A highly sensitive, real-time centrifugal microfluidic chip for multiplexed detection based on isothermal amplification.

A real-time centrifugal microfluidic chip with a companion analyzer was developed for highly sensitive, multiplexed nucleic acid detection based on RPA (recombinase polymerase amplification) isothermal amplification. In order to improve the detection sensitivity, two different optimization strategies were systematically studied. Witnessing the high viscosity of RPA reagent, one way was to improve the amplification efficiency by intentionally introducing active mixing based on centrifugal actuation. While the other way was to improve the detection sensitivity by utilizing two-stage amplification. The templates were pre-amplified in the first-stage amplification chamber before they were aliquoted and distributed into a couple of second-stage ones for multiplexed detection. Different mixing methods relative to different actuation time were studied and compared. Similarly, different two-stage amplification modes relative to different time protocols were compared as well. Totally four different amplification modes including with or without mixing, and with or without two-stage amplification, were systematically analyzed and compared. It was found that, the detection sensitivity could be significantly improved by the two-stage amplification with active mixing. Furthermore, as a proof of concept, the performance of the developed microfluidic chip was demonstrated by successfully detecting different genes of African swine fever virus (ASFV) in parallel with high sensitivity.