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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Multiresistant Salmonella Typhimurium Dt104 Using Multiplex and Fluorogenic Pcr

Authors
item Carlson, Steven
item Bolton, Lance
item Briggs, Constance
item Hurd, Howard
item Sharma, Vijay
item Cray, Paula
item Jones, B - UNIVERSITY OF IOWA

Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 10, 1999
Publication Date: N/A

Interpretive Summary: Salmonella infections continue to cause fatal disease throughout the world. Salmonella typhimurium DT104 (DT104), a specific Salmonella subtype, further represents a major health problem due to its resistance to many antibiotics. The rapid detection of DT104 would facilitate strategies aimed at interrupting the infectious cycle of this pathogen. We developed a specific and sensitive DNA amplification-based assay that promotes the detection of a segment of DNA that is unique to DT104. To provide further specificity for this diagnostic test, we simultaneously detected two other gene fragments that are present in DT104. This three-pronged test resulted in the identification of DT104 exhibiting the two most common multiple antibiotic resistance patterns. The three indicative segments of DNA can be visualized within 5 hours of bacterial isolation. A minor modification of the test enabled the detection of other multiple antibiotic resistant Salmonella. We also designed a fluorescence-based assay that can detect the DNA of multiresistant DT104 in the presence of excess contaminating bacterial DNA. The latter assay can give results within 3 hour of bacterial isolation and thus represents an appropriate complement to the DNA visualization assay. These results provide a method by which multiple antibiotic resistant DT104, or potentially the next emerging multiple antibiotic-resistant Salmonella, can be accurately detected. This method could be adapted to allow for large-scale rapid detection of DT104 in food, water, animal feeds, feces, etc. That is, this test may have clinical and food safety applications.

Technical Abstract: Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further represents a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant Salmonella typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive PCR assay that amplifies a segment of DNA that is conserved in multiresistant Salmonella typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in Salmonella typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from Salmonella typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other multiresistant Salmonella such as Salmonella typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant Salmonella typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant Salmonella typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected.

Last Modified: 7/23/2014