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Remnant diagnostic samples for high resolution genotyping of Chlamydia trachomatis

用于沙眼衣原体高分辨率基因分型的残留诊断样本

基本信息

批准号：
10301367
负责人：
DANIEL D ROCKEY
金额：
$ 7.43万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-11-12 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10301367
关键词：
Address Affect Amino Acids Bar Codes Bioinformatics Biological Biological Assay Biology Cervical Chlamydia Infections Chlamydia trachomatis Climate Clinic Clinical Collection Communities Complex DNA Data Diagnosis Diagnostic Diagnostic Procedure Diagnostic tests Disease Ectopic Pregnancy Environment Evolution Eye diseases Female genitalia Generations Genes Genetic Genetic Variation Genome Genomics Genotype Goals Health High-Throughput Nucleotide Sequencing Human Individual Infection Infertility Investigation Laboratories Libraries Liquid substance Metagenomics Modeling Monitor Nature Nonsense Mutation Nucleic Acid Amplification Tests Oligonucleotide Primers Organism Outcome Patients Pelvic Inflammatory Disease Persons Procedures Process Public Health Publishing Reaction Reproductive Health Research Personnel Resolution Robotics Running Sampling Scientist Serotyping Source System Technology Testing Trachoma Variant Woman Women&apos s Health Work base clinical material experience experimental study flexibility genetic analysis genetic strain high throughput technology host-microbe interactions human disease individual patient insertion/deletion mutation microorganism microorganism culture next generation sequencing novel pathogen patient population reproductive tract sample collection tissue tropism tool

项目摘要

Summary Chlamydia trachomatis infections in humans cause diseases of the eye and genital tract that affect millions of people annually. Our understanding of C. trachomatis biology is complicated by its obligate intracellular nature and a developing, but limited genetic tool kit. Because of the high level of conservation among C. trachomatis strains found in patients, next-gen sequencing has evolved into a straightforward tool for analysis of genetic variability of the pathogen. Our laboratory has a goal of monitoring genomic changes in C. trachomatis isolates circulating in patient populations. This monitoring is significantly affected by the lack of recent cultured strains, which are becoming increasingly rare as clinics move completely to nucleic acid amplification testing for chlamydial diagnosis. To address our goal in the current diagnostics climate, we propose to develop a robotics-based approach for evaluating contemporary strain variability, with deidentified remnant clinical samples as a source of material. Bioinformatic analysis of all published C. trachomatis genomes has identified a large library of candidate oligonucleotide primers that can be used to generate material for sequencing all variable regions of the genome, and we have demonstrated that remnant diagnostic material is suitable for a PCR-based approach. Draft analyses indicate that all indels and over half of all nonconservative base changes can be assessed in a set of 96 individual PCR reactions. In Aim 1, we will carefully evaluate different primer pairs to identify a collection that will cover a maximum fraction of the genome in a single tray-based amplification format. In Aim 2, we will test our identified primer set in three increasingly complicated robotics-based assays to assess the utility of the system for examining global genetic variation in a single high-throughput experiment. The final product of this project will be a flexible process that can amplify and sequence large collections of specimens and will take approximately one week to complete. We anticipate that this platform will be useful to basic researchers and clinician scientists exploring the connections between chlamydial genotype and clinical presentation, and will serve as a model for employing the approach with other challenging microorganisms for which diagnosis of infection does not include a culture step.

总结