Cargando…
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...
Autores principales: | , , , , , , , , , , , , , , , , |
---|---|
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 |
_version_ | 1784639937682866176 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8790172 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT wanglei developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT wangyan developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT wangfang developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT zhaomengdi developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT gaoxuzhu developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT chenhuimin developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT lina developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT zhuqing developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT liulipin developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT zhuwenjun developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT liuxia developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT chenyujiao developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT zhouping developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT luyingzhi developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT wangkun developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT zhaoweiguo developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip AT liangwei developmentandapplicationofrapidclinicalvisualizationmoleculardiagnostictechnologyforcryptococcusneoformanscgattiibasedonrecombinasepolymeraseamplificationcombinedwithalateralflowstrip |