Instituto de Investigaciones Biotecnológicas, Universidad de San Martín, San Martín, Buenos Aires, Argentina.
PLoS Negl Trop Dis. 2012;6(7):e1777. doi: 10.1371/journal.pntd.0001777. Epub 2012 Jul 31.
Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.
METHODOLOGY/PRINCIPAL FINDINGS: We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI-TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.
CONCLUSIONS/SIGNIFICANCE: Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.
克氏锥虫是恰加斯病的病原体。该寄生虫具有复杂的种群结构，包含六个主要的进化谱系，其中一些显然是由祖先杂交事件产生的。由于这些谱系之间存在重要的生物学差异，因此菌株分型方法对于研究克氏锥虫物种至关重要。目前，有许多用于克氏锥虫的分型方法，每种方法都有其优点和缺点。然而，这些方法大多基于可变数量的基因座的扩增。
方法/主要发现：我们提出了一种基于单个多态性基因座扩增的克氏锥虫简单分型方法：TcSC5D 基因。在分析该基因（一种假定的羊毛甾醇/甾醇氧化酶）的序列时，我们观察到了一些有趣的多态性位点，包括 1 个四等位基因和一些信息丰富的三等位基因和二等位基因 SNP。此外，这些 SNP 中的一些位于两种商业上可用的限制酶的识别序列内。用这些酶进行双酶切会产生一种独特的酶切模式，允许将菌株简单地分为六个主要组，对应于 DTU TcI-TcIV、最近提出的 Tcbat 谱系以及 TcV/TcVI（作为一组）。通过分析少数几个有区别的多态性位点，对扩增子进行直接测序，可将菌株分为七个组，包括目前公认的六个进化谱系。
结论/意义：基于这些发现，我们提出了一种简单的克氏锥虫分型方法，该方法需要进行一次 PCR 扩增，然后进行限制片段长度多态性分析或直接测序。在测试的菌株组中，基于测序的方法显示出与最近的多位点序列分型方法相当的谱系间分辨率。由于其简单性和低成本，所提出的方法是快速筛选菌株集的良好替代方法，为开发稳健的分型策略提供了基础。
