Sybr green qpcr based quantification of wssv between seasons in southwest Bangladesh

Abstract

Shrimp aquaculture is a progressive economic growth sub-sector of Bangladesh. The main impediment of shrimp farming is the outbreak of White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) which causes almost 100% mortality within 3-10 days. The prevalence and viral load of WSSV is not similar across all seasons of the year. SYBR green based qPCR provides an effective way for quantification of viral load. Sequencing gives the strain variation of WSSV circulating in Bangladesh. The present study, therefore, was designed to investigate the prevalence and viral load of WSSV in south-west coastalregion in Bangladesh. Total 48 dead shrimp samples were drawn from four sampling sites such as Satkhirasadar, Tala, Debhata and Assasuniupazillas of Satkhira district. After DNA extraction, the samples were analyzed by VP28 gene specific primers. The SYBR green PCR was performed by using 124bp amplicon for VP28 gene. The results were analyzed by SPSS and Microsoft Excel. The computational bioinformatics tools were used to perform molecular analysis. Conventional PCR showed that the prevalence in three seasons (Immediate Before Monsoon: 75%; During Monsoon: 81.25% and Immediately After Monsoon: 93.15%) had relationship with the viral load 3.52 ± 0.22 × 108 copy/g of tissue, 5.43 ± 0.25 × 109 copy/g of tissue 8.63 ± 0.23 × 1011copy/g of tissue, respectively. Protein BLAST of sequenced WSSV positive amplicon of VP28 amplicon (BAN_323_S3_2015_VP28) showed a mutation with NCBI reference sequences. Phylogenetic analysis of WSSV positive amplicon of VP28 indicates that WSSV Bangladeshi strain is phylogenetically related to India. WSSV infections are widely distributed in Satkhira and towards the coast the prevalence and viral load could be increased. Therefore, proper farm management is required to improve yield of shrimp. This study has indicated that SYBR Green qPCR technique can be successfully applied in detecting WSSV load 1000 times sensitive than conventional PCR method. WSSV load can vary between seasons and within sampling sites. WSSV prevalence had similar trend. Discovery of a new mutation in the VP28 gene denotes genetic persistent change in the genome.