基于探针熔解曲线分析的多位点序列分型技术研究

Multilocus sequence typing (MLST) is based on identification of sequence variants among multiple gene loci and is characteristic of unambiguity and portability for its typing results. Currently, MLST has become a reference typing method for increasing number of bacteria. However, the requirement of sequencing of multiple targets has increased the material cost and lowered the throughput, and therefore hindering its wide acceptance in different settings. Several improved approaches have been proposed but all of them suffer the loss of the discrimination power of MLST. We previously established a universal multicolor melting curve analysis (MCA) approach, which significantly improved the capacity of mutation detection on the real-time PCR platform. This approach was also successfully used in a preliminary study of identification of variant sites in multiple housekeeping genes of Vibrio cholerae. In this proposal, we will take advantage of MCA to achieve accurate genotyping of all the alleles of the selected housekeeping genes after extracting their specific variant sites. A new typing method named McMLST will be established with uncompromised discrimination power and extra advantages in throughput, ease of use and cost. Using Staphylococcus aureus as a model bacterium, we will establish and validate the McMLST system, and compare its typing results with MLST and pulsed field gel electrophoresis. McMLST should improve the typing efficiency and ease of use while keeping the unambiguity and portability of the typing results of MLST, and therefore might find wide applications in the molecular typing areas.

多位点序列分型（MLST）利用细菌多个基因座位的序列信息进行分型，具有无歧义和可传输的优势，成为越来越多细菌的标准分型模式。然而，MLST需要多个目标片段的测序分析，成本高，通量低，限制了其应用。已报道的改进模式都不同程度地造成分型分辨能力下降。申请者前期提出多色探针熔解曲线分析技术，显著提高了基于实时PCR平台的多位点突变检测水平，已成功用于霍乱弧菌多个管家基因的变异位点分析。本项目拟利用该技术，通过提取等位基因的特异变异位点，实现各等位基因的分型，建立起分辨能力等同于MLST，而在通量、操作简便性和成本上都更具优势的新型分型技术- - McMLST。本项目以金黄色葡萄球菌为模型，建立并评价McMLST技术，并分别与MLST和脉冲场电泳分型结果进行对比，探讨进一步提升其性能的可能性。鉴于McMLST保持了MLST优点，又提高了分型的效率和易用性，在分型领域应得以广泛应用。

病原微生物引起的传染性疾病是全球公共卫生领域面临的严峻课题。通过病原微生物分型，可实现对病原微生物的流行病学调查、跟踪和阻断传播途径以遏制其暴发流行。理想的病原微生物分子分型技术，应具有分型能力强、操作简便、重复性好、准确快速、高通量自动化、成本低廉的特点。本项目以金黄色葡萄球菌为模型，建立了一种新型的数字化病原微生物分子分型技术-基于熔解曲线的最小化多位点序列分型 (minim McMLST)。.本项目以金黄色葡萄球菌为模型，建立了三管三重的金黄色葡萄球菌分型体系。该体系检测15个SNPs位点，理论上可将2151个序列型分成288种MTs。本项目检测了823份标本，获得36种MTs，而MLST方法获得了40种STs。相对于MLST方法，计算出D值为0.9884。进一步利用goeBURST对128份菌株进行了克隆谱系分析，本项目方法未区分的菌株在MLST中均被划分到了相同的克隆群中，其溯源分析能力与MLST开始趋于一致。标本的分型正确率为97.19%。另外，通过对823标本的耐药状态检测，本项目观察到熔解类型与菌株的甲氧西林耐药性表现出明显的相关性，因此，基因分型对临床用药具有潜在的指导价值。.最后，完成了生物信息学分析软件——McMLST分析软件的编程与测试并投入使用。该软件通过已知SNP位点将原始基因序列数据转化为0/1位点编码的熔解型数据，并提供分型效率（Simpson指数）、总的分型数目（total-MT）以及一一对应的分型数目（unique-MT）等信息，实现从原始数据到基因型的转化和自动判读，从而大大加快分析速度，并提高分析结果判断的准确性。此外，该软件的完成，实现了本项目的最终目标——提供一套完整的包括试剂、软件和操作规程的分型系统。