Rapid Detection of vanA Resistance Gene from E. faecalis Clinical Isolates Using Duplex Loop-Mediated Isothermal Amplification and Triplex PCR Assay

Abstract

Today, the spread of vancomycin-resistant strains isolated from Enterococcus faecalis (E. faecalis) has become a major health concern worldwide. Therefore, it is essential to provide a rapid and sensitive assay for identifying vanA gene for timely and appropriate antimicrobial control of resistant enterococcal infections. For this purpose, a cross-sectional study was performed on different clinical specimens of enterococci from Imam Reza hospital, Kermanshah, Iran. The antimicrobial susceptibility testing was determined by disk diffusion and MIC methods. Triplex-PCR and duplex-LAMP assays were also used to identify vanA E. faecalis resistance gene isolates. The results of this study shown that out of 108 Enterococcus isolates, 86, 18, 2, 1, and one isolates of E. faecalis, E. faecium, E. avium, E. psudoavium, and E. raffinosus were identified, respectively. On the other hand, E. faecalis was confirmed in 87 and 88 isolates using duplex-LAMP and triplex PCR, respectively. The LAMP primer set designed in this study can reliably identify seven distinct regions of the vanA gene, and finally the sensitivity, specificity, and the positive and negative predictive values of LAMP assay were shown to be 94.19%, 72.73%, 76.19%, and 93.10%, respectively. In general, sample processing, isothermal reaction and result reporting were completed using the LAMP assay in 75 minutes. Our findings suggest that LAMP assay has been approved as an alternative to the vancomycin resistance Enterococcus genotype (vanA and vanB) compared to other methods and has the advantage of being rapid, time-consuming, and easy for diagnosis.

Figure 1

(a) PCR product electrophoresis of Enterococcus isolates. Lane 1: 100 bp DNA ladder; lane 2: negative control; and lane 3: positive control with 305 bp product for E. faecalis, 195 bp product for vanA, and 109 bp product for Enterococcus. (b) LAMP electrophoresis of amplified Enterococcus isolates. Lane 1: 8: 50 bp DNA ladder; lane 2: single reaction for detection of E. faecalis; lane 3: the duplex-LAMP for detection of E. faecalis and vanA gene; lane 4: the single reaction for detection of vanA; lane 5: EcoRI digested single reaction for detection of E. faecalis; lane 6: EcoRI digested of duplex-LAMP for detection of E. faecalis and vanA gene; and lane 7: EcoRI digested of single reaction for detection of vanA.

Figure 2

Result of analytical specificity of the LAMP primers. (a) Positive and negative reaction of the LAMP method. Naked eyes visual detection of LAMP reaction with purified DNA of non-E. faecalis. 1: positive control; 2: negative control; 3: E. faecium; 4: E. avium; 5: E. raffinosus; 6: E. psudoavium; 7: E. hirae; 8: E. durans; 9: S. pneumoniae; and 10: S. aureus. (b) PCR product for analytical specificity of primers. Lane 1: 100 bp DNA ladder; lane 2: negative control; and lane 3: positive control with 109 bp product for enterococcus, 195 bp product for vanA gene, and 305 bp product for E. faecalis.

Figure 3

(a) LAMP primers targeting the VanA (E. faecalis) gene were used in the serial dilutions to determine limit of detection. 1: negative control; 2: positive control; 3: 1 ng; 4: 0.01 ng; 5: 1 pg; 6: 0.01 pg; 7: 1 fg (LOD of E. faecalis); 8: 0.1 fg; 9: 0.01 fg; 10: 1 Ag from purify DNA of E. faecalis. (b) LOD of E. faecalis by PCR assay. 1: 100 bp DNA ladder; 2: positive control (109 bp Enterococcus, 195 bp vanA, and 305 bp E. faecalis); 3: negative control; 4: 10 ng; 5: 1 g; 6 : 0.1 ng; 7: 10 pg (LOD of vanA); 8: 1 pg; 9: 0.1 pg (LOD of E. faecalis); 10: 10 fg (LOD of Enterococcus); 11: 1 fg; 12: 0.1 fg; and 13: 0.01 fg from purify DNA of E. faecalis.