title: Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile.
abstract: Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.
authors.affiliation: BioHelix Corporation, 32 Tozer Road, Beverly, MA 01915, USA.
authors.forename: Wing Huen A
authors.initials: WH
authors.lastname: Chow
authors.forename: Cindy
authors.initials: C
authors.lastname: McCloskey
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Tong
authors.forename: Lin
authors.initials: L
authors.lastname: Hu
authors.forename: Qimin
authors.initials: Q
authors.lastname: You
authors.forename: Ciarán P
authors.initials: CP
authors.lastname: Kelly
authors.forename: Huimin
authors.initials: H
authors.lastname: Kong
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Tang
authors.forename: Wen
authors.initials: W
authors.lastname: Tang
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology / Q000633:toxicity; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification*; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004768:Enterotoxins / Q000032:analysis* / Q000276:immunology / Q000633:toxicity; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D004265:DNA Helicases