Abstract

Two different approaches, real-time PCR and in silico analysis, were used to study the abundance of bacterial genes encoding the enzymes producing biomarker metabolites in metagenomes of gut microbiota of children with autism spectrum disorders (ASD) and healthy children. As a result of the analysis of the published data, the following biomarker metabolites of ASD were chosen for the research: p-cresol, indole, propionic acid, and D-lactic acid. For the real-time PCR and the transcriptomic analysis, primer sequences were used specifically developed for conservative regions of the genes involved in production of these bacterial metabolites. Nucleotide sequences of the conservative regions were also united into a catalog and were searched in metagenomic assemblies as part of in silico analysis. Comparison of the results revealed a decrease in abundance of the genes in the metagenomes of children with ASD during both PCR and bioinformatics analyses. In addition, real-time PCR made it possible to detect a significant increase in abundance of the genes encoding p-hydroxyphenylacetate decarboxylase and D-lactate dehydrogenase in Bacteroides fragilis and Alistipes finegoldii species and methylmalonyl-CoA decarboxylase in B. fragilis, Alistipes shahii, and Eubacterium rectali species. A low level of the transcripts in total RNA was found in microbiota of a child with severe form of ASD.

中文翻译：

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幼儿肠道菌群自闭症谱系障碍代谢物生物标志物的细菌基因：实时PCR和计算机模拟分析

摘要

两种不同的方法，即实时PCR和计算机分析，用于研究自闭症谱系障碍（ASD）儿童和健康儿童肠道菌群的基因组中编码产生生物标志物代谢物酶的细菌基因的丰富性。分析已发表数据的结果，选择了以下ASD生物标志物代谢物进行研究：p-甲酚，吲哚，丙酸和D-乳酸。对于实时PCR和转录组学分析，使用引物序列专门开发用于涉及这些细菌代谢产物生产的基因的保守区域。保守区的核苷酸序列也被合并到一个目录中，并作为计算机模拟分析的一部分，在宏基因组中进行搜索。结果比较表明，在PCR和生物信息学分析过程中，ASD儿童基因组中基因的丰度降低。另外，实时PCR使得检测脆弱类拟杆菌和对羟基苯乙酸脱羧酶和D-乳酸脱氢酶编码基因的丰度大大提高。脆弱的拟杆菌，拟南芥和直肠真细菌中的细小金丝藻种和甲基丙二酰辅酶A脱羧酶。在患有严重形式的ASD的儿童的微生物群中发现总RNA的转录水平较低。