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 falsenegative 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. (J Mol

Detection of Severe Fever with Thrombocytopenia Syndrome Virus by Reverse Transcription–Cross-Priming Amplification Coupled with Vertical Flow Visualization

“…The end of the amplicon labeled with biotin binds streptavidinconjugated color particles for visualization. The other end, labeled with FITC, is captured by an anti-FITC antibody located on the test line of the strip, and the test results are shown as a colored line visible by the naked eye in 5 to 10 min (2,6,8,15). Importantly, the VF strip is housed in an enclosed, sealed plastic device to prevent the leakage of amplicons which may cause cross-contamination and false-positive results.…”

Erratum to: An integrated disposable device for DNA extraction and helicase dependent amplification

“…We saw the appropriate threshold value increases in our quantitative PCR, but not in our HDA. We accepted these results as real based on literature reports that stated that HDA is not always linear in this respect (Chow et al 2008;Goldmeyer et al 2007Goldmeyer et al , 2008Tong et al 2008). These explanations were not appropriate for the observed discrepancies in our data.…”

Thermophilic helicase-dependent DNA amplification using the IsoAmp™ SE experimental kit for rapid detection of Streptococcus equi subspecies equi in clinical samples

“…For instance, the analytical sensitivity of the assay for detection of C. difficile was 20 copies of genomic DNA per reaction. 7 Comparison of the tHDA test with assays currently used for identification of S. equi reveals that the specificity of tHDA is similar to that of culture and PCR when applied to nasal swabs and washes. Sensitivity and diagnostic accuracy of tHDA were 13% higher than those of culture.…”

“…27 Further simplification of the assay could be 416968X XXXXX10.1177/1040638711416968Artiu shin et alThermophilic helicase-dependent DNA amplification From the Gluck Equine Research Center, University of Kentucky, attained by using a disposable lateral flow cassette for detection of the amplicon. 12,27 Such a platform has been recently developed and commercialized a in a kit format and utilized for identification of toxigenic Clostridium difficile in stool samples, 7 Helicobacter pylori, 17 Vibrio cholera, Bacillus anthracis, 26 and Human immunodeficiency virus-1. 21 The assay takes less than 2 hr and can be performed in a minimally equipped laboratory or office setting.…”