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

Yu,Lan; Li,Huan; Zhao,Xiangna; Wang,Xuesong; Wei,Xiao; Lin,Weishi; Li,Puyuan; Cui,Lihong; Yuan,Jing

doi:10.3892/etm.2017.5178

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

Authors:
- Lan Yu
- Huan Li
- Xiangna Zhao
- Xuesong Wang
- Xiao Wei
- Weishi Lin
- Puyuan Li
- Lihong Cui
- Jing Yuan
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Affiliations: Department of Gastroenterology, Navy General Hospital, Beijing 100048, P.R. China, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, P.R. China
Published online on: September 21, 2017 https://doi.org/10.3892/etm.2017.5178
Pages: 4781-4788
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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.

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