Identification of Two Phylogenetically Related Organisms from Feces by PCR for Detection ofSalmonellaspp

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Abstract
Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detectingSalmonellaspp. from feces. Bovine and equine feces were enriched overnight in brain heart infusion broth and assayed using PCR methods and primer sets described by other investigators. A total of 774 fecal specimens were tested using a primer set (invE-Aprimer set) that amplifies a region spanning the invasin E and A genes ofSalmonella entericaserovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJprimer set) that amplify a portion of the histidine transport J gene. The PCR required 24 h to obtain results, whereas it took 5 to 7 days to identifySalmonellaspp. by culture. PCR detection ofSalmonellaspp. using thehisJprimers and theinvE-Aprimers had a sensitivity of 93.3 and 80%, respectively, and a specificity of 85.6 and 98.6%, respectively, compared with bacterial culture. Amplification of 42 culture-negative fecal specimens (of 306 total specimens) generated a DNA fragment that corresponded to the molecular weight of the amplifiedhisJgene. ThehisJ-generated amplicons from six culture-negative and six culture-positive specimens were sequenced and analyzed using DNA sequence alignment and phylogenetic analysis software. A neighbor-joining dendrogram of the DNA sequences of both sets ofhisJamplicons revealed two distinct groups—one group of amplicons from culture-positive specimens identical to thehisJgene ofS. entericaserovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related tohisJofS. entericaserovar Typhimurium than to otherhisJsequences present in nucleotide databases.