Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2022

Development and evaluation of a novel Multiple Cross Displacement Amplification assay for detection of toxigenic Clostridioides difficile as a Point-of-care testing (82401)

Dazhi Jin 1 2 3 , Xiaomei Zhang 4 , Yun Luo 4 , Xiaojun Song 2 , Shan Lin 1 3 , Jun Yang 1 3 , Yu Chen 1 3
  1. School of Laboratory Medicine, Hangzhou Medical College, Hangzhou, Zhejiang, China
  2. Centre of Laboratory Medicine, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China
  3. Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang province, Hangzhou, Zhejiang, China
  4. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia

Clostridioides difficile is recognized as the leading cause of antibiotic associated diarrhea worldwide. A definitive diagnosis relies on the rapid and easy-to-use detection of C. difficile tcdB production, which allows for prompt therapy and effective isolation to prevent further nosocomial spread of infection. Herein, we developed and evaluated a novel Multiple Cross Displacement Amplification (MCDA) assay targeting C. difficile tcdB gene for detection of toxigenic C. difficile using SYBR Green fluorescent dye conducted in a portable instrument.

The MCDA primers sets were designed and the reaction temperature were optimized. The limit of detection of MCDA assay was performed on serially diluted genomic DNA template. The fastest detection time was compared to real-time PCR. The sensitivity and specificity of MCDA were evaluated on 60 consecutively collected stool specimens from patients with suspected C. difficile infection. Each stool specimen was tested in parallel by the MCDA and real-time PCR assays, both of which were compared with toxigenic culturing, the “golden standard”.

The MCDA assay successfully amplified tcdB gene with no cross-reactions with non-toxigenic C. difficile and other diarrhea-associated pathogens at the reaction temperature of 63℃. The MCDA assay possessed an analytical sensitivity of 5.0 copies per reaction of genomic DNA. The MCDA assay was faster than the real-time PCR assay by over 10 min with fast detection time of 5.7min at 5.0 ng concentration. The MCDA average detection time of producing the test results at the concentration of genomic DNA 5.0 copies was 21.35±2.2 min, approximately half of detection time (40.7±1.5 min) cost by the real time PCR. Compared with the specificity (92.5%) and sensitivity (100%) of real-time PCR, the MCDA presented a higher specificity of 98.1%, but a lower sensitivity of 85.7% when validated on clinical specimens (Fisher’s exact test, P > 0.05).

This study demonstrated that the MCDA assay provided a rapid, portable, easy-to-use diagnostic point-of-care tool with promising performance for assessment of C. difficile infection in clinical stool specimens.

  1. 1. Bartlett JG, Chang TW, Gurwith M, et al. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978 Mar 9;298(10):531-4. 2. Ryder AB, Huang Y, Li H, et al. Assessment of Clostridium difficile infections by quantitative detection of tcdB toxin by use of a real-time cell analysis system. J Clin Microbiol. 2010 Nov;48(11):4129-4134. 3. Wang Y, Wang Y, Ma A-J, Li D, Luo L, Liu D, Jin D, Liu K, Ye C-Y. Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by multiple cross displacement amplification. Sci Rep. 2015 Feb; 5, 11902.