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Campylobacter jejuni and Campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment

Merchant-Patel, Shreema, Blackall, Patrick J., Templeton, Jillian, Price, Erin P., Tong, Steven Y. C., Huygens, Flavia and 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):493-499.

Document type: Journal Article
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IRMA ID 10603xPUB30
Title Campylobacter jejuni and Campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment
Author Merchant-Patel, Shreema
Blackall, Patrick J.
Templeton, Jillian
Price, Erin P.
Tong, Steven Y. C.
Huygens, Flavia
Giffard, Philip M.
Journal Name Applied and Environmental Microbiology
Publication Date 2010
Volume Number 76
Issue Number 2
ISSN 0099-2240   (check CDU catalogue  open catalogue search in new window)
Start Page 493
End Page 499
Total Pages 7
Place of Publication Washington, United States
Publisher American Society for Microbiology
HERDC Category C1 - Journal Article (DEST)
Abstract 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 the amplify the fragment used for flaA 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 analysed 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, binary markers, CRISPR HRM, and flaA RFLP. flaA HRM analysis 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.
Keywords Campylobacter jejuni
Campylobacter coli
flaA gene
DOI http://dx.doi.org/10.1128/AEM.01164-09   (check subscription with CDU E-Gateway service for CDU Staff and Students  check subscription with CDU E-Gateway in new window)


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