Rapid detection of mcr-1 by recombinase polymerase amplification.

Abstract

The plasmid-mediated mcr-1 gene conferring colistin resistance has a strong ability to spread. The objective of this study was to establish a rapid and sensitive recombinase polymerase amplification (RPA) method for plasmid-mediated polymyxin-resistant gene mcr-1 detection. Using the reported sequence of the mcr-1 gene, we designed specific primers and probes for RPA. Twenty mcr-1-positive strains carrying IncI2/IncHI2/IncX4/IncP plasmids were screened by RPA in this study. The performance of this new assay was compared to that of PCR, TaqMan probe real-time PCR and SYBR Green-based real-time PCR. Twenty mcr-1-positive samples and three negative samples were tested by RPA and the positive detection rate for the mcr-1-positive samples was 100 %. The detection limit of RPA was approximately 100 fg. Compared with real-time PCR, the RPA assay was more effective due to shorter reaction times, simpler instruments and higher sensitivity, while it had the same high specificity as real-time PCR. RPA detection based on the mcr-1 gene was successfully applied in our study. The plasmid-mediated mcr-1 gene conferring colistin drug resistance has a strong ability to spread, suggesting the need to further strengthen the detection of this resistance gene in surveillance. Therefore, we require more sensitive detection methods than have previously been available and the RPA assay established in this study meets these detection needs.