Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification

Baek, Changyoon; Li, Yun Guang; Yoo, Hyun Jin; Cui, Wen Ying; Kang, Jia; Son, Jun‐Hyeok; Min, Junhong

doi:10.1007/s00216-021-03557-4

Anal Bioanal Chem

2021

DOI: 10.1007/s00216-021-03557-4

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Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification

Changyoon Baek

Yun Guang Li

Hyun Jin Yoo

et al.

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2023

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Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

Yin,

Song,

Wang

et al. 2023

FEMS Microbiology Letters

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In the surveillance of outbreaks of Clostridioides difficile infection, the rapid detection and diagnosis of C. difficile remain a major challenge. Polymerase spiral reaction (PSR) is a nucleic acid amplification technique that uses mixed primers and the strand displacement activity of Bst DNA polymerase to achieve a pair of primers and a single enzyme in an isothermal environment. The primer design is simple, the reaction is efficient, and a color indicator can be used to visualize the result. In this study, we developed a rapid and visually interpretable PSR to detect C. difficile by analyzing artificially contaminated feces samples and clinical isolates from patient feces samples. We designed two pairs of primers for a PSR that specifically targeted the conserved tcdB gene of C. difficile. The amplification results were visualized with the chromogenic dye hydroxynaphthol blue. The entire process was accomplished in 50 min at 64°C, with high specificity. The limit of detection of C. difficile with PSR was 150 fg/μL genomic DNA or 2 × 10 CFU/mL in artificially contaminated feces samples. With this method, we analyzed four clinical isolates and also compared the PSR with an isolation-and-culture detection method, PCR, and the Sanger sequencing. The four clinical isolates were found positive for tcdB, which confirmed the high specificity of the primers. The positive rates of tcdB in toxigenic C. difficile detected with PSR, PCR, and Sanger sequencing were 100%. The proportions of toxin types in these clinical C. difficile strains were 50% tcdA+tcdB+CDT− and 50% tcdA+tcdB+CDT+. The assay described should extend our understanding of the incidence of C. difficile. This may allow the rapid diagnosis and screening of C. difficile-related disease outbreaks in the field.

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Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

Yin,

Song,

Wang

et al. 2023

FEMS Microbiology Letters

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show abstract

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Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification

Cited by 1 publication

References 19 publications

Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures

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