Multiplex Real-Time Polymerase Chain Reaction and Recombinase Polymerase Amplification: Methods for Quick and Cost-Effective Detection of Vancomycin-Resistant Enterococci (VRE).

Background/

Objectives

Vancomycin-resistant enterococci (VRE) are one of the leading causes of antibiotic-resistant infections in the hospital setting worldwide, and this has become a major issue, because most patients infected with this strain are difficult to treat. Multiplex real-time polymerase chain reaction (RT PCR) is an advantageous technique that can amplify multiple targets in a single reaction, and can be used to quickly detect specific targets in VRE within two hours, starting from suspected colonies of bacterial cultures, without sample preparation.

Methods

In this study, we selected the glycopeptide/vancomycin resistance genes that are most common in clinical settings, vanA and vanB, in combination with the species markers ddl_faecium and ddl_faecalis for the most common VRE species-Enterococcus faecium and Enterococcus faecalis.

Results

DNA from forty clinical VRE strains was prepared using a fast and economic heat lysis method, and a multiplex real-time PCR assay was optimized and carried out subsequently. The results were in concordance with the results from recombinase polymerase amplification (RPA) of the same VRE samples.

Conclusions

Multiplex RT PCR and RPA for VRE detection proffers a second method for the confirmation of vancomycin resistance, and it can be developed as a fast screening assay for patients before admission into high-risk settings.