Rapid visual detection of binary toxin producing Clostridium difficile by loop-mediated isothermal amplification.

Abstract

The binary toxin Clostridium difficile transferase (CDT) is frequently observed in C. difficile strains and is associated with an increased severity of C. difficile infection. CDT-producing C. difficile infections cause higher fatality rates than infections with CDT negative isolates. Thus, the rapid and accurate identification of a CDT positive C. difficile infection is critical for effective treatment. The present study demonstrates how loop-mediated isothermal amplification (LAMP) can be used to detect CDT-producing C. difficile based on visual observation. This is a low complexity, rapid molecular method that has the potential to be used within a point of care setting. The specificity and sensitivity of the primers in the LAMP reactions for CDT detection were determined using two different methods, a real-time turbidity monitor and visual detection after the addition of calcein to the reaction tube. The results revealed that target DNA was amplified and visualized by these two detection methods within 60 min at a temperature of 60°C. The sensitivity of the LAMP assay was identified to be 10-fold greater than that of polymerase chain reaction analysis. When 25 alternative bacterial strains lacking CDT were tested, the results of the amplification were negative, confirming the specificity of the primers. In conclusion, the visual LAMP method established in the present study may be a rapid, reliable and cost-effective tool for detecting CDT-producing C. difficile strains at the point of care.