The Central Region Laboratory, Center for Research and Diagnostics, Centers for Disease Control, Taichung City 40855, Taiwan.
BMC Microbiol. 2011 Sep 30;11:217. doi: 10.1186/1471-2180-11-217.
Polymerase chain reaction (PCR) ribotyping is one of the globally accepted techniques for defining epidemic clones of Clostridium difficile and tracing virulence-related strains. However, the ambiguous data generated by this technique makes it difficult to compare data attained from different laboratories; therefore, a portable technique that could supersede or supplement PCR ribotyping should be developed. The current study attempted to use a new multilocus variable-number tandem-repeat analysis (MLVA) panel to detect PCR-ribotype groups. In addition, various MLVA panels using different numbers of variable-number tandem-repeat (VNTR) loci were evaluated for their power to discriminate C. difficile clinical isolates.
At first, 40 VNTR loci from the C. difficile genome were used to screen for the most suitable MLVA panel. MLVA and PCR ribotyping were implemented to identify 142 C. difficile isolates. Groupings of serial MLVA panels with different allelic diversity were compared with 47 PCR-ribotype groups. A MLVA panel using ten VNTR loci with limited allelic diversity (0.54-0.83), designated MLVA10, generated groups highly congruent (98%) with the PCR-ribotype groups. For comparison of discriminatory power, a MLVA panel using only four highly variable VNTR loci (allelic diversity: 0.94-0.96), designated MLVA4, was found to be the simplest MLVA panel that retained high discriminatory power. The MLVA10 and MLVA4 were combined and used to detect genetically closely related C. difficile strains.
For the epidemiological investigations of C. difficile, we recommend that MLVA10 be used in coordination with the PCR-ribotype groups to detect epidemic clones, and that the MLVA4 could be used to detect outbreak strains. MLVA10 and MLVA4 could be combined in four multiplex PCR reactions to save time and obtain distinguishable data.
聚合酶链反应（PCR）核糖体分型是定义艰难梭菌流行克隆和追踪与毒力相关菌株的全球公认技术之一。然而，该技术产生的模棱两可的数据使得难以比较来自不同实验室的数据；因此，应该开发一种可以替代或补充 PCR 核糖体分型的便携式技术。本研究试图使用新的多位点可变串联重复分析（MLVA）面板来检测 PCR 核糖体型群。此外，评估了使用不同数量可变串联重复（VNTR）位点的各种 MLVA 面板来区分艰难梭菌临床分离株的能力。
首先，从艰难梭菌基因组中筛选出 40 个 VNTR 位点，以筛选最合适的 MLVA 面板。使用 MLVA 和 PCR 核糖体分型来鉴定 142 株艰难梭菌。比较了具有不同等位基因多样性的连续 MLVA 面板的分组与 47 个 PCR-核糖体型群。使用具有有限等位基因多样性（0.54-0.83）的十个 VNTR 位点的 MLVA 面板（MLVA10）生成与 PCR-核糖体型群高度一致（98%）的组。为了比较区分力，使用仅四个高度可变 VNTR 位点（等位基因多样性：0.94-0.96）的 MLVA 面板（MLVA4）被发现是保留高区分力的最简单 MLVA 面板。将 MLVA10 和 MLVA4 组合使用来检测遗传上密切相关的艰难梭菌菌株。
对于艰难梭菌的流行病学研究，我们建议使用 MLVA10 与 PCR-核糖体型群一起用于检测流行克隆，并且 MLVA4 可用于检测爆发菌株。MLVA10 和 MLVA4 可以组合在四个多重 PCR 反应中，以节省时间并获得可区分的数据。
