Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains

Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains

Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of <i>vanA</i> and <i>vanB</i> genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by...

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Autores principales: Hanane Zerrouki, Sid-Ahmed Rebiahi, Linda Hadjadj, Jean-Marc Rolain, Seydina M. Diene
Formato: article
Lenguaje:EN
Publicado: MDPI AG 2021
Materias:
real-time PCR assay
simultaneous detection
<i>Enterococci</i>
<i>vanA</i>
<i>vanB</i>
Medicine (General)
R5-920
Acceso en línea:https://doaj.org/article/e5046a47ba044f1b92f8de98b7730ef0
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spelling oai:doaj.org-article:e5046a47ba044f1b92f8de98b7730ef02021-11-25T17:21:28ZReal-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains10.3390/diagnostics111120812075-4418https://doaj.org/article/e5046a47ba044f1b92f8de98b7730ef02021-11-01T00:00:00Zhttps://www.mdpi.com/2075-4418/11/11/2081https://doaj.org/toc/2075-4418Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of <i>vanA</i> and <i>vanB</i> genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by in silico PCR and by BlastN analysis. The genomic DNA of 255 bacterial isolates, including <i>Enterococcus</i> spp., Gram-negative, and Gram-positive strains, as well as a collection of 50 stool and 50 rectal swab samples, were tested to evaluate the specificity of the new real-time PCR (RT-PCR) system. The results of the designed RT-PCR were 100% specific and 100% positive on tested vancomycin resistant isolates harboring either the <i>vanA</i> or <i>vanB</i> gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive <i>Enterococci</i> and <i>Enterococcus</i> strains harboring <i>vanC</i> genes. The limit of detection of <i>vanA</i> and <i>vanB</i> genes by RT-PCR assay was 47 CFU/mL and 32 CFU/mL, respectively. The rapid and accurate detection of vancomycin-resistant Enterococci is the cornerstone for minimizing the risk of nosocomial transmissions and outbreaks. We believe that this assay will strengthen routine diagnostics and surveillance programs.Hanane ZerroukiSid-Ahmed RebiahiLinda HadjadjJean-Marc RolainSeydina M. DieneMDPI AGarticlereal-time PCR assaysimultaneous detection<i>Enterococci</i><i>vanA</i><i>vanB</i>Medicine (General)R5-920ENDiagnostics, Vol 11, Iss 2081, p 2081 (2021)
institution DOAJ
collection DOAJ
language EN
topic real-time PCR assay
simultaneous detection
<i>Enterococci</i>
<i>vanA</i>
<i>vanB</i>
Medicine (General)
R5-920
spellingShingle real-time PCR assay
simultaneous detection
<i>Enterococci</i>
<i>vanA</i>
<i>vanB</i>
Medicine (General)
R5-920
Hanane Zerrouki
Sid-Ahmed Rebiahi
Linda Hadjadj
Jean-Marc Rolain
Seydina M. Diene
Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
description Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of <i>vanA</i> and <i>vanB</i> genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by in silico PCR and by BlastN analysis. The genomic DNA of 255 bacterial isolates, including <i>Enterococcus</i> spp., Gram-negative, and Gram-positive strains, as well as a collection of 50 stool and 50 rectal swab samples, were tested to evaluate the specificity of the new real-time PCR (RT-PCR) system. The results of the designed RT-PCR were 100% specific and 100% positive on tested vancomycin resistant isolates harboring either the <i>vanA</i> or <i>vanB</i> gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive <i>Enterococci</i> and <i>Enterococcus</i> strains harboring <i>vanC</i> genes. The limit of detection of <i>vanA</i> and <i>vanB</i> genes by RT-PCR assay was 47 CFU/mL and 32 CFU/mL, respectively. The rapid and accurate detection of vancomycin-resistant Enterococci is the cornerstone for minimizing the risk of nosocomial transmissions and outbreaks. We believe that this assay will strengthen routine diagnostics and surveillance programs.
format article
author Hanane Zerrouki
Sid-Ahmed Rebiahi
Linda Hadjadj
Jean-Marc Rolain
Seydina M. Diene
author_facet Hanane Zerrouki
Sid-Ahmed Rebiahi
Linda Hadjadj
Jean-Marc Rolain
Seydina M. Diene
author_sort Hanane Zerrouki
title Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
title_short Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
title_full Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
title_fullStr Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
title_full_unstemmed Real-Time PCR Assay for Rapid and Simultaneous Detection of <i>vanA</i> and <i>vanB</i> Genes in Clinical Strains
title_sort real-time pcr assay for rapid and simultaneous detection of <i>vana</i> and <i>vanb</i> genes in clinical strains
publisher MDPI AG
publishDate 2021
url https://doaj.org/article/e5046a47ba044f1b92f8de98b7730ef0
work_keys_str_mv AT hananezerrouki realtimepcrassayforrapidandsimultaneousdetectionofivanaiandivanbigenesinclinicalstrains
AT sidahmedrebiahi realtimepcrassayforrapidandsimultaneousdetectionofivanaiandivanbigenesinclinicalstrains
AT lindahadjadj realtimepcrassayforrapidandsimultaneousdetectionofivanaiandivanbigenesinclinicalstrains
AT jeanmarcrolain realtimepcrassayforrapidandsimultaneousdetectionofivanaiandivanbigenesinclinicalstrains
AT seydinamdiene realtimepcrassayforrapidandsimultaneousdetectionofivanaiandivanbigenesinclinicalstrains
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