Rapid Multiplex Real Time PCR Assay for the Identification of ESBL Genes in Urinary Tract Infections Without the Need for Melting Curve Analysis

Background: Extended-spectrum β-lactamases (ESBLs) are a major public health concern and have therefore become one of the most studied topics within the field of antibiotic resistance. Current standard detection methods for ESBLs are time-consuming and their accuracy has been called into question. This study aimed to develop a rapid, accurate method to detect ESBLs (blaTEM, blaSHV, blaOXA, and blaCTX-M) using real-time PCR, without the need for a melting curve analysis.

Materials/methods:The Plexor® qPCR system was chosen for it high multiplex ability. The system requires a florescent reporter, adjacent to an iso-dC label, to be attached to one of the primers in each pair. Florescence is then quenched by incorporation of a Dabcyl-iso-dGTP label contained in the mastermix, when bound to another DNA strand. Therefore, as product increases, florescence decreases. Monitoring this process each cycle allows us to calculate the Tm of each product, similar to what occurs during the high-resolution melting curve analysis, shown in fig 1. 90 patient isolates from urinary tract infections were obtained from the Leicester Royal Infirmary and tested using the new assay. DNA was extracted by means of boiling colonies for ten minutes at 95⁰C. Results were confirmed by multiplex end-point PCR.

Fig 1. (A) Initial denaturation - the primers are unbound, and florescence is high. (B) Annealing/extension - florescence decreases as primers bind and incorporate the Dabcyl-iso-dGTP label, quenching the reporter. (C) Denaturation - as temperature rises between the annealing step and the denaturation step, primers dissociate from the accompanying DNA strand, and the reporter is no longer quenched.

Results: The assay correctly identified 97.7% isolates tested, with a sensitivity and specificity of 98.7% and 83.3% respectively. The positive and negative likelihood ratio was 5.92 and 0.02 respectively.

Conclusions: The aim was to develop a rapid real-time PCR method to detect ESBL genes. It was found that the method could produce accurate results, without the need for high skill in real-time PCR analysis. The ability to rapidly and accurately detect ESBL genes is an important step in improving antimicrobial stewardship and reducing morbidity and mortality as a result of ESBL-producing pathogenic infections.