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A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

BACKGROUND: The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5’ untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate...

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Autores principales: Tsai, Jih-Jin, Liu, Wei-Liang, Lin, Ping-Chang, Huang, Bo-Yi, Tsai, Ching-Yi, Lee, Pei-Yu Alison, Tsai, Yun-Long, Chou, Pin-Hsing, Chung, Simon, Liu, Li-Teh, Chen, Chun-Hong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619671/
https://www.ncbi.nlm.nih.gov/pubmed/31291289
http://dx.doi.org/10.1371/journal.pone.0218139
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author Tsai, Jih-Jin
Liu, Wei-Liang
Lin, Ping-Chang
Huang, Bo-Yi
Tsai, Ching-Yi
Lee, Pei-Yu Alison
Tsai, Yun-Long
Chou, Pin-Hsing
Chung, Simon
Liu, Li-Teh
Chen, Chun-Hong
author_facet Tsai, Jih-Jin
Liu, Wei-Liang
Lin, Ping-Chang
Huang, Bo-Yi
Tsai, Ching-Yi
Lee, Pei-Yu Alison
Tsai, Yun-Long
Chou, Pin-Hsing
Chung, Simon
Liu, Li-Teh
Chen, Chun-Hong
author_sort Tsai, Jih-Jin
collection PubMed
description BACKGROUND: The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5’ untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system. METHODOLOGY/PRINCIPAL FINDINGS: Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems. CONCLUSIONS/SIGNIFICANCE: With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease.
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spelling pubmed-66196712019-07-25 A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos Tsai, Jih-Jin Liu, Wei-Liang Lin, Ping-Chang Huang, Bo-Yi Tsai, Ching-Yi Lee, Pei-Yu Alison Tsai, Yun-Long Chou, Pin-Hsing Chung, Simon Liu, Li-Teh Chen, Chun-Hong PLoS One Research Article BACKGROUND: The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5’ untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system. METHODOLOGY/PRINCIPAL FINDINGS: Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems. CONCLUSIONS/SIGNIFICANCE: With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease. Public Library of Science 2019-07-10 /pmc/articles/PMC6619671/ /pubmed/31291289 http://dx.doi.org/10.1371/journal.pone.0218139 Text en © 2019 Tsai et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Tsai, Jih-Jin
Liu, Wei-Liang
Lin, Ping-Chang
Huang, Bo-Yi
Tsai, Ching-Yi
Lee, Pei-Yu Alison
Tsai, Yun-Long
Chou, Pin-Hsing
Chung, Simon
Liu, Li-Teh
Chen, Chun-Hong
A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title_full A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title_fullStr A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title_full_unstemmed A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title_short A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos
title_sort fully automated sample-to-answer pcr system for easy and sensitive detection of dengue virus in human serum and mosquitos
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619671/
https://www.ncbi.nlm.nih.gov/pubmed/31291289
http://dx.doi.org/10.1371/journal.pone.0218139
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