The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step

Arunkumar Arumugam; Season S Wong

We first tested the feasibility using a very small amount of sample in RT-qPCR reactions by using aerosol generating vials to spray the samples over the uncapped PCR tubes. The material in vials containing influenza A (InfA), influenza B (InfB), or RSV clinical specimens (swabs in VTM) were sprayed into PCR tubes containing primer and probe sets targeting InfA, InfB, RSV and RNaseP (RP) in master mix prior to capping the tubes and performing RT-qPCR. Fig. 1 shows that there were positive PCR signals in the respective tubes that did not have any non-specific amplification. Though the unprocessed, sprayed samples have higher Ct values (36.5, 36.2 and 31 for InfA, InfB, and RSV, respectively) than the corresponding extracted RNA template samples (30.4, 32.6, 29.6 for InfA, InfB, and RSV). It is important to note that we were able to detect low viral load samples (Ct >30) when less than 1 µL of sample entered the tubes as measured by an analytical balance.