Detection of Tick-Borne Bacteria from Whole Blood Using 16S Ribosomal RNA Gene PCR Followed by Next-Generation Sequencing
- 20 April 2021
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 59 (5)
- https://doi.org/10.1128/jcm.03129-20
Abstract
Reported cases of tick-borne diseases have steadily increased for more than a decade. In the United States, a majority of tick-borne infections are caused by bacteria. Clinical diagnosis may be challenging as tick-borne diseases can present with similar symptoms. Laboratory diagnosis has historically relied on serologic methods, which have limited utility during the acute phase of disease. Pathogen-specific molecular methods have improved early diagnosis, but can be expensive when bundled together and miss unexpected or novel pathogens. To address these shortcomings, we developed a 16S ribosomal RNA (rRNA) gene PCR with next-generation sequencing approach to detect tick-borne bacteria in whole blood. A workflow was optimized by comparing combinations of two extractions platforms and two primer sets, ultimately pursuing DNA extraction from blood with the MagNA Pure 96 and PCR amplification using dual-priming oligonucleotide primers specific to the V1-V3 region of the 16S rRNA gene. The amplified product underwent modified Illumina 16S metagenomics sequencing library preparation and sequencing on a MiSeq V2 Nano flow cell, with data analysis using Pathogenomix RipSeq NGS software. Results with the developed method were compared to those from a V1-V2 16S rRNA gene primer set described by the Centers for Disease Control and Prevention (CDC). The V1-V3 assay demonstrated equivalent performance to the CDC assay, with each method showing concordance with targeted PCR results in 31 of 32 samples, and detecting 22 of 23 expected organisms. These data demonstrate the potential for using a broad-range bacterial detection approach for diagnosis of tick-borne bacterial infection from blood.Keywords
This publication has 11 references indexed in Scilit:
- Targeted Metagenomics for Clinical Detection and Discovery of Bacterial Tick-Borne PathogensJournal of Clinical Microbiology, 2020
- Detection of zoonotic human pathogens from Ixodes scapularis in WisconsinJournal of Vector Ecology, 2020
- Tick-Borne Diseases in the United StatesClinical Chemistry, 2020
- Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United StatesMMWR. Recommendations and Reports, 2016
- Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive studyThe Lancet Infectious Diseases, 2016
- National Surveillance of Spotted Fever Group Rickettsioses in the United States, 2008–2012The American Journal of Tropical Medicine and Hygiene, 2016
- Human Infection withEhrlichia muris–like Pathogen, United States, 2007–20131Emerging Infectious Diseases, 2015
- Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using SonicationJournal of Clinical Microbiology, 2012
- Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical SpecimensJournal of Clinical Microbiology, 2012
- Percent positive rate of Lyme real-time polymerase chain reaction in blood, cerebrospinal fluid, synovial fluid, and tissueDiagnostic Microbiology and Infectious Disease, 2008