Team Of Korean Researchers Develop “One-Pot,” Fluorescence-Based COVID-19 Assay

“One-Pot” Fluorescence-Based COVID-19 Test

Given that COVID-19 testing in the US can still be hard to find, with longer waiting times for results due to the long waits in line, experts have now agreed that there is a need for better COVID-19 testing in the country. Now, a team of Korean researchers has developed a novel assay that appears to be fast, simple, and sensitive, the right kind of diagnostic test currently needed. In the Nature Biomedical Engineering journal, the research team has reported their method.

The current gold standard for COVID-19 testing, the RT-PCR molecular tests has very high accuracy yet consists of a complicated preparation process for the virus extracting or refining. Additionally, this type of testing is not appropriate for use in places like drive-thru screening clinics, college campuses, airports, etc as the testing method requires expensive equipment as well as with skilled experts at such “pop-up” screening facilities.

A ligation-dependent isothermal reaction cascade that enables the rapid detection of RNAs with high sensitivity was established by a joint group of researchers at the Pohang University of Science and Technology (POSTECH). Anyone can quickly and easily diagnose COVID-19 through this method, which is based on the RNA sequence of the virus and is called sensitive splint-based one-pot isothermal RNA detection (SENSR).

In about 30-50 minutes of incubation, this “one-pot” assay can be carried out and has the capacity to reach a limit of detection of 0.1-attomolar RNA concentration, and it uses fluorescence-based detection of RNA from pathogens.

From POSTECH Department of Chemical Engineering, assistant professor, Jeong Wook Lee, Ph.D. said, “The RNA can be examined precisely without having to treat a patient’s sample using this fast and simple diagnostic technology.”

Two simple enzymatic reactions are involved in SENSR technology. It consists of a ligation reaction by SplintR ligase followed by T7 RNA polymerase’s transcription. An RNA aptamer is formed as a result and it gives rise to fluorescence only when target RNA is present in a sample, by binding to a fluorogenic dye.

For point-of-care detection of pathogens, the major innovative testing strategy has been the isothermal amplification methods for RNA, and the same has been used in SENSR.

Isothermal amplification methods have various advantages such as they can be carried out with equipment that is portable, cheap, and simple. This method needs one single, persistent temperature throughout the reaction which is another major advantage. For maintaining the required reaction temperature, a water bath or a simple heat block can be utilized. They are also faster than PCR-based methods as the time required for adjusting the temperature is eliminated by an isothermal reaction, thus reducing the overall reaction time.

The accuracy of SENSR was demonstrated by the POSTECH research in two bacterial pathogens, E. coli O157:H7 and V. vulnificus. After this, they modified the protocol for viruses, particularly the SARS-CoV-2 virus, and designed the test kit in such a way the only in the presence of the viral RNA, the kit would produce fluorescence.

The assay reached around 95% of positive predictive value and 100% negative predictive value in 40 nasopharyngeal SARS-CoV-2 samples. In just about 30 minutes, the SARS-CoV-2 virus RNA from a confirmed patient was identified by the researchers. The kit also proved its usability in detecting pathogens other than COVID-19 as it also detected five pathogenic viruses and bacterial RNAs additionally.

The SENSR technology might enable the development of a simple, easy-to-use-form, and portable diagnostic device. A more proactive response to COVID-19 would be enabled along with the possibility of onsite diagnosis if this method is introduced and employed.

Source
“One-Pot” Fluorescence-Based COVID-19 Test