Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

Abstract

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

Identification and differentiation of Campylobacter isolated from chicken meat using real-time polymerase chain reaction and high resolution melting analysis of hipO and glyA genes

“…Banowary et al . [ 9 ] conducted identification and differentiation study of Campylobacter isolates using multiplex PCR and HRM using the asp and hipO genes. This method identified and differentiated C. jejuni and C. coli species with a sensitivity and specificity of 100% and 92%, respectively.…”

“…The prevalence and contamination level of Campylobacter spp. in slaughterhouses and retailers can be used in the implementation of food safety policies and the evaluation of strategies to minimize risks to consumers [ 8 , 9 ].…”

“…Numerous studies in the food safety sector have demonstrated that qPCR-HRM analysis can be applied for screening genotypes and variants based on the melting points of DNA fragments of pathogenic foodborne bacteria [ 8 , 12 ]. The HRM technique has been used for genotyping C. jejuni and C. coli to detect and differentiate between the two species on the basis of visual interpretation of differences in the melting curve patterns [ 9 ].…”

Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis

“…Slight changes in the melting points of HRM curves were observed in different runs of PCR-HRM and on different days; however, the shape of conventional melt curves and normalized graphs were unchanged. The quality and quantity of genomic DNA are the two important factors in PCR-HRM curve analysis (31,(40)(41)(42). Therefore, to achieve comparable and reproducible HRM curves, the DNA concentrations of samples were adjusted.…”

Developments in Rapid Detection Methods for the Detection of Foodborne Campylobacter in the United States

“…Recent advances in multiplex qPCR technology have enabled even more sensitive and specific genotyping. Banowary et al. (2015, 2018) employed a high resolution melt (HRM) curve analysis method based on the use of fluorescent DNA binding dyes.…”