Campylobacter jejuni and campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment
Merchant-Patel, Shreema, Blackall, Patrick J. , Templeton, Elizabeth , Price, Erin P., Tong, Steven , Huygens, Flavia, & Giffard, Philip M. (2010) Campylobacter jejuni and campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment. Applied and Environmental Microbiology, 76(2), pp. 493-499.
The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaAHRManalysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.
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|Item Type:||Journal Article|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Bacteriology (060501)|
|Divisions:||Past > QUT Faculties & Divisions > Faculty of Science and Technology|
Current > Institutes > Institute of Health and Biomedical Innovation
|Copyright Owner:||Copyright 2010 American Society for Microbiology|
|Deposited On:||18 Mar 2010 08:04|
|Last Modified:||11 Aug 2011 03:54|
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