR•High sensitivity compared to current diagnostic PCR methods•Covering shrimps, bivalves and crabs•Faster than conventional two-tube nested PCR

White spot syndrome virus (WSSV) is a double-stranded DNA virus that causes severe mortality, leading to great economic loss for cultured shrimp farms. The Manual for Aquatic Animals published by the Office International des Epizooties (OIE, 2012) announced the nested PCR protocol, developed by Lo et al. in 1996 as the standard protocol for WSSV diagnosis. Because current detection tools are prone to carryover contamination and are labor-intensive, an accurate and convenient technique is required. Therefore, the aim of this study was to develop an improved diagnostic PCR method for WSSV that overcomes the known drawbacks of the conventional PCR method. We designed primers directed toward the nucleotide sequence encoding envelope proteins of WSSV, VP19 and VP28. By applying an antisense oligonucleotide technique, we converted this PCR assay into a non-stop one-tube system, subsequently enabling a faster and more sensible PCR assay. In practice, the newly developed protocol detected the WSSV genome from imported frozen shrimps, crabs and bivalves, with a shorter PCR running time and a higher sensitivity compared with other PCR methods.