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Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2

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 de...

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Autores principales: Gao, Lifen, Li, Lun, Fang, Bin, Fang, Zhiwei, Xiang, Yanghai, Zhang, Min, Zhou, Junfei, Song, Huiyin, Chen, Lihong, Li, Tiantian, Xiao, Huafeng, Wan, Renjing, Jiang, Yongzhong, Peng, Hai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10269707/
https://www.ncbi.nlm.nih.gov/pubmed/37098913
http://dx.doi.org/10.1128/spectrum.00206-23
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author Gao, Lifen
Li, Lun
Fang, Bin
Fang, Zhiwei
Xiang, Yanghai
Zhang, Min
Zhou, Junfei
Song, Huiyin
Chen, Lihong
Li, Tiantian
Xiao, Huafeng
Wan, Renjing
Jiang, Yongzhong
Peng, Hai
author_facet Gao, Lifen
Li, Lun
Fang, Bin
Fang, Zhiwei
Xiang, Yanghai
Zhang, Min
Zhou, Junfei
Song, Huiyin
Chen, Lihong
Li, Tiantian
Xiao, Huafeng
Wan, Renjing
Jiang, Yongzhong
Peng, Hai
author_sort Gao, Lifen
collection PubMed
description 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. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.
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spelling pubmed-102697072023-06-16 Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2 Gao, Lifen Li, Lun Fang, Bin Fang, Zhiwei Xiang, Yanghai Zhang, Min Zhou, Junfei Song, Huiyin Chen, Lihong Li, Tiantian Xiao, Huafeng Wan, Renjing Jiang, Yongzhong Peng, Hai Microbiol Spectr Research Article 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. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism. American Society for Microbiology 2023-04-26 /pmc/articles/PMC10269707/ /pubmed/37098913 http://dx.doi.org/10.1128/spectrum.00206-23 Text en Copyright © 2023 Gao et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Gao, Lifen
Li, Lun
Fang, Bin
Fang, Zhiwei
Xiang, Yanghai
Zhang, Min
Zhou, Junfei
Song, Huiyin
Chen, Lihong
Li, Tiantian
Xiao, Huafeng
Wan, Renjing
Jiang, Yongzhong
Peng, Hai
Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title_full Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title_fullStr Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title_full_unstemmed Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title_short Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2
title_sort carryover contamination-controlled amplicon sequencing workflow for accurate qualitative and quantitative detection of pathogens: a case study on sars-cov-2
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10269707/
https://www.ncbi.nlm.nih.gov/pubmed/37098913
http://dx.doi.org/10.1128/spectrum.00206-23
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