Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.
Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide 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 amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.
Carlson
S A
SA
Enteric Diseases and Food Safety Research Unit, National Animal Disease Center, Ames, Iowa 50010, USA. scarlson@nadc.ars.usda.gov
Bolton
L F
LF
Briggs
C E
CE
Hurd
H S
HS
Sharma
V K
VK
Fedorka-Cray
P J
PJ
Jones
B D
BD
D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012097:Repressor Proteins; D012235:Rhodamines; D012486:Salmonella typhimurium / Q000235:genetics*; D012680:Sensitivity and Specificity
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012097:Repressor Proteins; D012235:Rhodamines; C034811:tetracycline resistance-encoding transposon repressor protein; C024098:6-carboxyfluorescein