Fig. 4: Study of the inhibitory effect on reverse transcription recombinase polymerase amplification (RT-RPA) by fresh swab sample. | Communications Engineering

Fig. 4: Study of the inhibitory effect on reverse transcription recombinase polymerase amplification (RT-RPA) by fresh swab sample.

From: A gamepad-like nucleic acid testing device for rapid detection of SARS-CoV-2 via visible nested recombinase polymerase amplification

Fig. 4

a The experimental workflow (top) and the LFA result indicated the inhibitory effect of the swab sample on RT-RPA reaction (down). The “swab” indicated that pharyngeal swab sample was added into RPA reaction system. b Fluorescent RPA assay conducted with various combinations of DNA, RNA, and swab wash solutions. The results demonstrate effective RPA amplification in reactions containing DNA, even in the presence of swabs (labeled as PC/DNA/swab). However, the addition of swab wash solution inhibits amplification in reactions containing RNA (labeled as PC/RNA/swab). This suggests that the inhibitory effect may occur either during the RT reaction step or as a result of RNA degradation. The PC group contained 50 copies of nucleic acid templates, while the NC group did not contain contained samples any nucleic acid templates. c The inhibitory effect of fresh swab sample on inhibition RT-RPA using 2-F RNA (2’-Fluoro modificated RNA) template which can serve as normal RT template but is also stable against RNase. The lack of inhibitory effect using 2F-RNA as the RT-RPA template suggests the fresh swab sample did not affect the RT reaction. d The degradation of RNA template in different buffers analyzed by RT-qPCR. The Ct value increased dramatically only in the containing swab sample and RPA reagent (labeled as RNA-swab-RPA), indicating that the RNase activity can be strongly enhanced in RPA reaction buffer. Data are presented in term of the mean ± standard deviation with n = 3. e Inhibiting the RNase activity in swab samples by RNase Inhibitor can restore the RT-RPA amplification, studied by fluorescence (left) and LFA (right) readout assay. In both experiments, 50 copies of pseudovirus containing ORF1ab gene were utilized as the template and the “swab-RI” indicated the RNase inhibitor was added with swab sample in RPA reaction system. Besides, for LFA experiment, pseudovirus containing the Zs-green gene were spiked in as positive control to characterize the validity of the detection results.

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