Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform

Abstract

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 mL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Recombinase polymerase amplification: Basics, applications and recent advances

“…The chip consists of plates clamped together and contains 3 lanes used to place sample, RPA master mix and magnesium acetate, separately. Once each lane is loaded, the plates can slip in order to mix all the components, and amplification is followed using a real time machine [96]. The use of a programmable digital microfluidic platform based on an active matrix electrowetting-on-dielectric (AM-EWOD) for real time detection has also been described.…”

Recombinase Polymerase Amplification for Diagnostic Applications

“…RPA was successfully used to detect major human pathogens including bacteria (6,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), viruses (9, 24 -32 ), fungi (33 ), and parasites (34,35 ), as well as genetically modified organisms (36,37 ) and genetic alterations observed in cancer cells (38,39 ). RPA was also used for HIV diagnosis in low-resource settings (40,41 ) (Tables 2 and 3).…”

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform

“…Most standard platforms for miniaturized nucleic acid extraction and amplification utilize (plastic) chips with compartments or channels through which reagents are pumped using a series of electrically controlled pumps and valves. 31,32 Digital microfluidic micro-devices provide an alternative platform for miniaturised, fully automated assays, providing the basis of simple, programmable and portable test systems. 8,9,[28][29][30] DNA amplification has also been carried out using the Slip Chip platform.…”