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Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip

Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identific...

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Autores principales: Wang, Lei, Wang, Yan, Wang, Fang, Zhao, Mengdi, Gao, Xuzhu, Chen, Huimin, Li, Na, Zhu, Qing, Liu, Lipin, Zhu, Wenjun, Liu, Xia, Chen, Yujiao, Zhou, Ping, Lu, Yingzhi, Wang, Kun, Zhao, Weiguo, Liang, Wei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8790172/
https://www.ncbi.nlm.nih.gov/pubmed/35096653
http://dx.doi.org/10.3389/fcimb.2021.803798
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author Wang, Lei
Wang, Yan
Wang, Fang
Zhao, Mengdi
Gao, Xuzhu
Chen, Huimin
Li, Na
Zhu, Qing
Liu, Lipin
Zhu, Wenjun
Liu, Xia
Chen, Yujiao
Zhou, Ping
Lu, Yingzhi
Wang, Kun
Zhao, Weiguo
Liang, Wei
author_facet Wang, Lei
Wang, Yan
Wang, Fang
Zhao, Mengdi
Gao, Xuzhu
Chen, Huimin
Li, Na
Zhu, Qing
Liu, Lipin
Zhu, Wenjun
Liu, Xia
Chen, Yujiao
Zhou, Ping
Lu, Yingzhi
Wang, Kun
Zhao, Weiguo
Liang, Wei
author_sort Wang, Lei
collection PubMed
description Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection.
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spelling pubmed-87901722022-01-27 Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip Wang, Lei Wang, Yan Wang, Fang Zhao, Mengdi Gao, Xuzhu Chen, Huimin Li, Na Zhu, Qing Liu, Lipin Zhu, Wenjun Liu, Xia Chen, Yujiao Zhou, Ping Lu, Yingzhi Wang, Kun Zhao, Weiguo Liang, Wei Front Cell Infect Microbiol Cellular and Infection Microbiology Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection. Frontiers Media S.A. 2022-01-12 /pmc/articles/PMC8790172/ /pubmed/35096653 http://dx.doi.org/10.3389/fcimb.2021.803798 Text en Copyright © 2022 Wang, Wang, Wang, Zhao, Gao, Chen, Li, Zhu, Liu, Zhu, Liu, Chen, Zhou, Lu, Wang, Zhao and Liang https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Wang, Lei
Wang, Yan
Wang, Fang
Zhao, Mengdi
Gao, Xuzhu
Chen, Huimin
Li, Na
Zhu, Qing
Liu, Lipin
Zhu, Wenjun
Liu, Xia
Chen, Yujiao
Zhou, Ping
Lu, Yingzhi
Wang, Kun
Zhao, Weiguo
Liang, Wei
Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title_full Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title_fullStr Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title_full_unstemmed Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title_short Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip
title_sort development and application of rapid clinical visualization molecular diagnostic technology for cryptococcus neoformans/c. gattii based on recombinase polymerase amplification combined with a lateral flow strip
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8790172/
https://www.ncbi.nlm.nih.gov/pubmed/35096653
http://dx.doi.org/10.3389/fcimb.2021.803798
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