Scientists at the National Institutes of Health (NIH) have developed a new sample preparation method to detect SARS-Cov-2, the virus that causes COVID-19. The method bypasses the need to extract the genetic RNA material of the virus, simplifies sample purification, and has the potential to reduce testing time and cost. The method is the result of a collaboration between researchers at the National Eye Institute (NEI), the NIH Clinical Center and the National Institute of Dental and Craniofacial Research (NIDCR).
The diagnostic test remains an important tool in the fight against the COVID-19 pandemic. The standard test to detect SARS-CoV-2 involves amplifying viral RNA to detectable levels using a technique called quantitative reverse transcription PCR (RT-qPCR). First, however, RNA must be extracted from the sample. during the COVID-19 pandemic, manufacturers of RNA extraction kits had difficulty keeping up with demand, affecting the ability to test worldwide. With the emergence of new virus variants, the need for better and faster testing is greater than ever.
A team led by NEI Medical Genetics and Ophthalmic Genomics Group Leader Robert B. Hufnagel, PhD, and NEI Ophthalmic Genomics Laboratory Investigator Bin Guan, PhD, used Chelex 100 resin, a chelator manufactured by laboratory supply company Bio-Rad, to preserve SARS-CoV-2 RNA in samples for detection by RT- qPCR assay.
“We used nasopharyngeal and saliva samples with different virus concentrations to assess whether they could be used for direct RNA detection,” said Guan, lead author of a report on the technique published this week in iScience. “The answer is yes, with significantly high sensitivity. In addition, this preparation method inactivates the virus, making it safer for laboratory personnel to handle positive samples.”
Hufnagel’s team tested a variety of chemicals using synthetic and human samples to identify those that would preserve RNA in the samples with minimal degradation while allowing direct detection of the virus by RT-qPCR.
To validate the test, NIDCR’s Blake M. Warner and his team collected patient samples (per research protocol NIH IRB 20-D-0094) and stored them in either viral transport media or a newly developed chelating resin-buffer from the NIH Symptom Testing Facility.
Samples in viral transport media were tested by the NIH Clinical Center’s COVID-19 testing team, led by Karen M. Frank, MD, using routine RNA extraction and RT-qPCR testing. Samples in chelated resin buffer are heated and then tested for viral RNA by RT-qPCR. the new preparation method greatly increases the yield of RNA available for testing compared to standard methods.
We see clear benefits to this new method in terms of increased sensitivity, cost and time savings for the assay,” Hufnagel said. The method stabilizes RNA at room temperature for easier transport, storage and handling in the clinical setting.”