Sample collection and cDNA preparation
Nasal and oral samples were collected in the health care facilities in
the south-west part of Bangladesh and sent to the genome center, Jashore
University of Science and Technology, Bangladesh. RNA was extracted from
those samples using nucleic acid extraction kit, Invitrogen inc. The
extracted RNA was then tested for SARS CoV-2 using a commercial kit from
Sansure Biotceh Co., Ltd (China). The left-over RNA were preserved at
-40°C in the genome center lab. A total of 24 randomly selected SARS
CoV-2 positive samples were tested for the analysis
(supplementary Table s1 ). A representative SARS CoV-2 negative
sample (of 5 of the negative samples randomly selected) was used as a
negative control in this study (supplementary Table s2 ). cDNA
was prepared for each selected sample using the GoScript™ Reverse
Transcription System (Promega, USA) following the manufacturer’s
protocol. In brief, primer/ RNA mix was prepared by mixing 10µl of
extracted RNA with 1µl of Random primer and 1µl of
Oligo(dT)15 primer (total volume 12µl). Then the mixture
was heated at 70°C for 5 minutes, followed by immediate chilling on ice
for 5 minutes and a quick spin. The mixture for reverse transcription
reaction was prepared by making a cocktail of the components from
GoScript™ Reverse Transcription System in a sterile 1.5ml micro
centrifuge tube keeping on ice. The final reaction mix was 40μl for each
cDNA synthesis reaction to be performed.