Shrimp samples (Penaeus monodon): Shrimp samples were collected from different shrimp farms located on south-western region of Bangladesh. Shrimps were collected from grow out ponds, brought to the laboratory in iced boxes and stored at -20°C till analysis. Reference strains and cultures: The bacterial strains used in this experiment were obtained from culture collection pool of Industrial Microbiology Laboratory, IFST, BCSIR. Four reference strains were used for inoculation in shrimp homogenate as well as for spiking of shrimp samples; these are namely Salmonella typhi ATCC-65154, Vibrio parahaemolyticus ATCC-17802, Vibrio cholerae ATCC-15748 and E. coli O157:H7ATCC-12079. Bacterial strains were cultured in Trypticase Soy Broth (TSB,Oxoid) containing 0.6% yeast extract in a shaking incubator at 37°C overnight. The strains were maintained in Trypticase Soy Agar (TSA, Oxoid) slants at 4°C. Chemicals: PCR reagents were purchased from Promega (Madison, WI, USA). PCR primers were synthesized by 1st Base (Singapore). Bacteriological media and broths were purchased from Oxoid (Hampshire, England). The rest of the materials and chemicals and reagents used in this study were purchased from Sigma Chemical Co. (St Louis, MO, USA). Selection of genes and primers: Genes selected for this study were tdh for Vibrio parahaemolyticus, ompW for Vibrio cholerae, hlyEHEC for E. coli O157:H7 and sefA for Salmonella sp. Primer sequence and product size of the genes are recorded. DNA extraction from target organisms: DNA from target organisms was extracted by phenol/chloroform and ethanol precipitation method. Bacterial cells were grown overnight in nutrient broth at 37°C, aerated by shaking at 120 rpm in a shaking incubator. Harvesting was then performed by centrifuging the culture at 10000 rpm for 5 minutes. The supernatant was then discarded and remaining cell pellet was subjected to treatment with DNA extraction solution I (Tris HCl+EDTA+sucrose) for 30 minutes at 37°C on a water bath in order to disrupt cells. Then de-proteinization was done using DNA extraction solution II (proteinase K+SDS+NaCl) at 55°C for 1 h on a water bath. Phenol:chloroform:isoamylalcohol (25:24:1) solvent was used to precipitate proteins. The cell extract was mixed gently with the solvent and the nucleic acids were separated in the aqueous layer by centrifugation at 10000 rpm for 5 min. The aqueous solution of DNA was then removed using micropipette. The DNA was then concentrated by ethanol precipitation in the presence of Sodium acetate. After centrifuging and washing with 70% ethanol solution the final pellet was taken and suspended in TE buffer. This suspension was then stored at 4°C for further use. DNA extraction from spiked shrimp samples: Before spiking shrimps were peeled and autoclaved, the samples were then spiked with overnight grown bacterial culture (106 CFU mL-1). Following incubation at 37°C for 24 h, spiked shrimps were homogenized with sterile ringer solution in a stomacher. Three milliliter of stomached samples were inoculated into nutrient broth and incubated for 24 h. The overnight grown culture was subjected to DNA extraction and PCR. Quantification and purity of DNA: Quantification of genomic DNA was done using 1.0% agarose gel electrophoresis in 1X TAE buffer followed by staining with ethidium bromide. The concentration of extracted DNA was also estimated by visual comparison of the band with 100 bp marker DNA. The purity and concentration of the extracted DNA was checked by measuring absorbances on T60 UV-VIS spectrophotometer at 260 and 280 nm. Purity was analyzed by absorbance ratio of 260/280 nm. Uniplex PCR amplification of tdh, ompW, hlyEHEC and sefA gene: PCR amplification was performed in a 30 μL reaction volume containing 50 ng of DNA template, 3 μL 10X PCR reaction buffer without MgCl2, 0.5 μL 20 mMMgCl2 , 1 μL of dNTP mixture, 1 μL each of forward and reverse primer and 1 unit of Taq polymerase. Same reaction mixture was used for all of the genes. Thermal cycling was done in a DNA engine, BIO-RAD (USA). PCR reactions were maintained for initial denaturation at 94°C for 3 min, followed by 30 cycles of 1 min at 94°C (denaturation), 1 min at 55°C for tdh and ompw, 58°C for sefA and hlyEHEC (annealing) and 1 min at 72°C (extension). The final extension for 9 min at 72°C and hold temperature was maintained at 4°C. PCR products were mixed with 3 μL of 10X loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 40% sucrose, w/v) and electrophoresis was carried on 1.5% agarose gel in 1X TAE buffer at 100v for 1.5 hr and stained with ethidium bromide (10 μg mL-1). A 50/100 bp DNA ladder was used as a standard molecular weight marker. Reference band sizes for Salmonella, V. parahaemolyticus, V. cholerae and E. coli O157:H7 were 470, 250, 588 and 889 bp, respectively. Multiplex PCR amplification of tdh, ompW, hlyEHEC and sefA gene: Multiplex PCR was performed in a total volume of 30 μL containing 2 μL of mixed template DNA and 28 μL of PCR master mix composed of 1x PCR buffer, 5.0 mMMgCl2, 25 μM of each of Salmonella sp. detection primers (A058 and A01), 25 μM concentration of each of V. parahaemolyticus detection primers (tdh D3 and tdh D5), 25 μM concentration of each of V. cholerae detection primers (ompW F and ompW R), 25 μM concentration of each of E. coli O157:H7 detection primers (HlyF and HlyR), 200 μMdATP, dCTP, dGTP and dUTP, 0.5 U of Ampli Taq Gold DNA polymerase (Invitrogen, USA) with a DNA thermal cycler. The thermocycler was programmed as 50°C for 2 min for carryover treatment and initial denaturation at 95°C for 5 min. The samples were then subjected to 30 cycles of 95°C for 1 min, 58°C for 1 min, 55°C for 1 min, 54°C for 1 min, 72°C for 1 min and then 72°C for 9 min. The amplified products were then analyzed by 2.0% agarose gel electrophoresis. Reference band sizes for Salmonella, V. parahaemolyticus, V. cholerae and E. coli O157:H7 were 470, 250, 588 and 889 bp, respectively. Two different multiplex set up have been standardized, one for targeting sefA and tdh genes and another for all four genes. Specificity of the multiplex PCR setting: To investigate the specificity of the multiplex PCR method and to observe that the method do not amplify closely related genes present in other related organisms, Bacillus cereus-ATCC 10876, Shigella flexneri ATCC-12022 and Staphylococcus aureus ATCC-25923 were included in the study. DNA extracted from these organisms was subjected to PCR according to the multiplex settings.