Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains

Abstract

Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide-range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. A multiplex PCR designed for the detection of E. coli cdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT-producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type-specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. The results obtained from this study revealed that so-far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross-reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.