Presentation Category
Research
Introduction/Context/Diagnosis
This research was done to validate and test the current viral RNA detection techniques and to determine sensitivity and optimal temperature of RT-qPCR technique.
Methods/Treatment Plan
In Primer/probe validation, N and RP gene containing plasmids were prepared with corresponding to primer/probe labeled N1, N2, and RP. qPCR method was used to amplify the gene in interest. In gel electrophoresis, 5uL of DNA products from each set of primer/probe validation experiment were ran in 2% agarose gel with TAE buffer at 90 volts. In PCR temperature gradient, qPCR was ran with amplification temperature altered for each set ranging between 55 and 63C. In Plasmid Serial Dilution, stock DNA plasmids with 200,000 copies/uL were prepared in a test tube. Dilution was repeated with 0.1x decrease until the final tube contained 2 copies/uL. In Viral RNA Detection with Dilution, concentrate of 10 cVRNA with 250 uL of wastewater is added into a concentrate of 1250 uL of wastewater (250 uL each from five sources) without viral RNA. The combined mixture is then concentrated for RNA extraction and followed by one-step RT-qPCR.
Results/Outcome
Cq values ranged 24-25.1 for all genes. Approximately 100BP was shown for all amplicons. 62.6C had lowest average Cq and subsequently 60C. Plasmid concentration and average Cq value had an inverse correlation. 10 uL VRNA sample showed lowest Cq value, and both of the diluted 10 uL VRNA+250 uL wastewater and 10 uL VRNA + 1250 uL wastewater showed similar Cq values.
Significance/Conclusions
Each primer and probe sets showed effective detection among different plasmids. Agarose gel electrophoresis of DNA products correlated to the expected range of base pairs of ~100BP. Based on published data, 60C is proposed to be the ideal temperature for primer annealing. Greater number of samples should be included in the future for greater reproducibility and reliability. Inverse correlation of plasmid concentration to Cq value in a linear trend indicates the sensitivity of qPCR to be highly sensitive. Viral RNA detection was possible in a diluted solution with multiple pool of wastewater. However, there were no difference between the Cq value of highly diluted (+1250 uL WW) sample and less diluted (+250 uL WW) sample. Further study with greater number of samples would bring more insight into determining optimal lab set-up for RT-qPCR techniques.
Format
Event
COVID-19 Wastewater Project: Validating SARS-CoV-2 Detection Techniques
This research was done to validate and test the current viral RNA detection techniques and to determine sensitivity and optimal temperature of RT-qPCR technique.
Comments/Acknowledgements
This research was completed under the supervision of Dr. Ojcius and Dr. Thor from Biomedical Sciences.