Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

Abstract

Aim: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S–23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. Methods and Results: Campylobacter species‐specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. Conclusions: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. Significance and Impact of Study: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.

Impacts of Migratory Sandhill Cranes (Grus canadensis) on Microbial Water Quality in the Central Platte River, Nebraska, USA

“…PCR products (Linton et al 1996), (2) PCR assays used for the speciation of pathogenic Campylobacter (Stucki et al 1995;Gonzalez et al 1997;Khan and Edge 2007), and (3) Campylobacter spp. qPCR assays (Lund et al 2004).…”

Molecular Detection of Campylobacter spp. in California Gull (Larus californicus) Excreta

“…Sequences were analyzed using the Basic Local Alignment Search Tool (http: //blast.ncbi.nlm.nih.gov/Blast.cgi) and the software program MEGA 4.1 (32). For extracts showing positive amplification results, species-specific PCR assays for C. jejuni, C. coli, and C. lari were conducted using 16S-23S rRNA gene primer sets (20). In addition, PCR assays targeting the functional genes mapA and ceuE were used to further confirm the presence of C. jejuni and C. coli, respectively (13,31).…”

The Internal Transcribed Spacer Region, a New Tool for Use in Species Differentiation and Delineation of Systematic Relationships within the Campylobacter Genus

“…jejuni, C. coli, and C. lari has been reported to be highly variable in size and/or sequence composition and contains different types of sequences encoding tRNA molecules (8,29). The diversity of the ITS region observed previously in these Campylobacter species suggests that this region may be useful for differentiation and identification of other nonthermotolerant Campylobacter species.…”

“…Although the potential of using the 23S rRNA gene and the ITS region for species differentiation and systematics has not been extensively investigated in the majority of Campylobacter species, previous studies have examined the ability of these regions to differentiate a small number of thermotolerant Campylobacter species, including C. jejuni, C. coli, Campylobacter lari, and Campylobacter upsaliensis (16,18,29). While the 23S rRNA genes in a number of strains of C. jejuni and C. coli share a similar sequence identity (41), previous studies have reported the presence of strain-specific intervening sequences (IVS) within the 23S rRNA gene of C. jejuni, C. coli, C. fetus, and C. upsaliensis (26,53).…”