Recently, new methods for high throughput DNA sequencing have been introduced. Consequently, the method is expected to reveal only the dominant members of the bacterial community. In spite of these recent advances, technical difficulties and high cost associated with the cloning and Sanger sequencing approach make it difficult to analyze a large number of clones from a large number of samples. Cataloguing the bacterial taxa found in healthy and diseased conditions by broad-range methods has allowed further closed-ended molecular studies to target both cultivable and as-yet-uncultivated taxa in multiple samples, revealing several new candidate pathogens involved as for instance with caries ( 7), periodontal diseases, ( 8) and endodontic infections ( 9). Studies using this approach have revealed that the oral microbiota is more diverse than previously demonstrated by culture methods and that 40–60% of the bacterial taxa found in the mouth have not yet been cultivated and validly named ( 1– 6). Specifically, the adoption of 16S rRNA gene amplification by polymerase chain reaction (PCR), followed by cloning and Sanger sequencing, allowed an even more comprehensive broad-range investigation of oral bacterial communities. These methods have substantially refined and redefined the knowledge of the microbial diversity in the different oral habitats and expanded the list of candidate pathogens associated with oral infectious diseases ( 1– 4). Further advances in technology hold the perspective to have important implications in terms of accurate diagnosis and more effective preventive and therapeutic measures for common oral diseases.Ĭulture has been traditionally used for identification of bacteria in the oral cavity, but over the last two decades culture-independent molecular biology methods have been increasingly used. Because most oral infectious diseases are currently regarded as biofilm-related polymicrobial infections, high-throughput sequencing technologies have the potential to disclose specific patterns related to health or disease. The results brought about by pyrosequencing studies have significantly contributed to refining and augmenting the knowledge of the community membership and structure in and on the human body in healthy and diseased conditions. This article reviews several aspects of one of these technologies, the pyrosequencing technique, including its principles, applications, and significant contribution to the study of the human microbiome, with especial emphasis on the oral microbiome. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.Next-generation sequencing technologies have revolutionized the analysis of microbial communities in diverse environments, including the human body. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens.
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