A multiplex real-time polymerase chain reaction (qPCR) kit targeting VP664 and VP28 genes of white spot syndrome virus (WSSV)

The implementation of a precise and sensitive detection method is crucial to prevent the spread of the white spot syndrome virus (WSSV) within and between regions. The present study developed and validated the analytical and diagnostic performances of a multiplex real-time polymerase chain reaction kit (AccuPower (R) WSSV detection qPCR kit: AWqPCR kit) that simultaneously detects two distinct genomic sequences of WSSV (VP664 and VP28). The analytical sensitivity of the kit, defined as 95% limit of detection (LoD(95%)), was 1.15 copies of WSSV plasmid. No cross-reactivity was found against five shrimp pathogens (yellow head virus genotype 1, Taura syndrome virus, infectious myonecrosis virus, infectious hypodermal and hematopoietic necrosis virus, and Vibrio parahaemolyticus). Moreover, no significant PCR inhibition was observed while using nine potential interfering materials (antibiotics and immunostimulants). Diagnostic performance was evaluated using 200 known WSSV-infected whiteleg shrimp (Litopenaeus vannamei). Compared to the IQ2000 (TM) WSSV Detection and Prevention System (IQ2000 (TM) WSSV DPS), the diagnostic sensitivity (Dsn) and specificity (Dsp) of the AWqPCR kit were found to be 100% (95% confidence interval (CI): 96.38-100%) and 100% (95% CI: 96.38-100%), respectively. In addition, using 50 shrimp samples of unknown WSSV infection status, the Dsn and Dsp of the AWqPCR kit were compared to the two available gold standard WSSV detection methods, including the qPCR assay using WOAH primers and probe (WOAH qPCR) and IQ2000 (TM) WSSV DPS. The evaluated Dsn and Dsp of the kit were 100% (95% CI: 87.66-100%) and 72.73% (95% CI: 49.78-89.27%) compared to the WOAH qPCR, whereas the Dsn and Dsp were 100% (95% CI: 25-100%) and 32.65% (95% CI: 19.95-47.54%) when compared to the IQ2000 (TM) WSSV DPS. All six samples that were detected as WSSV-positive by the AWqPCR kit and confirmed by sequencing were found to be WSSV-negative by WOAH qPCR. Furthermore, the blind sample analysis of 50 shrimp samples carried out by three laboratories showed no significant difference in the test results. Overall, AWqPCR kit was found to be a simple and reliable method for WSSV detection with high sensitivity and specificity. Therefore, it could be applied in quarantine, surveillance, and monitoring programmes as well as for the confirmation of WSSV in apparently healthy shrimps.