Thermal effect on the persistence of SARS-CoV2
A novel coronavirus (SARS-CoV-2) also known as COVID-19 has recently emerged and first recorded in Wuhan city, China in December 2019 with more than 5.7 million infected patients and deaths toll exceeds 357,000 by the end of May 2020. The first case reported of COVID-19 pandemic in Egypt confirmed on 14 February 2020. By the end of May 2020, there have been about 13,000 confirmed cases, and nearly 1000 deaths. SARS-CoV-2 is a member of Beta coronaviridae, single stranded positive sense RNA viruses which are enveloped viruses that possess extraordinarily large single-stranded RNA genomes ranging from 26 kilobases-32 kilobases in length. Growing evidence of the limitations of qRT-PCR prompts further consideration of the limitations of this diagnostic test. First, there are already over 7 different SARS-CoV-2 nucleic acid PCR tests, yet many variation in sensitivity have been seen which might be extrinsic to the kit construction.
Thus information on virus stability with special regards to its thermal stability and viral integrity of the SARS-CoV-2 in the environment at different temperature conditions is important for understanding virus transmission, stability, integrity and handling the spacemen’s properly for molecular diagnoses of the disease. In this study, we reported the thermal stability of different SARS-CoV-2 isolates at different temperatures/time combination points ranging from 20ºC to 70°C for 2, 4 and 6 hours as measured by quantitative real time RT-PCR. RNA from thermally treated and untreated samples was extracted using Resay Mini Kit (Qiagen Cat#52904) according to the manufacture instruction. Briefly, samples were lysed with 560 µl of buffer AVL at room temperature for 10 min, then 560 µl of absolute ethanol was added and the whole solution was then placed in the QIA amp Mini column provided with the kit. The spin columns were centrifuged at 8000 RPM/2 min and washed with 500 of washing solution AW1 then AW2. The RNA was eluted in 50 μl of the AVE elution buffer and stored at till used.
It was done using SARS-CoV-2 Real Time PCR detection KIT high profile. According to the manufacture instructions, briefly, the master mix was rehydrated with 15 µL of rehydration Buffer and 5 µL of RNA of thermally treated and untreated samples were added (samples were run in duplicates), positive and negative controls (provided with the kit ) were included in each test to judge the quality of amplification. Real time RT-PCR was done using Aram thermal cycler (Agilent, Germany) with the following parameters, reverse transcription step at minute, followed by initial denaturing and enzyme activation step at minute, then 45 cycled of denaturing at sec and annealing/extension seconds with florescence collected at the end of this step. Results presented as the mean values of the triplicated for each sample (mean Cq of the thermally treated sampleMean of the thermally untreated sample) and considered negative when there is no amplification which means that there is no amplification and the virus completely inactivated (lost its integrity).
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Journal of Biochemistry & Biotechnology