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Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification.

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Rapid diagnostic tests for Clostridioides difficile infection (CDI) are crucial. New recombinase polymerase amplification (RPA) assays can detect key toxins A and B in just 15 minutes, enabling faster treatment.

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Area of Science:

  • Molecular biology
  • Microbiology
  • Biotechnology

Background:

  • Clostridioides difficile infection (CDI) diagnosis relies on toxin detection, often with delays.
  • Rapid and direct diagnosis is essential for effective CDI treatment and preventing severe outcomes.

Purpose of the Study:

  • To develop rapid, isothermal recombinase polymerase amplification (RPA) assays for detecting Clostridioides difficile toxins A and B.
  • To adapt these assays to a 3D-printed microreactor for potential point-of-care use.

Main Methods:

  • Developed singleplex and duplex fluorescent 15-minute isothermal RPA assays targeting tcdA and tcdB genes.
  • Adapted singleplex RPA assays to a 3D-printed microreactor, optimizing primer/probe concentrations and reaction volumes.
  • Determined analytical sensitivity using DNA standards and specificity with various bacterial strains.

Main Results:

  • The duplex RPA assay achieved simultaneous detection of both tcdA and tcdB in 15 minutes.
  • Detection limits (LOD) were comparable between the duplex tube format and the 3D-printed microreactor format.
  • The 3D-printed microreactor enabled a significant reduction in reaction volume.

Conclusions:

  • The developed duplex RPA assay offers a timely and reliable method for diagnosing CDI by detecting key toxins.
  • The 3D-printed microreactor adaptation shows promise for developing a point-of-care microfluidic system for CDI diagnosis.