Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with p...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Aleksandr V. Ivanov, Irina V. Safenkova, Anatoly V. Zherdev, Boris B. Dzantiev
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	RNA virus RPA probe with tetrohydrofuran lateral flow test nfoRPA Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/a0cdb7d007bc4587a97575f0b1772452
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:a0cdb7d007bc4587a97575f0b1772452
record_format	dspace
spelling	oai:doaj.org-article:a0cdb7d007bc4587a97575f0b17724522021-11-11T17:18:11ZRecombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe10.3390/ijms2221118851422-00671661-6596https://doaj.org/article/a0cdb7d007bc4587a97575f0b17724522021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11885https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 10<sup>3</sup> copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.Aleksandr V. IvanovIrina V. SafenkovaAnatoly V. ZherdevBoris B. DzantievMDPI AGarticleRNA virusRPA probe with tetrohydrofuranlateral flow testnfoRPABiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11885, p 11885 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	RNA virus RPA probe with tetrohydrofuran lateral flow test nfoRPA Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	RNA virus RPA probe with tetrohydrofuran lateral flow test nfoRPA Biology (General) QH301-705.5 Chemistry QD1-999 Aleksandr V. Ivanov Irina V. Safenkova Anatoly V. Zherdev Boris B. Dzantiev Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
description	The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 10<sup>3</sup> copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.
format	article
author	Aleksandr V. Ivanov Irina V. Safenkova Anatoly V. Zherdev Boris B. Dzantiev
author_facet	Aleksandr V. Ivanov Irina V. Safenkova Anatoly V. Zherdev Boris B. Dzantiev
author_sort	Aleksandr V. Ivanov
title	Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
title_short	Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
title_full	Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
title_fullStr	Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
title_full_unstemmed	Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe
title_sort	recombinase polymerase amplification assay with and without nuclease-dependent-labeled oligonucleotide probe
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/a0cdb7d007bc4587a97575f0b1772452
work_keys_str_mv	AT aleksandrvivanov recombinasepolymeraseamplificationassaywithandwithoutnucleasedependentlabeledoligonucleotideprobe AT irinavsafenkova recombinasepolymeraseamplificationassaywithandwithoutnucleasedependentlabeledoligonucleotideprobe AT anatolyvzherdev recombinasepolymeraseamplificationassaywithandwithoutnucleasedependentlabeledoligonucleotideprobe AT borisbdzantiev recombinasepolymeraseamplificationassaywithandwithoutnucleasedependentlabeledoligonucleotideprobe
_version_	1718432135628455936

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

Ejemplares similares