New COVID-19 RNA test returns results in minutes
Researchers at the University of Birmingham have invented a COVID-19 test that is said to reduce testing time from 30 minutes to under five, while still delivering accurate results. The method is described in a preprint paper published on MedRxiv, where the researchers also demonstrate its rapidity and sensitivity using patient sample RNA provided by Public Health England.
The current gold standard for COVID-19 testing is a reverse transcription polymerase chain reaction (RT-PCR) test, which takes more than an hour per sample and has two steps. The first, which takes 30 minutes, uses a reverse transcriptase enzyme to convert RNA to DNA. The second uses a DNA polymerase enzyme to copy the DNA and amplify it to detectable levels, and requires time-consuming cycles of heating and cooling. Lateral flow tests, which measure the presence of antibodies, can take up to 30 minutes.
The Birmingham researchers have now created a novel single-step approach for converting viral RNA into DNA and combined it with a known technique called exponential amplification reaction (EXPAR), which increases DNA concentration to detectable levels at a constant temperature. They call their new method Reverse Transcriptase Free EXPAR (RTF-EXPAR) testing.
The method uses a DNA sequence (called Binder DNA) that recognises and binds to SARS-CoV-2 viral RNA and an enzyme (BstNI) that recognises the Binder DNA, and cuts a short section from it when viral RNA is present. Once this cleavage has occurred, the viral RNA is free to bind to more Binder DNA and the cycle is repeated. The test detects the output of this cycle.
The researchers tested the method by introducing BstNI, Binder DNA and SARS-CoV-2 samples to EXPAR reagents and incubating at a constant temperature of 50°C. These conditions yielded test results in 4 ± 0.72 minutes for the positive sample. No signal was observed for the negative (control) sample after 10 minutes of incubation. The concentration of SARS-CoV-2 RNA in the positive sample was 72.7 copies/µL, a value up to 10 times lower than the average viral load found in COVID-19 patient samples.
“We have designed a new method for testing that combines the ease of use and speed of lateral flow testing with the inherent sensitivity of an RNA test,” said Professor Tim Dafforn, a co-author on the study. “It features reagents that can be used in existing point of care devices and meets the need for testing in high-throughput, near-patient settings where people may be waiting in line for their results.”
The method was developed to reduce time and increase throughput in COVID-19 testing; however, it could be applied to any RNA-based infectious agent or disease biomarker (including cancer) and so is expected to extend beyond SARS-CoV-2. University of Birmingham Enterprise has filed a patent application covering the method and its use in diagnostic equipment, and is now seeking to license the patent for rapid product development.
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