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Abstract

Introduction:The human body harbors bacteria, archaea, viruses, and eukaryotic microbes that inhabit interactive interfaces exposed to the external environment. This collection of microorganisms consists of about 100 trillion of microbes called the human microbiota and the total genes encoded by these microbes collectively called as human microbiome. They are strongly associated with the development of non-communicable diseases (NCDs) in addition to the role they play in communicable infectious diseases. Changes in microbiota has been reported in various NCDs including obesity, cancer, diabetes, metabolic syndrome, inflammatory bowel disease (IBD), asthma, cardiovascular disease (CVD), kidney disease (KD) and others. Advances in DNA sequencing and bioinformatics had enabled researchers the exploration of the genetic diversity of the uncultured component of host-associated microbial communities. The aim of this work is to test different DNA extracting protocols to characterize human microbiota of biological samples collected from adult volunteers. Materials & Methods: Biological samples such as human saliva, vaginal swabs and stool were collected from well-characterized adult participants. Total DNA were extracted from Vaginal swabs (n = 5) using DNeasy Blood & Tissue Kit and Mobio power Soil protocol; Stool samples (n = 17) using MoBio Power Fecal and Qiamp fast stool kits; Saliva samples (n = 8) using QIASymphony DNA Midi kit. DNA quantity and quality was assessed by Nanodrop spectrophotometer. Integrity of the DNA was reviewed on LabChip. Human microbiota diversities of different biological samples through different extraction protocols will be determined using MiSeq-Illumina high-throughput sequencing of bacterial 16S rDNA V1-V3 fingerprint. Sequenced data will be analyzed using QIIME pipeline. Results DNA measurement revealed that DNeasy Blood & Tissue Kit gave higher DNA yield with good integrity (2-8 ng/μL) than Mobio power Soil protocol (0.6-2 ng/μL) for Vaginal swabs. Likewise, MoBio Power Fecal kit (1.8-41.5 ng/mL) gave higher DNA yield than Qiamp fast stool kit (5-21 ng/mL) for stool samples. On the other hand, DNA Qiasymphony DSP kit gave (3-241 ng/μL) for saliva. Expected Outcome: From the previous studies by other groups, it is expected that Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria are the most abundant phyla in human microbiota. The abundance of the mentioned phylum may differ with respect to body sites. Actinobacteria members will be abundant on skin, Firmicutes and Bacteroidetes will be more abundant in gut. Firmicutes, Proteobacteria, and Bacteroidetes will be the more abundant in saliva and Firmicutes in Saliva. In addition, Each DNA extraction protocol has different strategies to lyse the human microbes. We speculate that the microbial diversity of same body site can be different with different protocols. Characterization of healthy human microbiome gives a better understanding of their role in human health during diseased conditions. Especially, Human gut microbiota influence the metabolism of host and it links to several metabolic disorders such as obesity, Type 2 diabetes and Metabolic syndrome when there is an imbalance in microbiota. Exploring Vaginal microbiome can guide as to find new therapeutic targets to treat several infections and to treat complications with preterm birth to pregnant women. This work will be a promising candidate to choose the adequate protocol based on the nature of the sample, which can guide us to explore the human microbiome in right path.

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/content/papers/10.5339/qfarc.2018.HBPD823
2018-03-15
2020-09-23
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