To detect genomic variation of white spot syndrome virus (WSSV) isolates from different geographical regions of India, the variable number of the tandem repeat (VNTR) region of the ORF 94 (Thailand WSSV isolate - GeneBank Accession No. AF369029) was analysed using five specific sets of primers. Analysis of 70 WSSV-positive samples showed the presence of 14 different genotypes of WSSV with VNTRs ranging from 2 to 16 tandem repeats with the majority (85.47%) having 6-12 tandem repeats. Occurrence of different genotypes of WSSV was found to be neither correlated to any specific geographical region nor to the different growth stage of the tiger shrimp, Penaeus monodon. Pathogenicity studies conducted with 25 isolates of WSSV revealed the presence of virulent and avirulent strains of WSSV in Indian shrimp farms. However, an unambiguous link could not be established between the different genotypes and their virulence.

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.

A multiplex PCR based assay was developed for the identification of the genus Salmonella. Five sets of primers from different genomic sequences such as fimA, himA, hns, invA and hto genes were selected for the identification of serogroups of Salmonella enterica such as S. Typhi, S. ParatyphiA, S. Typhimurium, S. Enteritidis and S. Weltevreden. The selected primers amplified products with the sizes of 85, 123, 152, 275 and 496 bp, respectively, for the genus Salmonella. This assay was found to be highly sensitive, as it could detect 5 cells of Salmonella and 1,000 fg of genomic DNA. Amplification of DNA extracted from other genera viz. V. cholerae and E. coli yielded negative results. This assay provides specific and reliable results and allows for the cost-effective detection of Salmonella in one reaction tube in mixed bacterial communities.

A multiplex PCR (MPCR) assay was developed for the detection of Staphylococcus aureus using selected primers designated on the genus-specific gap gene, speciesspecific nuc gene and enterotoxin-producing EntC1 gene. The internal regions amplified had a product size of 933 bp, 273 bp and 531 bp, respectively. This MPCR assay had a sensitivity to detect 10 cells of S. aureus and 100 pg of genomic DNA of S. aureus. The MPCR assay was found to be specific for S. aureus, as it yielded negative results with other tested bacterial pathogens such as Salmonella typhimurium, Vibrio cholerae and Escherichia coli. Therefore, this developed MPCR assay could be used for the simultaneous detection of S. aureus enterotoxin Cproducing strains from clinical and environmental samples.

A rapid and sensitive multiplex PCR (MPCR)-based assay was developed for the detection of Salmonella serovars in shrimps within 8 h of pre-enrichment. Five sets of primers from different genomic sequences such as fimA, himA, hns, invA and hto genes were selected for the detection of serovars of Salmonella enterica such as S. Typhi, S. Paratyphi A, S. Typhimurium, S. Enteritidis and S. Weltevreden. The selected primers amplified products with sizes of 85 bp, 123 bp, 152 bp, 275 bp and 496 bp, respectively, for the genus Salmonella. The specificity and sensitivity of the assay were tested by contaminating shrimp homogenate artificially with viable cells of Salmonella. The MPCR assay could detect up to five Salmonella cells within 8 h. Amplification of DNA extracted from other genera, viz. Vibrio cholerae and Escherichia coli, yielded negative results. This assay allows for the cost effective and reliable detection of serovars of Salmonella enterica in one reaction tube from mixed bacterial communities occurring in products like shrimp.