AJVR, Vol 63, No. 9, September 2002 1265 S almonella infections are an important cause of mor- bidity and mortality in domestic animal species. 1-3 Clinical infections with Salmonella organisms can manifest as gastroenteritis that can lead to septicemia. 4 It is critical to rapidly identify active infections within a herd or in patients at a veterinary hospital, because the organism can be easily disseminated among patients through the fecal-oral route and result in envi- ronmental contamination. There have been a number of reports 5-7 of outbreaks of salmonellosis in large-ani- mal patients in veterinary teaching hospitals during recent years, with many of these outbreaks resulting in temporary closure of these facilities. The costs associ- ated with mitigation of the problem can be a large financial burden. Many laboratories currently consider the criterion standard for Salmonella spp detection to be conven- tional bacterial culture methods that require 3 to 7 days of laborious sample handling for positive identi- fication. 8 The polymerase chain reaction (PCR) assay is a rapid molecular identification technique that has been adapted to diagnostic applications for many microorganisms. In 1 study, 9 a Salmonella organism- specific assay that targeted the invE-A genes was test- ed on 16 clinical specimens and provided a sensitivity and specificity of 100%. 9 In another study, 10 a second assay that targeted the hisJ gene was tested on feces from 110 horses; 71 horses had positive PCR assay results whereas only 11 horses had positive bacterial culture results. 10 These PCR assays, by use of invE-A and hisJ primers, were used in our laboratory on clin- ical fecal specimens (774 total) and had relative sensi- tivities of 80 and 93.3% and relative specificities of 98.6 and 85.6%, respectively, when compared with bacterial culture results. 11 The reduced sensitivity and specificity of these assays, compared with the use of routine bacterial culture, are potentially problematic when the organism involved can be so easily transmit- ted and the consequences of infection can be life- threatening. Use of real-time PCR technology has the potential to improve on results obtained from a standard PCR assay. The use of a sequence-specific fluorogenic probe in addition to standard PCR primers allows for greater specificity for detecting a target sequence. 12 The fluoro- genic nature of the probe also allows for rapid detec- tion, because a fluorescent signal can be detected as the reaction progresses, negating the need for post-PCR assay sample processing. The objective of our study was to use real-time PCR technology to develop a high- ly sensitive and specific diagnostic assay for the detec- tion of Salmonella spp in fecal specimens. Materials and Methods Study overview—Using real-time PCR technology, 3 primer-probe sets (sipC, invE, and spaQ) were tested on puri- fied genomic DNA from various Salmonella serotypes and a number of non-Salmonella gram-negative and gram-positive bacterial spp to determine which set most accurately ampli- fies the Salmonella serotypes while failing to amplify the non- Salmonella spp. The optimal primer-probe set from these 3 was used to compare 3 commercially available DNA extrac- tion methods for the recovery of genomic DNA from enriched fecal specimens. The procedure using the optimal primer-probe set and DNA extraction method was tested on clinical fecal specimens to determine the relative sensitivity and specificity of the assay, compared with bacterial culture results. Received Feb 7, 2002. Accepted Apr 9, 2002. From the Departments of Microbiology (Kurowski, Gentry-Weeks), Clinical Sciences (Traub-Dargatz), and Environmental Health (Morley), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. Supported by the College of Veterinary Medicine and Biomedical Sciences College Research Council. The authors thank Denise Bolte and Steve Burns for technical assis- tance. Address correspondence to Dr. Gentry-Weeks. Detection of Salmonella spp in fecal specimens by use of real-time polymerase chain reaction assay P. Brett Kurowski, BS; Josie L. Traub-Dargatz, DVM, MS; Paul S. Morley, DVM, PhD; Claudia R. Gentry-Weeks, PhD Objective—To use real-time polymerase chain reac- tion (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Sample Population—299 fecal specimens from cat- tle, horses, and dogs. Procedure—Enrichment of fecal specimens was fol- lowed by genomic DNA extraction by use of com- mercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results—Use of the spaQ primer-probe set resulted in a relative sensitivity of 100% and a specificity of 98.2%, compared with bacterial culture results when tested on 299 clinical fecal specimens. Conclusions and Clinical Relevance—A rapid, sen- sitive, and specific assay for the detection of Salmonella spp from enriched clinical fecal specimens was developed. This technique would be highly valu- able in clinical settings to help avoid or mitigate the complications arising from an outbreak of salmonel- losis in a herd or among patients of a veterinary hos- pital. (Am J Vet Res 2002;63:1265–1268) Unauthenticated | Downloaded 08/13/22 01:59 PM UTC