DNA detection of Clostridium difficile infection based on real-time resistance measurement.
We used a newly developed electrochemical method, real-time resistance measurement, based on loop-mediated isothermal amplification (LAMP), with real-time resistance monitoring and derivative analysis. DNA extracted from specimens was amplified through LAMP reaction. The 2 products of LAMP, DNA and pyrophosphate, both are negative ions; they combine with positive dye (crystal violet) and positive ions (Mg(2+)), which leads to an increase in the resistivity of the reaction liquid. The changes of resistivity were measured in real-time with a specially designed resistance electrode, to detect Clostridium difficile DNA. We found that electrochemical detection of C. difficile could be completed in 0.5-1 h, with a detection limit of 10(2) CFU/mL, with high accuracy (95.0%), sensitivity (91.1%), and specificity (97.3%) compared to PCR methods. C. difficile is commonly associated with antibiotic-induced diarrhea. Due to the difficulty in performing anaerobic culture and cytotoxicity neutralization assays, a simple, rapid, sensitive, and accurate method is preferred. We conclude that real-time resistance measurement is a rapid, sensitive, and stable method for the diagnosis of C. difficile infection that could be applied to gene chips and pocket instruments.
Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
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D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D003015:Clostridium Infections / Q000175:diagnosis* / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D006801:Humans; D057230:Limit of Detection; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial