Establishment of diagnostic test for enteric diarrhea in pigs using efficient multiplex polymerase chain reaction

Abstract

Present study was planned to develop and validate a new methodology using multiplex PCR (M-PCR) assay for the detection of maximum number of diarrhea causing bacteria from pig faeces samples in a single test. Two M- PCR assays were designed to identify the diarrheic fecal bacteria and amplified the target genes including control group. Moreover, the unique primers were designed for the simultaneous detection of enterotoxigenic E. coli (ETEC; F4, F5, F6, F41, LT, STa, STb), Salmonella Enteritidis and Salmonella Typhimurium. The designed primers showed higher specificity and sensitivity to detect the targeted genes. The M-PCR method was able to detect at the 1 µg concentration of microbial DNA for all the genes detected by the designed primers. Further, the efficacy of M-PCR was investigated using 3 non-E. coli pathogenic strains L. intracellularis, B. hyodysenteriae, and Salmonella from different pig farms. Results sufficiently revealed that the developed M-PCR assay is an efficient tool for detection of ETEC and other non-E. coli strains from naturally infected pigs. The ability of M-PCR to identify the many bacteria simultaneously without biochemical identification would facilitate early diagnosis and treatment of diarrhea in pigs.

Keywords

• Collibacillosis
• Multiplex-PCR
• Pig diarrhea
• Salmonellosis