Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site...

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Autores principales:	Fang Wang, Lei Wang, Huimin Chen, Na Li, Yan Wang, Yan Li, Wei Liang
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Carbapenemase Enterobacterales recombinase polymerase amplification rapid detection false positive Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6338e94877f54ca0bede7c56ba092a07
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spelling	oai:doaj.org-article:6338e94877f54ca0bede7c56ba092a072021-12-02T10:28:13ZRapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip2235-298810.3389/fcimb.2021.772966https://doaj.org/article/6338e94877f54ca0bede7c56ba092a072021-12-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fcimb.2021.772966/fullhttps://doaj.org/toc/2235-2988The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.Fang WangLei WangLei WangHuimin ChenNa LiYan WangYan LiWei LiangFrontiers Media S.A.articleCarbapenemaseEnterobacteralesrecombinase polymerase amplificationrapid detectionfalse positiveMicrobiologyQR1-502ENFrontiers in Cellular and Infection Microbiology, Vol 11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Carbapenemase Enterobacterales recombinase polymerase amplification rapid detection false positive Microbiology QR1-502
spellingShingle	Carbapenemase Enterobacterales recombinase polymerase amplification rapid detection false positive Microbiology QR1-502 Fang Wang Lei Wang Lei Wang Huimin Chen Na Li Yan Wang Yan Li Wei Liang Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
description	The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.
format	article
author	Fang Wang Lei Wang Lei Wang Huimin Chen Na Li Yan Wang Yan Li Wei Liang
author_facet	Fang Wang Lei Wang Lei Wang Huimin Chen Na Li Yan Wang Yan Li Wei Liang
author_sort	Fang Wang
title	Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
title_short	Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
title_full	Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
title_fullStr	Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
title_full_unstemmed	Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip
title_sort	rapid detection of blakpc, blandm, blaoxa-48-like and blaimp carbapenemases in enterobacterales using recombinase polymerase amplification combined with lateral flow strip
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/6338e94877f54ca0bede7c56ba092a07
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Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

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