Cargando…

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

Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB 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....

Descripción completa

Detalles Bibliográficos
Autores principales: Zerrouki, Hanane, Rebiahi, Sid-Ahmed, Hadjadj, Linda, Rolain, Jean-Marc, Diene, Seydina M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618848/
https://www.ncbi.nlm.nih.gov/pubmed/34829428
http://dx.doi.org/10.3390/diagnostics11112081
_version_ 1784604846661304320
author Zerrouki, Hanane
Rebiahi, Sid-Ahmed
Hadjadj, Linda
Rolain, Jean-Marc
Diene, Seydina M.
author_facet Zerrouki, Hanane
Rebiahi, Sid-Ahmed
Hadjadj, Linda
Rolain, Jean-Marc
Diene, Seydina M.
author_sort Zerrouki, Hanane
collection PubMed
description Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB 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 Enterococcus 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 vanA or vanB gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive Enterococci and Enterococcus strains harboring vanC genes. The limit of detection of vanA and vanB 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 Online
Article
Text
id pubmed-8618848
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-86188482021-11-27 Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains Zerrouki, Hanane Rebiahi, Sid-Ahmed Hadjadj, Linda Rolain, Jean-Marc Diene, Seydina M. Diagnostics (Basel) Article Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB 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 Enterococcus 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 vanA or vanB gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive Enterococci and Enterococcus strains harboring vanC genes. The limit of detection of vanA and vanB 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. MDPI 2021-11-10 /pmc/articles/PMC8618848/ /pubmed/34829428 http://dx.doi.org/10.3390/diagnostics11112081 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zerrouki, Hanane
Rebiahi, Sid-Ahmed
Hadjadj, Linda
Rolain, Jean-Marc
Diene, Seydina M.
Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title_full Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title_fullStr Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title_full_unstemmed Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title_short Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains
title_sort real-time pcr assay for rapid and simultaneous detection of vana and vanb genes in clinical strains
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618848/
https://www.ncbi.nlm.nih.gov/pubmed/34829428
http://dx.doi.org/10.3390/diagnostics11112081
work_keys_str_mv AT zerroukihanane realtimepcrassayforrapidandsimultaneousdetectionofvanaandvanbgenesinclinicalstrains
AT rebiahisidahmed realtimepcrassayforrapidandsimultaneousdetectionofvanaandvanbgenesinclinicalstrains
AT hadjadjlinda realtimepcrassayforrapidandsimultaneousdetectionofvanaandvanbgenesinclinicalstrains
AT rolainjeanmarc realtimepcrassayforrapidandsimultaneousdetectionofvanaandvanbgenesinclinicalstrains
AT dieneseydinam realtimepcrassayforrapidandsimultaneousdetectionofvanaandvanbgenesinclinicalstrains