Rapid and Simple Method for Detecting the Toxin B Gene of Clostridium difficile in Stool Specimens by Loop-Mediated Isothermal Amplification

Abstract

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A ؉ B ؉ ) and TcdA-negative, TcdB-positive (A ؊ B ؉ ) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A ؉ B ؉ or A ؊ B ؉ C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.

Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity

“…Six false-positive results were obtained by the illumigene assay, an incidence similar to that noted by other studies (3,6). The mechanism of these false positives is unclear, although some have suggested that loop-mediated isothermal amplification is more sensitive than toxigenic culture, and these may thus represent true-positive results, albeit at very low levels of organism presence (20,21). However, others have reported that the sensitivity of the Xpert C. difficile assay, which was used as an arbiter test in our study, is greater than that of the illumigene assay (22).…”

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection

“…However, the prevalence of C. difficile infection was not statistically different (P ϭ 0.12; Fisher) between PPI users (19%) and nonusers (7%). It is possible that the illumigene assay was more sensitive than TC, as suggested by others (12,19); however, this is unlikely, given the multiple and extended culture protocols used during discrepant analysis and the extended clinical follow-up on these patients. The one false-negative result with the illumigene assay was positive by GeneOhm, the GDH component of the Quik Chek Complete, and TC (discrepant analysis) and negative by both toxin assays.…”

“…The illumigene assay is based upon LAMP (12,17,18), in which primers qualitatively amplify a 204-bp region of the conserved 5= sequence of the tcdA gene within the PaLoc of toxigenic C. difficile via continuous isothermal amplification. Magnesium pyrophosphate is an amplification byproduct that forms a white precipitate that is detected via turbidimetric measurement.…”

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of Bordetella pertussis Infection

“…The significant advantages of the LAMP method are (i) high amplification efficiency under isothermal conditions (63 to 65°C) and (ii) visual judgment based on the turbidity or fluorescence of the reaction mixture, which is kept in the reaction tube (18). Although it has thus emerged as a powerful tool to facilitate genetic testing for the rapid diagnosis of several viral and bacterial infectious diseases in clinical laboratories (5,11,13,14), the LAMP method has not been evaluated for the diagnosis of B. pertussis infection. In the present study, we developed a LAMP method for diagnosis of B. pertussis infection and evaluated its sensitivity, specificity, and applicability for clinical specimens.…”