A New Rapid Method for Clostridium difficile DNA Extraction and Detection in Stool: Toward Point-of-Care Diagnostic Testing

• Autores: Alison G. Freifeld, Kari A. Simonsen, Christine S. Booth, Xing Zhao, Scott E. Whitney, Teresa Karre, Peter C. Iwen, Hendrik J. Viljoen
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 14, Nº. 3, 2012, págs. 274-279
• Idioma: inglés
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• Resumen

  • We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH+/toxin+, n = 48; GDH-/toxin-, n = 81). All 69 stool samples with discordant EIA results (GDH+/toxin-) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases.