School of Applied Sciences, Building 223, Level 1, Bundoora Campus RMIT University, PO Box 71, Bundoora 3083, Australia.
J Microbiol Methods. 2013 Jun;93(3):257-72. doi: 10.1016/j.mimet.2013.02.013. Epub 2013 Mar 29.
Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1's from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.
艰难梭菌可引起感染性腹泻的爆发，最常见于医疗机构。最近，人们对社区获得性而不是医疗机构获得性的低危人群中艰难梭菌疾病的报道感到担忧。这提高了人们对跟踪爆发的必要性的认识，聚合酶链反应（PCR）核糖体分型已被广泛用于阐明具有流行病学关联的分离株。PCR 核糖体分型使用 16S rRNA 基因和 23S rRNA 基因的保守区域作为引物结合位点，由于多个操纵子的基因间 spacer（ITS1）区域的存在，产生不同的 PCR 产物。随着全基因组序列数据的爆炸式增长，人们可以分析 23S rRNA 基因的起始部分，以更准确地选择靠近 ITS1 末端的区域。然而，仍需提出以下问题：（i）艰难梭菌菌株的 rrn 操纵子的染色体结构是否存在差异？（ii）引物结合区域的保守程度如何？对 8 个已发表的艰难梭菌基因组进行了比对，以生成 rrn 操纵子 ITS1 缺失的详细数据库。构建了一个名为 RiboTyping（RT）的 iPad Filemaker Go 应用程序。它包含了序列、核糖体分型、菌株编号、GenBank 编号和基因组位置编号等详细信息。该数据库提供了对不同层次数据库的访问，以便打印详细信息。该数据库已分析了 rrn 操纵子的三个主要区域，并通过菌株、核糖体分型和操纵子相关联：（1）16S 基因；（2）ITS1 缺失；（3）23S 基因。这使得在每个三个基因组区域内，以菌株、核糖体分型和操纵子（等位基因）为单位，能够进行直接的种内和种间基因组比较。这是首次进行这样的分析。通过使用 RT 应用程序和搜索标准，将有可能为与诊断、分型和操纵子重组相关的实验选择特定菌株/核糖体分型/ rrn 操纵子的探针组合。在 GenBank 中记录了更多正在进行的不完整的艰难梭菌全基因组测序项目，当这些项目可用时，也可以将 rrn 操纵子信息添加到 RT 应用程序中。由于艰难梭菌的 ITS1 非常复杂，即使在单个基因组中也是如此，并且由于其他等位基因特异性区域（16S 和 23S 基因）的可变性也可以相关联进行比较，以设计额外的探针来提高灵敏度，因此 RT 应用程序将有助于简化探针选择。
