Mass PCR-testing has been a key component of the response to the SARS-COV-2 (COVID-19) pandemic to inform quarantine measures and infection rates. However, this relies on having the capacity to test at the scale needed, which can be a problem in countries lacking that infrastructure.
Some of the major challenges to developing this infrastructure include accessing necessary equipment and reagents at times of high global demand, and testing in remote and low-resource communities that lack molecular laboratory facilities.
A group of researchers from Peru, a country that has particularly suffered from lack of COVID-testing infrastructure, have proposed a low cost, open-source solution. They also investigated if Bento Lab could be used as part of these workflows in remote situations lacking molecular testing facilities, such as small rural towns and villages.
Importantly, they suggest that the same protocols and approaches could potentially be adapted for any forthcoming pathogenic threat.
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The team, from Universidad Peruana de Ciencias Aplicadas and Instituto de Investigacion Nutriciona, Lima, Peru, developed and validated open-source protocols for local production of biological reagents and enzymes needed for COVID-19 testing. They then developed and validated workflows for COVID testing suited for a range of different laboratory scenarios.
They aimed to allow any country to locally mass-produce the necessary reagents and enzymes needed for COVID-testing. This would mean groups could run testing programmes wherever required, either in conventional molecular laboratories or where such facilities may be lacking.
Bento Lab was used to test whether the effectiveness of these workflows could be undertaken in remote and low-resource scenarios. With its 32 well PCR capacity, centrifuge, electrophoresis unit, and blue-light transilluminator, the lab contains all the hardware required for that workflow.
By using open access protocols, the authors designed a workflow where the reagents and enzymes could be freely replicated by anyone with sufficient technical skill and laboratory equipment within a country, removing the obstacle of lack of available testing reagents during a global pandemic.
The team’s detection workflows used reverse transcriptase PCR to read COVID-19 viral RNA and transcribe it into DNA. Once transcribed and amplified with PCR using specific primers, DNA could be detected either by agarose gel electrophoresis, or by CRISPR-Cas12a induced fluorescence detected by a plate reader or by UV or blue light transilluminators.
Reverse transcriptase reagents and enzymes were produced using the “BEARmix” open access protocols developed by researchers at University of California, Berkeley, and plasmids obtained directly from the authors of this protocol. Plasmids for producing the CRISPR-Cas12a enzymes were obtained from Addgene, a nonprofit plasmid repository.
The team then optimised and standardised their protocols and designed suitable primers by examination of ninety-six COVID genomes. They validated the protocols using 100 clinical samples that had already been examined using the gold standard qRT-PCR test.
The researchers found that the RT-PCR coupled to CRISPR-Cas12-mediated fluorescence detection indicated an overall sensitivity of 80%-88% infections detected, with middle and high viral load samples having a detection success rate of 92-100%.
Detection using agarose gel electrophoresis was less effective with low viral loads (50-66%) but was much more competitive in medium to high viral loads, and was still considered a viable alternative to laboratories only with a thermocycler and transilluminator. Overall, specificity for both assays was 96-100%.
After validation, the team considered that their fully described molecular toolkit showed high sensitivity to mid to high viral loads, as well as versatility for adaptation to different contexts and different PCR-based methods, and with reagents which could be mass produced in a local capacity.
This project shows exciting potential for future decentralised testing, and we are so proud that Bento Lab was featured in this study, with the authors concluding:
“We show that the Bento Lab is capable of amplifying SARS-CoV-2 genetic material […] and allows the visualization of results with our molecular toolkit [..]. Furthermore, a comparison of the amplification of target RNA between the Bento Lab and a regular thermocycler by quantitatively measuring Cas12a-dependent fluorescence shows that the portable laboratory can be a solid alternative. Thus, the molecular toolkit described here can potentially unlock the molecular detection of pathogenic agents even in resource-limited regions.”
Read the open access paper here: