Multiplex PCR Protocol Development for Rapid Screening of White Spot Syndrome Virus (WSSV) in Shrimp

Abstract

One of the most serious problems confronted by the shrimp farming industry is the disease caused by white spot syndrome virus (WSSV). For definite diagnosis and certification of the virus, nested polymerase chain reaction (PCR) technology based imported kit has been used that are expensive, time consuming and vulnerable for cross-contamination. Therefore, this study aimed to develop a non-stop, faster,single tube, single step multiplex PCR protocol using locally available reagents for the simultaneous detection of WSSV with its host(shrimp) as internal positive control.In the multiplex PCR procedure, two sets of WSSV specific primer and one set of shrimp specific primerwere tested. A total of eight (08) primer combinations based on the WSSV specific primer (Lo F1R1, Lo F2R2, Lo F1R2, Lo F2R1, Lo F1R1F2, Lo F1R1R2, Lo F2R2F1, Lo F2R2R1)being incorporated with one (01) pair of the shrimp specific primer (16S rRNA) were evaluated and optimized in a single tube to find out the most suitable protocol. All the primer combinations yielded their expected amplicon sizes with stronger band resolution intensity. Any one of the developed multiplex PCR protocols with its reagent composition and thermal cycling conditions could be applicable for routine diagnosis of the virus with simultaneous presence of the host. Among the protocols, it was suggested to follow the protocol based on 16S rRNA and Lo F2R2 considering time, cost, effort, sensitivity and specificity. If sensitivity is not the major problem, this study recommended the multiplex protocol with the primer combination of 16S rRNA and Lo F1R1. The study also recommended one of the semi nested multiplex PCR protocols with the primer combination of 16S rRNA and Lo F1R1R2 (combination-6) that would detect both of the target DNA as well as the severity of the infection.The developed multiplex protocols could be used for saving time and effort, eliminating chance for cross-contamination and it ensured thirty five (35) times reduced cost in comparison with a renowned imported kit named ‘IQ2000 WSSV detection System’ certified by Office International des Epizootics (OIE).