ArticleMicrobiology spectrum2023
Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.
Article in Microbiology spectrum, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Bioinformatic identification of lentivirus transfer plasmid contamination causing false-positive HIV NAT results in a high-throughput molecular screening laboratory.Microbiology spectrum · 2026Article
- MGV-seq: a sensitive and culture-independent method for detecting microbial genetic variation.Frontiers in microbiology · 2025Article
- Article
- Characterization of Heat-labile Uracil-DNA Glycosylase fromProtein and peptide letters · 2024Article
Corrections and comments
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Authors and funding
14 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases.
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