2.1. Ethical Clearance
Swabs samples of patients were collected from routine COVID-19 testing purpose at Kangra district, Himachal Pradesh, India. The Institute Ethics Committee of CSIR-Institute of Himalayan Bioresource and Technology, Palampur, India, approved the collection of human swab samples. All the experiments were done according to the Ethical Guidelines for Biomedical Research on Human Subjects, Indian Council of Medical Research and Government of India.
2.2. Chemicals used for this study
Tween-20 and Triton X-100 were purchased from Hi Media, India. EDTA disodium sulphate, Tris-B, Acrylamide, Ammonium per-sulphate and TEMED were purchased from Hi Media, India and Sigma Aldrich, USA, respectively. Nuclease free water was purchased from Promega. RNA isolation and qPCR testing kits (LabSystem, COVID-Sure) were procured form Post Graduate Institute of Medical education and Research, Chandigarh, India. Carrier RNA [Poly (A)] was purchased from Qiagen, Germany.
2.3. Preparation of Carrier Molecule [Linear Polyacrylamide (LPA)]
Linear polyacrylamide solution was prepared according to the protocol of Gaillard and Strauss, 1990 [10]. Briefly, 0.25 g acrylamide dissolved in 5 ml of 40 mM Tris-cl (pH8), 1 mM EDTA and 20mM Sodium acetate (pH 7.5) followed by addition of APA (0.1% final conc.) and 5 µl of TEMED and allowed solution to polymerise for 30 min at RT. Viscous solution was further precipitated with 2.5 volume of absolute ethanol. A large precipitated pellet was obtained after centrifugation, followed by washing with 70% ethanol and dry for 10 min at RT. The pellet was dissolved in 50 ml of Tris-EDTA buffer pH 8. The final concentration was now to be 5mg/ml.
2.4. Preparation of glycogen and PolyA carrier molecules
Ten microgram/microlite of glycogen was purchased from Promega. Poly (A) carrier RNA (Qiagen, Germany) was dissolved in nuclease free water to make final concentration 5 µg/ml.
2.5. Preparation of lysis buffer, Swabs/lysis (V/V), and detergent ratio in lysis solution
Lysis buffers were prepared using 5mM Tris-Cl (pH 7.2) and 1mM EDTA (pH 8.0) and supplemented with different concentration of detergents in preheated nuclease free water (about 450C) and vortex gently for a moment. For initial optimization, in determining the appropriate volume of VTM swabs in lysis solutions, the lysis solution was augmented with 0.1% triton x-100 and 0.1% tween-20, and various samples/Lysis volume ratios (100:100, 100:300, 100:400, 200:100,200:400, 400:100, 400:200 in µl) were taken and incubated at room temperature for 15 minutes. For optimizing the concentration of detergents in lysis solution, the lysis solutions were supplemented with different final concentration of detergents in tritonx-100/tween-20 ratios (0.1:0.1, 0.1:0.2, 0.1:0.4, 0.2:0.1, 0.2:0.3, 0.3:0.2; 0.4:0.1 in % ), and incubated for 15 min at room temperature. To assess the effect of carrier molecules on realtime-PCR efficiency, the lysis buffers were supplemented with final concentrations of glycogen (50, 100, 150, 200) µg, LPA molecules (10, 15, 20, 25, 30, 40, 50) µg and Poly (A) carrier (5, 10, 15, 20) µg/ml concentration were used as indicated in individual experiments.
2.6. Reverse transcriptase-real time-Polymerase chain reaction (RT-qPCR)
Reactions were carried out using a commercial COVID Sure kit (Lab system diagnostics, Trivitorn Healthcare), which contain both reverse transcription and DNA-polymerase enzyme activities. All reactions were carried out on 96-well plates in a CFX-96 Real-Time System (BioRad, USA) and each plate included samples RNA (positive controls), and a non-template (negative control). Each 15 µl reaction consisted of 10 µl of 2X hydrolysis mixes, 2.0 µl of primer probe mix [ORF-1ab-5′-FAM, E- HEX, and Internal Control (RNase P)-ROX] and 3 µL of water as needed, 6.5 µl of lysate was added in the final reaction mix. The cycling profile PCR was performed on an automated system that involved reverse transcription at 46 oC for 15 min, initial activation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 sec and 58°C for 30 sec. Fluorescence was measured at the combined annealing–extension step and results were interpreted using Bio-Rad CFX Maestro software (supplied with thermocycler). Cases were dichotomized as either positive or negative for SARS-CoV-2, based on Ct values and RFU curves of confirmatory gene, ORF-1ab.
2.7. Sample characterisation
RNA of VTM swabs were isolated using commercial RNA isolation kit according to the manufacturer’s protocol and reverse-transcriptase real time PCR was performed. Positive individuals were classified based on Ct values of SARS-CoV-2 gene obtained from routine RT-qPCR testing. Furthermore, group 1 defined with Ct value ≥ 25 and group 2 defined with ≤ 25–30 were characterised and experiments were performed to check the sensitivity and specificity of the methods design.
2.8. Estimation of sensitivity and specificity of the reaction:
The appropriateness of diagnostic methods is determined through measuring the sensitivity and specificity of testing procedures for the diagnosis of the disease. The sensitivity and specificity were calculated through ignoring calculation defined by Genders et al., 2012 [11, 12]. Briefly, test positivity (test+) and test negativity (test-) are defined based on the Ct values of routine validated PCR protocol for diagnostic of SARS-CoV-2. A positive or negative test result might be based either on one observation randomly picked from each patient or, it could be based on a summary measure of all the observations. When it comes to COVID-19 disease, a patient's test result is normally classified as true-positive, if the validated qPCR showed a substantial Ct value and RFU curve for a patient sample. If the validated qPCR showed no significant Ct value or RFU curve at all, but the method used showed significant values, the patient's test result is frequently counted as false-positive. If the validated qPCR indicated any significant Ct value or RFU curve at all, but the method used yielded no significant results, the patient's test result is usually considered false-negative. If the number of patients with a true-positive, true negative, false- negative and false-positive test and the result are respectively true positive, true negative, false negative and false positive, then the calculation for sensitivity and specificity is done as follows:
2.9. Statistical analysis
The results were statistically analysed using the GraphPad PRISM software. The student's t-test was used to determine statistical significance (P-value). P-values of less than 0.05 were considered statistically significant. *p value 0.05, **p value 0.01 and ***p value 0.001 were deemed statistically significant. DeLong methodology was used for Receiver Operator Characteristic (ROC) curve analysis using MedCalc software [13]. ROC curve was used to measure the AUC of diagnostic test with the following equation;
Where, ROC (t) represents sensitivity and t represents, 1-specificity. The area under ROC determined the accuracy of the test, larger area represent high accurate diagnostic test.