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

Sherif A El-Kafrawy,Mai M El-Daly,Esam I Azhar,Mohammad A Kamal,Adel M Abuzenadah,Reham M Kaki,Ahmed M Hassan

doi:10.3390/healthcare9010037

Sherif A El-Kafrawy, Mai M El-Daly + Show 5 more

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https://doi.org/10.3390/healthcare9010037

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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.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China and quickly spread globally
In response to the shortage of nucleic acid extraction kits needed for real-time reverse transcription PCR assays, we developed a method to perform the molecular assay without the need for nucleic acid extraction
We report a direct rtRT-PCR protocol to detect SARS-CoV-2 that is based on diluting the clinical samples, pretreating them with heat, and amplifying the nucleic acids with rtRT-PCR

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China and quickly spread globally. It causes a disease called coronavirus disease 2019 (COVID-19) that includes acute pneumonia [1,2]. The genome sizes range from 26 to 32 kb. There are four subfamilies of coronaviruses; alpha, beta, gamma, and delta. Alpha and beta originate from mammals, especially bats, while gamma and delta originate from birds and pigs. SARS-CoV-2 belongings to the B strain of beta-coronaviruses, and is closely related to SARS-CoV virus [3]. SARS-CoV-2 shows a 96% sequence similarity to the genome of a bat coronavirus [3]

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