Study Area and Sampling: How the farm and premises conditions influences on the E. coli contamination was one of the objectives of this study. During collection of samples, therefore, several criteria was set to mark the farm as traditional sanitation and improved sanitation. Shrimp, water and sediment samples were collected from four different locations (Dumuriasadar = F1, Gutudia = F2, Kharnia = F3 and Sobhana = F4) in Khulna district, Bangladesh. Preparation of samples in sterile condition and separate instrument and time ensured its status of not being secondary and cross contamination, followed by storing at 4 ºC. The Experiment was conducted at Genetics and Molecular Laboratory of Fisheries and Marine Resource Technology Discipline of Khulna University. Experimental Design: Water, sediment and shrimp samples were collected at first from four shrimp farms designed as F1, F2, F3 and F4. One positive (P) control (water of Khulna University Lake, which is proved to have E. coli and one negative (N) control (absent DNA template into PCR tube) were taken for the experiment. Six pairs of designed oligonucleotide primers (APHO-F, APHO-R; HST1-F, HST1-R; HLT1-F, HLT1-R; HLT2-F, HLT2-R; VT-F, VT-R and EAE-F, EAE-R) were used for the amplification and then detection of six different target genes. The second step involved the extraction of DNA from the bacterial cell. In third step, qualitative and quantitative analyses of extracted DNA were done by using agarose gel electrophoresis and spectrophotometric analysis. The fourth step involved the amplification of target gene by PCR technology. In the final step, Gel electrophoresis was performed to detect enterovirulent E. coli strains by observing clear bands. Bacteria Culture and DNA Extraction: Two grams Luria-Bertani (LB) broth was added with 100 ml of distilled water followed by the autoclaving at 121 oC for 20 minutes. Autoclaved 5 ml LB broth and 100 μl samples, which may contain environmental bacteria, were then mixed followed by placing on shaking incubator (VS-8480SL, Korea) at 37oC with 200 rpm for overnight to enrich E. coli culture. DNA extraction was carried out from amplified bacterial cells grown in culture media using DNAZOL® Reagent (Invitrogen Life technologies, USA), ethanol and sodium hydroxide (Difco Laboratories, MI, USA). DNA Integrity, Concentration and Purity: To check the integrity of extracted DNA, agarose gel electrophoresis technique was applied (2% agarose gel containing 3 μl ethidium bromide, 6 V/cm, 1X TAE buffer). DNA bands were visualized and photographed by high performance UV transilluminators (Ultra-Violet Products Ltd., UK). The concentration of DNA samples were determined from the absorbance at A260 (absorbance at 260 nm) using a double beam spectrophotometer (Hitachi U-2910 spectrophotometer, Japan) against NaOH blank. The protocol used in this experiment was designed for a double-beam spectrophotometer. The DNA concentration (=A260×50×500) purity (=A260/280) was measured for further assessment. Target Genes: The target genes chosen of this experiment were: Alkaline phosphate (phoA), housekeeping gene (present in all E. coli); the hlt1, hlt2 and hst1 genes of ETEC; the vt of EHEC and eae virulence genes of EPEC. Six pairs of specific primers were chosen from gene sequence to amplify the target DNA fragments of genes. To compare the size of double stranded DNA from 100 to 2,000 base pairs, 100 bp designed DNA markers were used. The DNA marker consists of 13 double stranded DNA fragments ranging in sizes from 100 to 1,000 (Bioneer, Korea). Target Gene Amplification in Thermal Cycler: At first, the concentrations of all the extracted DNA samples were adjusted. The reactions were performed in a 20 μl reaction mixture containing 1 μl DNA sample (having 20-25 ngof template DNA), 2 μl (10 pmole/μl) oligonucleotide primers (Bioneer, Korea), 2 μl 10X reaction buffer, 2 μl 10 mM dNTPs mixture, 2 μl Taq DNA polymerase (1 unit) and 11 μl de-ionized distilled water. The reaction mixtures were then placed in a DNA thermal cycler (C1000TM, BIO-RAD, USA) for PCR. The PCR conditions for target DNA amplification were: initial extended step of denaturation at 94 ?C for 2 minutes followed by 35 cycles of denaturation at 94 ?C for 1 minute, primer annealing at 58 ?C for 1 minute and elongation at 72 ?C for 1 minute. A positive control, constituting highly E. coli contamination and a negative control, having no DNA template in the reaction mixture were kept. After the completion of thermal cycling, 8 μl of each PCR products was analyzed electrophoretically by running through a 2% agarose gel and the amplified product size was determined by comparing with a 100 bp DNA size marker.