RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses

Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herei...

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Autores principales:	Guohao Fan, Ruiqing Zhang, Xiaozhou He, Fengyu Tian, Mingzhu Nie, Xinxin Shen, Xuejun Ma
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	respiratory viruses qPCR RAA highly sensitive clinical detection Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/cdc56d24b79d42efa2188dfa5acb76fe
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spelling	oai:doaj.org-article:cdc56d24b79d42efa2188dfa5acb76fe2021-11-05T08:43:02ZRAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses2296-418510.3389/fbioe.2021.766411https://doaj.org/article/cdc56d24b79d42efa2188dfa5acb76fe2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fbioe.2021.766411/fullhttps://doaj.org/toc/2296-4185Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application.Guohao FanRuiqing ZhangXiaozhou HeFengyu TianMingzhu NieXinxin ShenXuejun MaXuejun MaFrontiers Media S.A.articlerespiratory virusesqPCRRAAhighly sensitiveclinical detectionBiotechnologyTP248.13-248.65ENFrontiers in Bioengineering and Biotechnology, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	respiratory viruses qPCR RAA highly sensitive clinical detection Biotechnology TP248.13-248.65
spellingShingle	respiratory viruses qPCR RAA highly sensitive clinical detection Biotechnology TP248.13-248.65 Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
description	Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application.
format	article
author	Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma
author_facet	Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma
author_sort	Guohao Fan
title	RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
title_short	RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
title_full	RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
title_fullStr	RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
title_full_unstemmed	RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
title_sort	rap: a novel approach to the rapid and highly sensitive detection of respiratory viruses
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/cdc56d24b79d42efa2188dfa5acb76fe
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RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses

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