New test detects SARS-CoV-2 in less than 5 minutes

The COVID-19 pandemic is still strong a year after the new coronavirus originated in Wuhan, China, with the virus turning into more infectious variants. A rapid and accurate virus detection strategy is very crucial to reduce the rate of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for COVID-19. The global scientific research community has responded to this unprecedented demand for diagnostic tests by developing several detection platforms, the most sensitive of which detect viral RNA.

The current gold standard in testing – the reverse transcriptase polymerase chain reaction (RT-PCR) – is a 2-step test that takes over 60 minutes to test a sample. Reverse transcriptase is an enzyme that converts viral RNA into complementary DNA (cDNA), which takes almost 30 minutes. The cDNA is then amplified using quantitative PCR (qPCR), and the amplified DNA is detected using a fluorescent dye, which can take about an hour.

Reducing detection times and increasing sample throughput are critical to reducing the rate of virus spread. The research community has discussed several new approaches that help reduce testing times for the detection of SARS-CoV-2 over the past year.

A single elongated CCL-81 cell heavily infected with SARS-CoV-2 virus particles. The small spherical structures in the image are SARS-CoV-2 viral particles. The rod-shaped protrusions from the cells are cellular projections or pseudopodia. Image taken at the NIAID Integrated Research Center (IRF) in Fort Detrick, Maryland. Credit: NIAID

SARS-CoV-2 isothermal detection without reverse transcriptase

In a recent withRxiv* Preprinted research paper, UK researchers reported a rapid isothermal method for the detection of SARS-CoV-2. The researchers used a new approach to convert RNA to cDNA without using the enzyme reverse transcriptase. This new technique rapidly amplifies DNA using the Exponential Amplification Reaction (EXPAR).

Since the new reverse transcriptase-free (RTF-EXPAR) approach combines the conversion of RNA to DNA and amplification of cDNA using EXPAR in a single step, it enables detection of SARS- CoV-2 in a sample in less than 5 minutes.

How does the EXPAR approach work?

In the EXPAR technique, amplification of the cDNA occurs at a single temperature, which avoids time-consuming heating and cooling steps. In addition, the amplicon is relatively small – typically about 15-20 bases long – compared to PCR and isothermal loop-mediated AMP techniques (LAMP). Thus, once triggered, EXPAR can produce up to 108 strands of DNA in a matter of minutes. Similar to the COVID-19 RT-PCR assay, duplex formation is monitored in EXPAR spectroscopically using a fluorescent intercalating dye such as SYBR Green.

Data from the RTF-EXPAR assay for detection of SARS-CoV-2 RNA (72.7 copies / µL, n = 3), showing: (a) mean time for amplification reaction only and (b ) the average total time of the test between the RNA sample and signal. Each run time was calculated as the point at which the fluorescence signal was greater than 10 standard deviations from the baseline signal (10 sigma time). Error bars in datasets are standard deviations of 10 sigma time. Signals observed for negative samples at> 10 min are attributed to amplification resulting from non-specific interactions.

“A crucial element for the development of a successful EXPAR assay is the identification of the optimal nucleotide sequences in the target genome.”

The researchers applied this EXPAR approach in a two-step process. First, they performed an enzymatic digestion at 50 ° C for 5 minutes of binding DNA (1 µM) in the presence of a patient sample SARS-CoV-2 RNA (72.7 copies / µL). Then they added this solution to the mixture of EXPAR reagents to amplify the DNA obtained. Performed in triplicate, this step offered an amplification time of 3.17 ± 0.24 minutes.

The RTF-EXPAR assay could be modified for use in the detection of other RNA viruses

To sum up, the researchers used a new isothermal, reverse transcriptase-free amplification technique called RTF-EXPAR to demonstrate successful detection of SARS-CoV-2 RNA with a total test time of less than 5 minutes. . This approach is not only much faster than the RT-PCR test, which requires at least 60 minutes to test a sample, but also outperforms LAMP and lateral flow antigen techniques.

“This time is not only much faster than RT-PCR (test time of at least 60 minutes), but also outperforms the 30 minute LAMP and lateral flow antigen tests in the current deployment.”

According to the authors, RTF-EXPAR must be fully compatible and ready to deploy on the same equipment currently used for COVID-19 RT-PCR testing. In addition, the speed and simplicity of the assay allows this method to be customized for use in the detection of a range of other infectious diseases caused by RNA-based viruses such as Ebola and RSV.

“In conclusion, through the use of a novel isothermal amplification method without reverse transcriptase, RTF-EXPAR, involving a selective restriction endonuclease to DNA, we have demonstrated the successful detection of RNA from SARS-CoV-2 in a total test time of less than 5 minutes. “

*Important Notice

medRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as established information.