Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR

Salmonella infections continue to cause gastrointestinal and systemic disea se throughout the world. Salmonella typhimurium DT104 further poses a major hearth concern due to its acquisition of resistance to multiple antibiotic s. The rapid detection of multiresistant S. typhimurium DT104 would facilit ate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segmen t of DNA that is conserved in multiresistant S. typhimurium DT104. To provi de further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplificatio n of predicted size fragments from S. typhimurium DT104 exhibiting the ACSS uT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetrac ycline) or ASSuT resistance phenotypes. A minor modification of the multipl ex PCR enabled the detection of other related multiresistant Salmonella suc h as S. typhimurium U302. To augment the detection process, we also designe d a fluorogenic PCR assay that can detect the DNA of multiresistant S. typh imurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or p otentially the next emerging multiresistant Salmonella, can be accurately d etected in only 3-4 h.