On-chip two-step nucleic acid amplification for point-of-care and clinical molecular detection of multiple diseases and co-infections

Polymerase chain reaction methods (PCR) for nucleic acid amplification have revolutionized molecular diagnostics and genetics research. Nested PCR relies on two successive runs of thermal cycling and two sets of primers, reducing non-specific amplification that occurs with traditional PCR.  However, this method is not optimal for point-of-care applications because of its complexity and the risk of contamination that can occur upon manual transfer of the products from the first amplification round to the second round. Furthermore, thermal cycling requires specialized automated equipment that is not practical for resource-poor regions.

The Bau Lab has devised a two-step nucleic acid amplification method, termed Rapid Amplification (RAMP). The first amplification step uses recombinase polymerase amplification (RPA), and the second step relies on loop-mediated isothermal amplification (LAMP), amplifying secondary targets within the products of the first round of amplification. 

Both amplification and detection are carried out under isothermal conditions, simplifying or eliminating instrumentation.  Moreover, the researchers have designed and tested a microfluidic chip device by which RAMP and other nucleic acid amplification methods, including nested PCR and isoPCR, may be carried out. The device is comprised of multiple hydraulically connected reaction chambers; the products of the first amplification stage transfer to the second amplification reactor by diffusion.

• High sensitivity
• Rapid assay
• Detect multiple targets simultaneously without loss of specificity
• Simplify instrumentation
• Lower risk of sample contamination with entire process in closed system
• Method compatible with on-chip processing of designed microfluidic device
• Sixteen-plex assay demonstrated (higher level of multiplexing is possible)

Diagnostic testing in clinical and point-of-care settings