Fig 1.
Overview of the simplified SARS-Cov-2 test protocol.
The RNA is isolated from biological samples (NP, OP, NV or saliva) with a simplified TRIzol RNA isolation protocol. The RT-qPCR reactions utilize the CDC nCoV-N1, nCoV-N2 and RP primer/probe sets. Samples showing amplification for primer/probes nCoV-N1 and nCoV-N2 with a Ct of under 35 cycles are considered positive for SARS-CoV-2. Genomic viral RNA (gRNA) or an inactivated viral preparation can be added to the biological samples from healthy donors to obtain contrived samples.
Fig 2.
SARS-CoV-2 and RNAse P primer/probes RT-qPCR standard curves.
Primers-probes sets A) nCoV-N1 and B) nCoV-N2 standard curves were generated by amplifying 3-fold serial dilutions of purified viral genomic RNA (BEI resources, NR52285). 6 copies of viral genomic RNA were detected with a Ct lower than 34 cycles with both primer/probe sets. C) A titration curve for the endogenous housekeeping gene RNase P probe set (RP) was obtained utilizing plasmid DNA coding for the RNase P gene.
Fig 3.
SARS-CoV2 different RNA isolation methods.
10,000 copies of viral genomic RNA (gRNA) were added to a serial dilution of HeLa cells (from 24,300 to 900). Total RNA was isolated with TRIzol, the RNAqueous kit and the hypotonic freeze method. 1 μL of each RNA preparation was amplified utilizing the A) nCoV-N1, B) nCoV-N2 and C) RP primer sets. Following isolation, the RNA was resuspended in 25 μL of ddH2O. Isolation of the RNA with 100% efficiency will yield 400 copies / μL of gRNA. 400 copies of purified gRNA were directly amplified as a control for RNA isolation efficiency.
Fig 4.
Detection of SARS-CoV-2 in contrived samples from the upper respiratory tract.
NP, OP, NV and saliva samples from 7 healthy donors were spiked-in with a preparation of inactivated SARS-CoV-2 virus (isolate USA-WA1/2020) containing 10,000 viral genome copies. RNA extracted with the simplified TRIzol method was amplified with the nCoV-N1, nCoV-N2 and RP primer/probe sets. 400 copies of purified gRNA were directly amplified as a control for RNA isolation efficiency.
Fig 5.
SARS-CoV2 gRNA degradation in saliva samples.
10,000 copies of synthetic SARS-CoV-2 RNA (NR52286), purified SARS-CoV-2 RNA (NR52358, (NR52285, NR52347), inactivated viral preparations (NR52350, NR52287), and a control SARS-CoV-1 genomic RNA (NR52346) were added before (pre-) and after (post-) addition of TRIzol to a saliva sample or directly to the TRIzol sample without saliva (RNA). RNA was isolated with the simplified TRIzol protocol and amplified with the A) nCoV-N1 and B) nCoV-N2 primer sets.
Fig 6.
Stability of SARS-CoV2 and cellular RNA in transport media and TRIzol.
A time course was set up with samples containing 24,000 HeLa cells and a preparation of inactivated SARS-CoV-2 virus (isolate USA-WA1/2020) containing 10,000 viral genome copies. Samples were preserved in either A) TRIzol or B) viral transport media (VTM) at 4°C for up to 7 days. RNA was isolated with the simplified TRIzol protocol and amplified with the nCoV-N1 and RP primer/probe sets.