Development of Diagnostic Real-Time RT-PCR Assays for SARS-CoV-2 Using Polyvinyl Alcohol (PVA) Sponge as a Saliva Collection Tool

Given the pandemic of COVID-19, the diagnostic challenges using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) assays for SARS-CoV-2 are mainly concentrated in the operation, which is to timely process multiple samples and to judge false-positive/false-negative accurately. Here we suggest a unique real-time RT-PCR protocol using a polyvinyl alcohol (PVA) sponge for high-throughput screening against COVID-19. By collecting saliva using a PVA sponge, we have solved operational issues allowing for skipping of conventional sample preparation and purification procedures. While standard sample pooling methods can be time consuming, costly, and the expected positive rate of the sample can be low, our method allows for simplification of the procedures for pool testing. Moreover, the decrease in sensitivity due to dilution when the solution is added can be prevented when testing samples using pieces of dried PVA sponges. Ribosomal protein L-13A (RPL13A), a human-derived housekeeping gene of internal control, are included in the primers and probes of the assay kit for simultaneous detection. Therefore, the accuracy for avoiding false-positive or false-negative RT-qPCR assay results can be improved. In summary, we have developed a novel RT-qPCR testing procedure by uniquely using PVA sponge without purification, which can be utilized for the detection of SARS-CoV-2. Graphical Index O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/25340856v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@6a4209org.highwire.dtl.DTLVardef@1575b4org.highwire.dtl.DTLVardef@e284aborg.highwire.dtl.DTLVardef@1743073_HPS_FORMAT_FIGEXP M_FIG C_FIG