School of Applied Sciences, RMIT University, Bundoora, 3083, Australia.
J Microbiol Methods. 2012 May;89(2):87-94. doi: 10.1016/j.mimet.2012.02.011. Epub 2012 Mar 3.
The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.
产超毒 027 型艰难梭菌的流行率增加，要求快速鉴定分离株，以便及时实施感染控制策略。高分辨率熔解曲线（HRM）分析聚合酶链反应（PCR）产物可识别细菌属内的菌株变异。种间内基因（16S-23S rDNA）间隔区包含属内保守的序列区域和属内种间可变的其他序列区域。我们希望研究 HRM 分析 PCR 核糖体分型产物是否能鉴定产超毒 027 型艰难梭菌。对 93 株临床分离株和 5 株对照株进行核糖体分型，并用 GelCompar II（美国应用数学公司）分析条带模式。使用核糖体分型引物进行实时 PCR，并生成归一化熔解曲线。然后将 HRM 数据导入 ScreenClust 软件（QIAGEN），生成表示菌株聚类关系的主成分分析图。88/98 株试验分离株的核糖体分型产生了清晰的 PCR 条带。GelCompar 生成的系统发育树显示，这 88 株分离株的核糖体模式多样性丰富，有 18 个群，同源性为 70%。一株临床分离株与对照 027 株 100%同源。对 88 个 HRM 结果进行 ScreenClust 分析显示，分离株聚类，027 株可识别为一个独特的聚类。HRM 分析正确鉴定了对照 027 株和显示为 027 型的临床分离株。HRM 联合实时 PCR 产物 16S-23S rDNA 间隔区的 ScreenClust 分析成功鉴定了 027 型菌株。出于感染控制的目的，这可以在菌落分离后 2-3 小时内完成。
