Eight months after the SARS-CoV-2 virus first entered the U.S., diagnostic tests can still be hard to find, with long waits in line, and even longer wait times for results that vary in their accuracy. Experts agree that what is needed are diagnostic tests that are sensitive, simple, and fast. Now, a Korean team of researchers is reporting they have developed a novel assay meets these needs.
Their method is published in Nature Biomedical Engineering in a paper titled, “Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription.”
The RT-PCR molecular test that is currently the gold standard for COVID-19 testing has very high accuracy but includes a complex preparation process to extract or refine the virus. It is not suitable for use in “pop-up” testing centers that would be useful at airports, on college campuses, drive-thru screening clinics, etc., as it requires expensive equipment as well as skilled experts.
A joint research team at Pohang University of Science and Technology (POSTECH) developed a ligation-dependent isothermal reaction cascade that enables the rapid detection of RNAs with high sensitivity. The method, termed sensitive splint-based one-pot isothermal RNA detection (SENSR), allows anyone to easily and quickly diagnose COVID-19 based on the RNA sequence of the virus.
The “one-pot” assay that uses fluorescence-based detection of RNA from pathogens can be performed within 30–50 minutes of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration.
“This method is a fast and simple diagnostic technology which can accurately analyze the RNA without having to treat a patient’s sample,” commented Jeong Wook Lee, PhD, assistant professor, department of chemical engineering, POSTECH. “We can better prepare for future epidemics as we can design and produce a diagnostic kit for new infectious diseases within a week.”
SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that binds to a fluorogenic dye and produces fluorescence only when target RNA exists in a sample.
Isothermal amplification methods for RNA, like the one used in SENSR, have been at the center of innovative testing strategies for point-of-care detection of pathogens due to the multiple advantages.
First, isothermal methods can be performed with simple, portable, and cheap equipment. Another huge advantage is that they require one single, constant temperature throughout the reaction. A simple heat block or even water bath could be used to maintain the reaction temperature needed. In addition, an isothermal reaction can reduce the overall reaction time by eliminating the time necessary for temperature adjustment, making them faster than PCR-based methods.
The POSTECH researchers first demonstrated the accuracy of SENSR in two bacterial pathogens, V. vulnificus and E. coli O157:H7. They then adapted the protocol for viruses, and specifically the SARS-CoV-2 virus, designing the test kit to show fluorescence only when the viral RNA is present.
In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95% and 100%, respectively. The research team found the SARS-CoV-2 virus RNA from an actual patient sample in about 30 minutes. In addition, five pathogenic viruses and bacterial RNAs were detected which proved the kit’s usability in detecting pathogens other than COVID-19.
The SENSR technology may allow the development of a diagnostic device that can be developed into a simple, portable, and easy-to-use form. If this method is introduced, it not only would allow for onsite diagnosis, but would also allow for a more proactive response to COVID-19 by supplementing the current centralized diagnostic system.