A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

Abstract

Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.

Clinical profile, risk factors and outcomes of pediatric COVID-19: a retrospective cohort multicentre study in Saudi Arabia

“…Fever was the most observed symptom (69%), followed by cough (34.3%) and rhinorrhoea (29.2%). In comparison, previous reports noted fever as the most observed symptom, ranging between 41% and 59% cases, while cough was the second most prominent symptom (37%–46%) 26–34…”

“…In comparison, previous reports noted fever as the most observed symptom, ranging between 41% and 59% cases, while cough was the second most prominent symptom (37%-46%). [26][27][28][29][30][31][32][33][34] Only 3.85% patients in our cohort developed complications, with the most common being bacterial infection (40.0%), ARDS (13.3%) and shock (13.3%). Furthermore, Open access we found that dyspnoea, abnormal chest radiographic findings, lethargy, elevated initial ferritin and elevated D-dimer were significantly associated with the development of complications in our cohort.…”

Emergency SARS-CoV-2 variants of concern: rapidly direct RT-qPCR detection without RNA extraction, clinical comparison, cost-effective, and high-throughput

“…Our protocol increases the testing capacity during the period limited supplies. In another recent study to assess lab-developed direct RT-qPCR, only specific types of virus containers or other pretreatments were used [19,20].…”

Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples

“…Several studies have tried different thermal shock protocols as an extraction-free technique to detect SARS-CoV-2 ( Beltrán-Pavez et al, 2021 ; El-Kafrawy et al, 2021 ; Fomsgaard and Rosenstierne, 2020 ; Pearson et al, 2020 ; Smyrlaki et al, 2020 ). Based on these data, we tested two different thermal shock techniques.…”