Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification

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.