Clinical samples and sample lysis
A selection of upper respiratory tract specimens (Nasopharyngeal/Throat swab) sent to the Defence Research & Development Establishment, Gwalior Madhya Pradesh India through different medical health authorities for laboratory diagnosis of SARS-CoV-2 were used for the study. A total of 100 residual anonymized and de-identified swab samples including 40 SARS-CoV-2 positive and 60 negative by RT-qPCR were included in the study. The following steps were performed in a biosafety level 3 (BSL-3) laboratory according to standard microbiological and diagnostic practices. The experimental protocols of this study were approved by Defence Research and Development Establishment- Institutional Biosafety Committee, (DRDE-IBSC) vide no IBSC/VIRO-01/2021/PKD. Ethical approval and informed consent waiver for the study has been granted vide no. VCH/VEC/June-2021/04 of Vidya Ethics Committee, Gwalior, India. All methods were performed in accordance with the relevant national biosafety guidelines and regulations.
Magnetic bead particles were prepared using modified Stober’s process 14. Briefly, five hundred milligram of Fe2O3 nanoparticles (Aldrich, USA, Cat. No. 544884) were sonicated (30 min) in a solution having 320 ml of ethanol and 80 ml of water followed by addition of 10 ml of 30% NH4OH solution. To this solution was added 1 gm of tetraethyl orthosilicate (Aldrich, USA, Cat No. 86578) in 45 min and the contents were allowed to stir at room temperature for additional 24 h. Thus formed silica coated nanoparticles were then separated under magnetic field, washed repeatedly with ethanol and finally dried under vacuum (70oC, 24 h). The average size of particles was found 2.3 µm and BET surface area was 17 m2/g. Finally these particles were used as suspension of 50 mg of particles/ml nuclease free water.
Three buffer solutions were employed for extraction of viral RNA viz, lysis/binding buffer (3.96% Ammonium sulphate, 0.8% NP-40 in 0.2M Tris acetate, pH – 4), washing buffer (0.5% NP-40 in 0.01 M Tris-HCl, (pH - 6.8)) and elution buffer (10 mM Tris-HCl, (pH- 8.5)).
Extraction of viral RNA emolying silica coated magnetic bead Thirty micro liter of magnetic bead suspension were added to lysis buffer (760 µl) and sample (nasal/throat swab (140 µl), further the tubes were incubated at room temperature for 8 min. Subsequently, proteinase K (25µl) was added, to the tubes and further incubated for additional 2 min. The magnetic bead particles were separated and supernatant was discarded. Subsequently, washing buffer (500 µl) containing proteinase K solution (2.5 ml) (20mg/ml) was added, and the beads were mixed vigorously. Further the mix was incubated for 10 min with washing buffer, the magnetic beads particles were separated by applying magnetic field and the remaining supernatant was pipetted out. The washing step was repeated again following removal of washing buffer and elution buffer (50 µl) was applied to the bead and mixed for uniform suspension. This was further kept at room temperature for 10 min. Finally the beads were separated and supernatant was collected in RNase-free tube by applying magnetic field. The purity and concentration of eluted RNA was carried out in Qubit 4.0 Fluor meter (Thermo, USA). The suitability of extracted viral RNA for molecular assay was confirmed through SARS-CoV-2 specific TaqMan RT-qPCR21. The eluted RNA was stored at -80ºC.
Extraction of viral RNA employing Qiagen kit
The extraction of viral RNA was carried out from infected material using the Qiagen viral RNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. In brief, AVL buffer (560 µl) was added to nasopharyngeal/throat swab sample (140 µl ) and thoroughly vortexed (15 sec), then the mixture was further kept at room temperature (10 min). Subsequently, ethanol (560 µl) was applied to this mixture and the mixture was passed through QIAamp spin column, by centrifugation (6000rpm, 1 min), the spin column residuals were discarded. . Spin column were further sequentially washed using wash buffer AW1(500 µl) and AW2 (500 µl). Finally viral RNA was extracted in elution buffer (50 µl) added to the centre of the column followed by centrifugation at 8000 rpm for 2 min. The purity and concentration of viral RNA was analyzed with Qubit 4.0 Fluormeter (Thermo, USA). The suitability of extracted viral RNA for molecular assay was confirmed through SARS-CoV-2 specific TaqMan RT-qPCR2. Finally the eluted RNA (50 µl) was stored at –80 0C.
