Identification of two phylogenetically related organisms from feces by PCR for detection of Salmonella spp

Abstract

Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detecting Salmonella spp. 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-A primer set) that amplifies a region spanning the invasin E and A genes of Salmonella enterica serovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJ primer 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 identify Salmonella spp. by culture. PCR detection of Salmonella spp. using the hisJ primers and the invE-A primers 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 amplified hisJ gene. The hisJ-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 of hisJ amplicons revealed two distinct groups-one group of amplicons from culture-positive specimens identical to the hisJ gene of S. enterica serovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related to hisJ of S. enterica serovar Typhimurium than to other hisJ sequences present in nucleotide databases.

FIG. 1.

Agarose gels of amplicons generated by PCR amplification of enriched fecal specimens using the invE-A (457-bp amplicon) and hisJ (496-bp amplicon) primer sets. (A) Results using the invE-A primers. Lanes 1 and 6 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lane 3, DNA extracted from culture-negative clinical specimen; lane 4, negative control (distilled water); lane 5, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). (B) Results using hisJ primers. Lanes 1 and 9 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lanes 3 to 6, DNA extracted from culture-negative clinical specimens; lane 7, negative control (distilled water); lane 8, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). Arrows indicate amplicons produced in each reaction.

FIG. 2.

Relationship of hisJ genes from Salmonella culture-positive and culture-negative enriched fecal specimens obtained by phylogenetic analysis of hisJ sequence data. The numbers at each branch point indicate the percentage of bootstrap replications. E. coli hisJ homolog (AE000320 locus, accession no. AE000320.1 [6]) and E. coli hisJ (ECU47027 locus, accession no. U47027 [Joshi and Ames, unpublished data]) were designated as outgroups.