Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia.
Appl Environ Microbiol. 2010 Jan;76(2):493-9. doi: 10.1128/AEM.01164-09. Epub 2009 Nov 20.
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. 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.
高变异性的鞭毛蛋白编码基因 flaA 长期以来一直被用于空肠弯曲菌和大肠弯曲菌的基因分型。高分辨率熔解（HRM）分析作为一种高效且强大的方法，正在用于区分 DNA 序列变异。本研究的目的是将 HRM 分析应用于基于 flaA 的基因分型。最初的目的是确定合适的 flaA 片段。结果发现，用于扩增 flaA 短可变重复（SVR）的常见 PCR 引物产生了一种不适合 HRM 分析的混合 PCR 产物。然而，由用于扩增用于 flaA 限制片段长度多态性（RFLP）分析的片段的上游引物和用于 flaA SVR 扩增的下游引物组成的 PCR 引物对产生了非常纯净的 PCR 产物，并且该引物对用于本研究的其余部分。对 87 株空肠弯曲菌和 15 株大肠弯曲菌分离株进行了 flaA HRM 分析和部分 flaA 测序。有 47 种 flaA 序列变异，均通过 HRM 分析得到解决。所使用的分离株之前也使用单核苷酸多态性（SNP）、二进制标记物、CRISPR HRM 和 flaA RFLP 进行了基因分型。flaA HRM 分析提供了与 SNP、二进制标记物和 CRISPR HRM 相乘的分辨率，并与 flaA RFLP 基本一致。结论是 HRM 分析是一种很有前途的基于高度变异基因的基因分型方法。