Extraction of viral RNA employing commercial magnetic beads extraction kit
Viral RNA from clinical samples was extracted using MGI Kit, Wuhan, China using manufacture’s protocol. Briefly 200µl of sample was added to 481µl lysis buffer (containing 200 µl MLB, 250µl 100% ethanol, 15µl Proteinase K, 15µl Magnetic Beads and 1µl Enhancer buffer). It was mixed well and centrifuged at 1050 RPM for 2 min. The lysate was then allowed to incubate at 65⁰C for 5 minutes. Later it was mixed well and placed on magnetic stand for 1 minutes to achieve a magnetic bead pellet and the supernatant was discarded. Subsequently, 500µl of MW1 buffer followed by 500µl MW2 and 600µl of Absolute ethanol with the similar magnetic bead pelleting procedure as mentioned above was applied and supernatant was discarded. After this, ethanol was dried by subjecting the tube at 56⁰C for 1 minute. At the end, the viral RNA was eluted in 60µl of RNase free water at 65⁰C for 10 min and further stored at -80⁰C until use.
SARS-CoV-2 RNA detection by RT-qPCR
RT-qPCR detection of the SARS-CoV-2 was carried out using both by both screening and confirmatory assay by targeting (E/RNaseP) and (RdRP/ORF1ab gene) respectively 2. For the mastermix, 0.5 µl of Primer/Probe mix SARS and Wuhan CoV E-gene (Eurofins, India) and 0.5 µl of Primer/Probe mix Human RNaseP gene (Eurofins, India) or 0.5 µl of Primer/Probe mix SARS and Wuhan CoV RdRP-gene (Eurofins, India) and 0.5 µl of Primer/Probe mix Human ORF1ab gene (Eurofins, India) was mixed with 5.5 µl RNase-free water, 12.5 µl Invitrogen master mix (Thermo, USA), and 0.5 µl reverse transcriptase enzyme (Thermo, USA) per sample. RT-qPCR was performed in ABI 7500Dx Instrument (Thermo, USA) with 30 min of reverse transcription at 56 °C, initial denaturation at 95 °C for 5 min, and subsequent 45 amplification cycles with 95 °C for 5 sec, 60 °C for 30 sec. Cycle threshold (CT) was determined, where the fluorescence signal of the amplification reaction was above the background fluorescence using the ABI software (Thermo). Data analysis on raw CTs was performed in Excel and Correlation between three methods were calculated.
Estimation of the detection limit of magnetic bead RNA extraction using RNaseP
Five µl in vitro transcribed RNA targeting RNaseP gene (6.1 × 1010 copies) was ten fold serially diluted and used in RT-qPCR reaction. 5µl RNA template was added covering a range from 101 to 106 RNA molecules per reaction. RT-qPCR was performed for human RNaseP gene using similar primer/probe as described earlier for all the three methods i.e. in-house magnetic bead, Qiagen, commercial magnetic bead. RT-qPCR cycling program for RNaseP gene amplification was performed as described in earlier in method section.
Evaluation with clinical samples
One SARS-CoV-2 positive Naso-pharyngeal swab sample, was 10 fold serially diluted in RNase-free water prior to RNA isolation a 10-fold dilution series up to 105 fold, in order to compare the magnetic bead RNA extraction protocol with commercial Qiagen and magnetic bead extraction method. The extracted RNA from all three protocols were subjected to RT-qPCR using RNaseP gene primers that act as QC of sample. A panel of 40 SARS-CoV-2 positive and 60 SARS-CoV-2 negative were used for comparative evaluation of all three viral RNA extraction protocols in conjunction to RT-qPCR.