Development of Rapid and High-Throughput Genetic Fingerprinting Assay for the Identification of Specific Intestinal Microbes

人们越来越意识到，我们的微生物区系在包括肥胖、糖尿病、帕金森病、多发性硬化症和结直肠癌在内的各种病理疾病中发挥着重要作用。个性化医学将发展为包括对复杂样本(如粪便)中特定微生物的存在或不存在的分析。大多数微生物是不可培养的，目前的基因组测序技术分辨率高，但速度慢，成本高。人们将越来越需要有针对性的、廉价的、无快速培养的方法来从各种患者和环境样本中识别特定的微生物。Mass微阵列技术用于识别基因测试的基因多态。Massarray涉及对多达40个不同的基因座进行选择性聚合酶链式反应(多重聚合酶链式反应)扩增，然后用质谱仪(MALDI-TOF)鉴定特定的变异(突变)。北极诊断公司(ArcticDx，多伦多)在使用MassARRAY方面拥有丰富的经验，在过去5年中对患者进行了约10万次基因测试，以确定黄斑变性、囊性纤维化和妊娠期糖尿病的风险等位基因。鉴于最近有证据表明微生物区系在这些疾病中都发挥了作用，ArcticDx有动力扩大他们的Massarray应用程序，以识别患者样本中的特定微生物“指纹”。为此，北极希望与威廉·纳瓦尔(多伦多大学)博士建立合作关系，后者是一名微生物学家，过去三年一直在开发工具来培养和分析来自人类和老鼠的微生物群样本。我们已经制定了一项明确的、技术上可行的计划，以确定是否可以使用Massarray技术同时从复杂的(小鼠粪便)样本中识别多达20种特定微生物的存在或不存在。这将涉及开发针对单个微生物物种的多重引物组，并排除PCR、MALDI-TOF和计算参数的故障，以达到可接受的特异性和选择性水平。

There is an increasing awareness that our microbiota plays an important role in a variety of pathologicaldisorders including obesity, diabetes, Parkinson's disease, multiple sclerosis, and colorectal cancer.Personalized medicine will evolve to include assays for the presence or absence of particular microbes incomplex samples (e.g. stool). Most microbes are uncultivable and current genomic sequencing techniques arehigh resolution but slow and expensive. There will be an increasing need for targeted, inexpensive, and rapidculture-free methods to identify particular microbes from a variety of patient and environmental samples.MassArray technology is used to identify genetic polymorphisms for genetic testing. MassArray involvesthe selective PCR amplification of as many as 40 different loci (multiplex PCR), where specific variants(mutations) are subsequently identified by mass spectrometry (MALDI-TOF). Arctic Diagnostics Inc.(ArcticDx, Toronto) has extensive experience in the use of MassArray, performing ~100,000 genetic tests onpatients over the past 5 years to identify risk alleles for macular degeneration, cystic fibrosis, and gestationaldiabetes. Given recent evidence that microbiota play a role in each of these diseases, ArcticDx is motivated toexpand the application of their MassArray to identify specific microbial "fingerprints" in patient samples.Toward this end Arctic wishes to establish a collaboration with Dr. William Navarre (University ofToronto), a microbiologist who has spent the past three years developing tools to culture and analyzemicrobiota samples from both humans and mice. We have developed a clear and technically feasible plan todetermine if MassArray technology can be employed to simultaneously identify the presence or absence of upto 20 specific microbes from complex (mouse stool) samples. This will involve the development of multiplexprimer sets specific to individual microbial species and troubleshooting the PCR, MALDI-TOF, andcomputational parameters necessary to achieve acceptable levels of specificity and selectivity.