Diagnostic power of one-step and two-step RT-qPCR methods to SARS‑CoV‑2 detection

Abstract

Background Coronavirus-2019 (COVID-2019) is a novel coronavirus known as Acute Respiratory Syndrome (SARS-CoV-2). The premier standard test for SARS-CoV-2 diagnosis is a one-step RT-qPCR method, which requires specific probes and reagents. Therefore, detection on a large scale is expensive and cannot be very accurate. Methods A cost-effective technique based on SYBR green was evaluated in the current study. The specific primers for S and N genes were designed, then performed the cross-reactivity test with other coronavirus and respiratory viruses positive samples. Moreover, the analytical sensitivity test was carried out with 8 dilutions (1:10). Lastly, the SARS-CoV-2 clinical samples (n = 210) were tested by these two methods, and receiver operating characteristic (ROC) analysis was performed to investigate the incremental diagnostic value of each gene in the study methods. Results The two-step method detected up to 6th dilutions of the SARS-CoV-2 samples and did not show any amplification of the positive samples of other respiratory viruses. ROC analysis revealed a diagnostic ability of the two-step method for SARS-CoV-2 with an area under the ROC curve of ≥ 0.7 (P ˂ 0.05) and relatively high sensitivity and specificity. The combination of N and S genes increased the sensitivity up to 88%, specificity up to 86%, and area under the ROC curve up to 0.85 (95% confidence interval (95% CI) 0.72 to 0.93, P = 0.0461). Conclusion Our findings indicated that the two-step method has comparable sensitivity and specificity to the one-step method. Therefore, this method can be considered a potential diagnostic method for diagnosing and monitoring COVID-19 patients. It suggests that when the one-step RT-qPCR method is not available, the two-step RT-qPCR can be used to identify SARS-CoV-2.

Colorimetric Detection of SARS-CoV-2 Using Plasmonic Biosensors and Smartphones

“…2.2 × 10 –3 mol L –1 citrate and 25 × 10 –3 mol L –1 HAuCl 4 , seven times with a 30-min break. 1 The solution containing AuNPs was cooled by adding 50 mL of ultrapure water. The solution was kept at 4 °C in the dark for further work.…”

“…The growth of Au nanoparticles (AuNPs) occurred after mixing two solutions, v iz. 2.2 × 10 –3 mol L –1 citrate and 25 × 10 –3 mol L –1 HAuCl 4 , seven times with a 30-min break . The solution containing AuNPs was cooled by adding 50 mL of ultrapure water.…”

“…Most diagnostic tests require cheaper detection methods than prevailing molecular techniques, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), isothermal amplification-based methods, and CRISPR-based diagnostics. 1 Antigen-based detection (antigen test) has become commonplace in lateral flow immunoassays (LFI) or immunostrips, 2 but it achieves less sensitive responses than the reverse-transcription polymerase chain reaction (RT-PCR). 3 Current tests employ mostly blood and nasopharyngeal samples, which are uncomfortable for many patients.…”

“…Mass testing for viral diseases remains relevant given the persistence of contamination with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus). Most diagnostic tests require cheaper detection methods than prevailing molecular techniques, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), isothermal amplification-based methods, and CRISPR-based diagnostics . Antigen-based detection (antigen test) has become commonplace in lateral flow immunoassays (LFI) or immunostrips, but it achieves less sensitive responses than the reverse-transcription polymerase chain reaction (RT-PCR) .…”

A brief review on the validation of biology methods for COVID-19 detection

“…Method 1: performing the reaction in two steps by creating complementary DNA from the viral template RNA with reverse transcriptase, then amplifying the target gene segment by realtime PCR reaction to identify the virus. Method 2: one-step realtime RT-PCR method that combines reverse transcriptase and PCR in a single tube and uses a reactive enzyme that is both reverse transcriptase and DNA polymerase (13,32,33). Table 2 shows the characteristics that laboratory personnel should be aware of when performing one-step RT-PCR and two-step RT-PCR tests (34,35).…”