A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit

Abstract

The World Health Organization (WHO) has declared a pandemic caused by a new coronavirus named SARS-CoV-2. The growing demand for commercial kits used for automated extraction of SARS-CoV-2 RNA, a key step before rRT-PCR diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples. Although the recommendation is to use automated methods for nucleic acid extraction, alternatives are necessary to replace commercial kits. However, these alternatives should be as reliable as automated methods. This work describes a simple method to detect SARS-CoV-2 from specimens collected in different preservation media. Samples were previously inactivated by heating and precipitating with a PEG/NaCl solution before rRT-PCR assays for Orf1ab, N and S genes. The new method was compared with an automated protocol of nucleic acid extraction. Both procedures showed similar analytical results. Consequently, this simple and inexpensive method is a suitable procedure for laboratory diagnosis of SARS-CoV-2 infection.

Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

“…In Argentina, a trained lab technician processes approximately 24 samples every 1.5 hours. Additionally, the skyrocketing increment on the clinical use of RT-qPCR due to the COVID-19 pandemic led to a worldwide shortage of RNA purification reagents [4,5] thus, many research groups around the world have concentrated their efforts on either simplifying or eliminating the extraction step [6][7][8][9][10][11][12][13][14][15][16].…”

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

“…To achieve this goal, several studies have tackled this challenge and these efforts have yielded several diagnostic kits. Many approaches have been proposed to detect SARS-CoV-2 virus in nasopharyngeal fluids such as multiplex RT-PCR [ 2 , 3 ], CRISPR/Cas12 [ 4 , 5 ], and CRISPR-Cas3 [ 6 ], lateral flow immunoassay [ 7 ], paper-based biomolecular sensors [ 8 ], SHERLOCK testing in one pot [ 9 ], DNA aptamer [ 10 ], loop-mediated isothermal amplification (LAMP) [ 11 ], etc. Each of these methods has its own strong and weak points in terms of sensitivity and specificity.…”

“…Therefore, several viral genes could be targeted for the detection of SARS-CoV-2 by RT-PCR methods. These genes include such as RdRP and S genes (Yan et al 2020) [14], N and S genes [2], and E gene [15]. For the best RT-PCR performance, the combination of these potential gene targets should be optimized.…”

Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection

“…The COVID-19 pandemic is caused by SARS-CoV-2 ( Gorbalenya et al, 2020 ) and has been responsible for infecting more than 116,521,281 individuals and cause 2.589.548 deaths in more than 216 territories until March 8 th , 2021, 2020 ( WHO, 2020 ). The relevance and importance of mass diagnosis in order to find the asymptomatic individuals is widely recognized as a mandatory tool to reinforce the control measures for monitoring virus circulation and reduce the spreading of SARS-CoV-2 ( Ulloa et al, 2020 ; Vandenberg et al, 2020 ). Molecular assays for viral genome detection using RT-qPCR is considered the “gold standard” method in detecting SARS-CoV-2 infection.…”