Sensitive extraction-free SARS-CoV-2 RNA virus detection using a chelating resin

Abstract

Current conventional detection of SARS-CoV-2 involves collection of a patient's sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified preparation method using a chelating resin, Chelex, which obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital droplet PCR were compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high-throughput platforms.

Keywords: Biological sciences; Biological sciences research methodologies; Molecular biology experimental approach; Virology.

Graphical abstract

Figure 1

RT-ddPCR assays for simulated dry and wet swab using RNA-extraction-free methods for SARS-CoV-2 detection (A) 200,000 SARS-CoV-2 virions and 20,000 293FT cells were dried at room temperature in a SpeedVac and then resuspended in a 200 μL lowTE buffer to mimic the dry swab. The same amount of virions and 293FT cells directly added to the 200 μL lowTE buffer was used to mimic the wet swab. The samples expected to have 1,000 virion genome copies/μl were then used directly for RT-ddPCR (no heat), heated at 98°C for 5 min (heat), or heated with 5% Chelex (Heat_Chelex). The RT-ddPCR reactions were carried out in one well for N1 and cRPP30 and another well for N2 and RPP30. (B) Negative controls and virion samples were prepared as wet swabs. The mean genome copies/μl of N1 and N2 were less than 1.2 in negative controls without virions added. N1 and N2 target SARS-CoV-2. cRPP30 is specific for RPP30 cDNA, and RPP30 targets both genomic DNA and cDNA. Copies/μl refers to concentration in the samples used for RT-ddPCR. The error bars represent Poisson 95% confidence intervals. Dashed line indicates the threshold for the low detection limit of 1.8 copies/μl of SARS-CoV-2 virions. (C) Virions of 1,000 genome copies/μl and 100 293FT cells/μl were prepared in lowTE, H2O, MEM alpha, or TED10, treated and assayed by RT-ddPCR as in (A). (D) Virions of 1,000 genome copies/μl and 100 293FT cells/μl were prepared in HBSS with or without Ca²⁺ & Mg²⁺ supplemented with 2% FBS, M4, or PBS, treated and assayed by RT-ddPCR in for N1, N2, cRPP30, and chr5UC, a genomic DNA region on chromosome 5.

Figure 2

The limit of detection of SARS-CoV-2 using RT-qPCR or -ddPCR (A) RT-qPCR comparing Chelex-RNA and conventional RNA extraction. RNA refers to RNA prepared by simulating conventional method with RNA extraction: a swab with 200,000 to 200 genome copies of SARS-CoV-2 virions was added to 3 mL of VTM, of which 200 μL were used for RNA extraction, and RNA was eluted in 50 μL H2O. LowTE refers to simulating a swab with 200,000 to 200 genome copies of virions eluted in 200 μL lowTE and then heated in the presence of 5% Chelex. (B) RT-qPCR comparing Chelex and proteinase K methods for saliva samples. One thousand to 1 genome copies/μl of SARS-CoV-2 virions were prepared in saliva samples and subjected to the Chelex or proteinase K methods and RT-qPCR. The NEB Luna RT-qPCR kit and NEB-Luna-Program II were used with 2.5 μL samples in 10-μL reaction volumes. Samples with undetermined Ct values were plotted as Ct 40. (C) The limit of detection of SARS-CoV-2 using RT-ddPCR. Samples from (A) were used for RT-ddPCR. The sample of 5 μL was used for RT-ddPCR in 20-μL reaction volume. The mean genome copies/μl of N1 and N2 were less than 1.2 in negative controls without virions added. N1 and N2 target SARS-CoV-2. cRPP30 is specific for RPP30 cDNA, and RPP30 targets both genomic DNA and cDNA. Copies/μl refers to concentration in the samples used for RT-ddPCR. The error bars represent Poisson 95% confidence intervals. Dashed line indicates the threshold for the low detection limit of 1.8 copies/μl of SARS-CoV-2 virions. NTC, no-template control.

Figure 3

SARS-CoV-2 detection of patient samples prepared by the Chelex method (A) Patient NP swab samples were heated in the presence of 5% Chelex (S01–S16, and S19, S20) or 10% Chelex (S17 and S18). S19 and S20 are of 1:2 dilutions of S17 & S18 in LowTE, respectively. (B) 50 μL of patient saliva samples (Saliva 01 and 02) or negative patient saliva samples spiked with positive patient saliva samples (Salia 03–22) was mixed with 25 μL of 50% Chelex in TED99 and heated for 5 min in a ThermoMixer. (C) Paired NP swabs from seven patients (P1 to P7) and saliva-saturated swabs from four patients (P4 to P7) were collected in VTM or Chelex collection tubes. VTM samples were used for RNA extraction (EasyMag). Luna refers to the NEB Luna RT-qPCR kit and NEB-Luna-Program II with 2.5 μL samples in a 10-μL reaction volume. CL refers to a CDC assay performed in the clinical laboratory with 5-μL samples in a 20-μL reaction volume. Undetermined Ct values were plotted as Ct 40.

Figure 4

Viral and cellular RNA stability in lowTE by RT-ddPCR assays (A) Virions of 1,000 virion genome copies/μl and 100 cells/μl 293FT cells were prepared in lowTE, TED10, or MEM α and stored at room temperature. Samples were heated with 5% Chelex on the time points indicated and assayed. The RT-ddPCR reactions were carried out in one well for N1 and cRPP30 and another well for N2 and RPP30. (B) Virions of 1,000 virion genome copies/μl and 100 cells/μl 293FT cells were prepared in the buffers, heated with 5% Chelex on day 0, and assayed on a time series. Copies/μl refers to concentration in the samples used for RT-ddPCR. The error bars represent Poisson 95% confidence intervals.

Figure 5

Viral RT stability in saliva by RT-ddPCR assays (A) Virions were added to saliva samples at 1,000 virion genome copies/μl and stored at room temperature. Samples were heated with 1/5 volumes of 50% Chelex prepared in H2O or TED99 on the time points indicated and assayed. The RT-ddPCR reactions were carried out in one well for N1 and cRPP30 and another well for N2 and RPP30. (B) Virions of 1,000 virion genome copies/μl were prepared in saliva as in (A), heated with Chelex on day 0 or day 3, and assayed on the days indicated. cRPP30 data points were not plotted because of the low level detected. Copies/μl refers to concentration in the samples used for RT-ddPCR. The error bars represent Poisson 95% confidence intervals.