Comparative Analysis of the Effect of Different Nucleic Acid Extraction Methods on SARS-CoV-2 Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Results

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus diseases-2019 (COVID-19) disease. Rapid and accurate detection of the virus is vital to prevent transmission and effectively manage the pandemic. The gold standard diagnostic method for this agent is the real-time reverse transcription polymerase chain reaction (qrRT-PCR) test conducted on respiratory tract samples and one of the most critical steps affecting the sensitivity of this test is the nucleic acid extraction stage. However, restrictive factors such as reagent supply and storage conditions limit the testing capacity. Therefore, innovative and cost-effective alternatives are needed to speed up testing and minimize pre-processing steps. The aim of this study was to evaluate the impact and applicability of different methods to enhance the efficiency of the nucleic acid extraction stage in the SARS-CoV-2 qrRT-PCR test. As an alternative to the routinely used viral nucleic acid extraction buffer (vNAT), the modified vNAT method (MvNAT), which includes centrifugation, the R1-R2 kit and the heat treatment (HT) method, was applied to 118 respiratory tract samples. Samples determined with threshold cycle values of (Cq) of ≤ 35 (n= 10), > 35 (n= 42), indeterminate (n= 56) in routine results and negative controls (n= 10) were included in the study. The RNA quantities obtained after extraction for each method were measured and recorded using a spectrophotometric measurement device. All samples were processed using the SARS-CoV-2 qrRT-PCR kit targeting the RdRp region. The results were statistically analyzed using unpaired and paired t-tests and results with a p-value of < 0.05 were considered statistically significant. Excluding negative control samples, while the standard method yielded a Cq value of 48.1% (mean Cq value (Cqmean)= 39.5 ± 6.9) for a total of 108 samples, the MvNAT method produced a Cq value of 11.1% (Cqmean= 38.4 ± 5.2), the R1-R2 kit yielded 14.8% (Cqmean= 35.9 ± 7.1) and HT method resulted in 25% (Cqmean= 31.4 ± 6.3). When the variability in target gene Cq values was analyzed in all samples compared to the standard method, the HT method significantly provided lower Cq values (n= 16; p= 0.007; paired t-test) while the MvNAT method and R1-R2 kit yielded higher Cq values (n= 6; p= 0.025, n= 11; p= 0.004; paired t-test). Sensitivity rates were MvNAT= 31.6%, R1-R2= 57.9%, HT= 84.2%, with 100% specificity for all three methods. The HT method demonstrated a positive extraction efficiency because it is fast, easy and not dependent on reagents. Although this method provided lower Cq values than the standard method, especially in samples with a high viral load, it should be considered that it also has the potential to yield false-negative results in samples with Cq> 35. With this study, it was concluded that the extraction phase of the SARS-CoV-2 qrRT-PCR test can be carried out using various methods that do not require kits or reagents, such as the HT method. However, it is believed that multicenter studies involving a larger number of samples are necessary to standardize the test and assess the possibility of false negatives.