Introduction E. coli belonging to sequence type 131 clonal complex (ST131) have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole genome sequencing and multi-locus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high resolution melting (HRM) analysis to differentiate ST131 from non-ST-131 E. coli in large sample populations in the absence of sequence analysis.
Methods The method was optimized using DNA from twelve E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer and Multi-Locus Sequence Typing primers followed by multiplex PCR. Amplicon sizes ranged from 630-737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50-95°C on a Rotor-Gene Q 5-Plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli of ST131 and unknown sequence types validated this methodology.
Results This methodology returned 99.2% specificity (True Negative=124, False Positive=1) and 100% sensitivity (True Positive=66, False Negative=0).
Conclusion This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 hours in any laboratory with a HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.
http://ift.tt/2oGr4GB
Δεν υπάρχουν σχόλια:
Δημοσίευση σχολίου