A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock

Abstract

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol–samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

Emergence of SARS-CoV-2 Delta Variant, Benin, May–July 2021

“…Here, we report continuous genomic surveillance at the Benin reference laboratory on samples obtained from 4 sites in southern Benin during late April–mid-July 2021 ( Figure 1 , panel A). During the study period, routine testing at the reference laboratory and associated satellite laboratories in Benin averaged at 1,370 samples per day ( Figure 1 , panel B), a 900% increase from a comparable timeframe in 2020 ( 6 ) ( Figure 1 , panel B). The decentralization of diagnostic testing and simplification of extraction protocol contributed to increased testing ( 7 ).…”

Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification

“…Others found a proteinase K digest prior to heat lysis to be beneficial to detecting SARS-CoV-2 in patient samples [ 15 , 16 ]. In our hands, heat lysis of SARS-CoV-2 VPM gave significantly worse results than using a lysis buffer; therefore, that avenue was not pursued further for in vitro samples.…”

“…For SARS-CoV-2, several groups have developed direct RT-qPCR methods for detection aimed at patient swabs. The most commonly used method is direct use of swabs following a heating step: 30 min at 65 • C or increasingly shorter periods up to 95 • C. With or without addition of commercial buffers or detergents, a Ct difference between four and seven cycles was observed compared with extracted vRNA [8][9][10][11][12][13][14][15][16]. Other methods include the addition of proteinase K to patient swab samples, showing 4-6 cycle differences, but no proof of virus inactivation was shown for these samples [17,18].…”

True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

“…FDA and other authors have reported the optimal human endogenous genes in the SARS-CoV-2 RT-qPCR detection. The RNAse P and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) are among the genes that act as an excellent internal control by excluding the possibility of false results due to the presence of low quality and integrity of RNA samples [ 65–67 ].…”