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

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 culturepositive 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.

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

“…The results, which reveal a significant proportion of PCR-positive but culture-negative specimens were consistent with many other studies (Logan, et al 2001, Gentry-Weeks, et al 2002, Kulkarni, et al 2002. Taken together with the epidemiological data presented in the present study, supported the view that PCR-based testing is more sensitive than conventional culture for the detection of enteric bacterial pathogens in stool samples.…”

Simple and rapid detection of Salmonella strains by direct PCR amplification of the hilA gene

“…However, several compounds used in DNA extraction procedures have been found to have some inhibitory effect on PCR (Rossen et al, 1992). Some laboratories have described enrichment of faecal samples before performing PCR (Gentry-Weeks et al, 2002;Stone et al, 1994). However, in these protocols, DNA extraction from the enriched broth/faeces mixture is still required prior to PCR, using a commercially available kit, which is time-consuming and expensive.…”

“…PCR is the best known and most successfully implemented nucleic acid detection technology to date (Nissen & Sloots, 2002). Several PCR assays have been developed for the detection of Salmonella strains in faeces (Chiu & Ou, 1996;Gentry-Weeks et al, 2002;Stone et al, 1994;Widjojoatmodjo et al, 1992). However, the methods described are either too laborious and time-consuming, too expensive or not sufficiently sensitive.…”

A loop-mediated isothermal amplification method targets the HisJ gene for the detection of foodborne Salmonella

“…The HisJ gene was reportedly used successfully to detect Salmonella spp. and was demonstrated to be a promising target gene [16,17]. Using the regimen of selective enrichment of Salmonella, 200 market samples were screened.…”