Researchers confirm speed, simplicity and sensitivity for new COVID-19
test

Date:
August 17, 2021
Source:
University of Birmingham
Summary:
Researchers have published a three way comparison study to confirm
that the Reverse Transcriptase Free Exponential Amplification
Reaction (RTF- EXPAR) method is just as sensitive, but faster,
than both PCR and LAMP tests. The study revealed that the RTF-EXPAR
method converts under 10 strands of RNA into billions of copies of
DNA in under 10 minutes, using a one-pot assay that is compatible
with more basic, benchtop equipment than that used with current
testing methods.



FULL STORY ========================================================================== Researchers from the University of Birmingham, U.K., have confirmed the
speed, accuracy and simplicity of a novel, highly sensitive testing
method for COVID- 19 that can be deployed at entertainment venues,
airport arrival terminals, and in remote settings where clinical testing laboratories are not available.


==========================================================================
The scientists used a three way comparison study to confirm that the Exponential Amplification Reaction (EXPAR) method is just as sensitive,
but faster, than both PCR and LAMP tests which are currently used in
hospital settings. The Birmingham COVID-19 test, called RTF-EXPAR, gives
a sample-to- signal time of under 10 minutes, even for low viral levels
where current lateral flow tests are less effective.

Professors Tim Dafforn from the School of Biosciences, and Jim Tucker
from the School of Chemistry, worked with graduate student Jake Carter,
and Professor Andrew Beggs from the Institute of Cancer and Genomic
Sciences on the study, which was published today in PNAS.

Both PCR and LAMP tests detect viral RNA, which can be present in
extremely low levels in swabs taken from the mouth and nose. These tests
use a two-step process which involves first converting to RNA to DNA (a
process called reverse transcription) and then 'amplifying' the material
many times over so it can be detected in the sample.

Professor Tim Dafforn said: "Both the reverse transcription and
amplification steps slow down existing COVID assays that are based on
nucleic acid detection, compared to antigen tests, such as lateral flow,
which do not have these steps.

However, while this makes lateral flow tests faster than those based on
PCR and LAMP, in return they are typically less sensitive. An ideal test
would be one that is both sufficiently sensitive and speedy -- our test,
called RTF-EXPAR, achieves this goal.

RTF-EXPAR achieves this feat in two ways -- firstly the assay
team designed a new RNA-to-DNA conversion step that avoids reverse transcription, making it reverse transcription-free (RTF). Secondly
their amplification step to generate the read-out signal uses EXPAR,
an alternative DNA amplification process to PCR and LAMP.

Professor Dafforn added: "EXPAR amplifies DNA at a single temperature,
thus avoiding lengthy heating and cooling steps found in PCR. However,
while LAMP also uses a single temperature for amplification, EXPAR is
a simpler and a more direct process, in which much smaller strands are amplified. This makes EXPAR an even faster DNA amplification technique
than not only PCR but also LAMP." The study revealed that the RTF-EXPAR
method converts under 10 strands of RNA into billions ofcopies of DNA
in under 10 minutes, using a one-pot assay that is compatible with more
basic, benchtop equipment than that used with current testing methods.

RTF-EXPAR also demonstrated significant improvements over both PCR and
LAMP- based assays on time to signal detection. At low concentrations of
RNA (7.25 copies/myL), the time to signal detection was 42.67 (+/- 0.47) minutes for PCR, 11.25 (+/- 0.20) minutes for LAMP, and 8.75 (+/- 0.35)
minutes for EXPAR. At high (1450 copies/myL) concentrations of viral RNA,
the time to signal detection was 34.00 (+/- 0.00) minutes for PCR, 11.25
(+/- 0.20) minutes for LAMP, and 3.08 (+/- 0.42) minutes for EXPAR.

Identification of the optimal sequence was clearly an important step in
the development of the EXPAR method, and the sequence detected in the
study, which comes from the Orf1ab gene in the SARS-CoV-2 genome, has
been shown to be conserved in all current variants of COVID. However,
the RTF-EXPAR method can be quickly adapted should new variants emerge,
or for testing other viral pathogens such as Influenza, Respiratory
Syncytial Virus (RSV), or Ebola, where near-patient testing is required
to prevent more widespread transmission.

The assay has been tested at the Surgical Research Laboratory at the
University of Birmingham. Professor Andrew Beggs, whose team conducted
the testing, commented: "The testing used swabs containing a typical
range of viral loads seen during the pandemic, and had a six-minute
cut-off time. The analysis showed RTF-EXPAR's sensitivity is equivalent
to quantitative PCR testing, with a positive predictive value of 89%,
and a negative predictive value of 93%. We expect to publish the full
results of this testing in the near future." The team is now seeking commercial partners for rapid licensing, to make the RTF-EXPAR test as available as widely as possible.

========================================================================== Story Source: Materials provided by University_of_Birmingham. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Jake G. Carter, Lorea Orueta Iturbe, Jean-Louis H. A. Duprey, Ian R.

Carter, Craig D. Southern, Marium Rana, Celina M. Whalley, Andrew
Bosworth, Andrew D. Beggs, Matthew R. Hicks, James H. R. Tucker,
Timothy R. Dafforn. Ultrarapid detection of SARS-CoV-2 RNA using
a reverse transcription-free exponential amplification reaction,
RTF-EXPAR.

Proceedings of the National Academy of Sciences, 2021; 118 (35):
e2100347118 DOI: 10.1073/pnas.2100347118 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/08/210816191328.htm

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