08 February 2022

This is not an opera

Microfluidic CARMEN detects dozens of viruses in hundreds of samples in hours

Elena Kleshchenko, PCR.news

CARMEN's diagnostic platform — a microfluidic chip that uses CRISPR-Cas and fluorescence detection — is adapted for the clinic. Now the reaction is carried out on commercially available Fluidigm chips and devices. In addition to detecting more than two dozen respiratory viruses, the platform distinguishes between coronavirus strains and can be adapted to determine the viral load.

Scientists from the USA presented the microfluidic CARMEN (mCARMEN) platform for cost-effective and fast detection of viruses and their variants, which combines CRISPR-based diagnostics with microfluidics and is optimized for clinical use. The authors developed a panel for testing 21 respiratory viruses, including SARS-CoV-2, other coronaviruses and influenza A and B viruses, and demonstrated its operability in real clinical conditions. Article by Welch, N.L., et al. The multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants is published in Nature Medicine (see also the preprint on medRxiv).

The development has a long history. In May 2020, researchers from the Broad Institute (MIT and Harvard) They published an article in Nature about the multiplexed pathogen detection platform, which they called CARMEN (Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids). It uses droplets with reagents with a volume of about a nanoliter, which self-organize in an array of microlunks. The idea arose even before the appearance of SARS-CoV-2, and when the pandemic began, the authors added this virus to the panel.

CARMEN-Cas13 is a version of the SHERLOCK platform (Specific High Sensitivity Enzymatic Reporter UnLOCKing). The idea is that the CRISPR-Cas system simultaneously cuts the target RNA and the nucleic acid reporter molecule - for example, a polyuracil with a fluorescent molecule at one end and a fluorescence extinguisher at the other; when the ends move away from each other, a fluorescent signal occurs. As for the microfluidic technique, it was created in 2018 in the laboratory of Paul Blaney, an associate professor at MIT and co-author of a new paper. Smartphone-sized chips contain tens of thousands of microlunks, each of which is designed for a couple of nanoliter drops. Two merging droplets are used for detection: one contains amplified genetic material from the sample, and the other contains reagents designed to detect the virus. Since the platform used the Cas13 nuclease, guide RNAs were not needed for its operation, shorter CRISPR RNAs (crRNAs) were enough. 

Each sample was tested for the presence of RNA complementary to one of the 169 crRNAs contained in reagent drops. This development allowed detecting up to 169 viruses in 8 samples simultaneously.

However, the first version of the technology was poorly suited for clinical conditions: specially made chips and reading equipment were needed, the process required manual work and took 8-10 hours. Now the authors have presented an updated version that uses commercially available microfluidic and Fluidigm measuring devices. As a result, mCARMEN can test samples from 188 patients for more than 20 respiratory viruses at a cost of less than $10 per sample. The processing time was reduced to five hours. RNA extraction is automated, RT-PCR is carried out in one stage.

The diagnostic effectiveness of the platform was tested on 525 patient samples in a scientific laboratory and 166 samples in real hospital conditions (in the laboratory of Clinical Microbiology at Massachusetts General Hospital).

Both mCARMEN and CARMEN v1 demonstrated 100% analytical specificity, that is, they did not give false positive results. But mCARMEN showed 100% sensitivity at a viral RNA concentration of 100 copies/ml and 98.4% sensitivity to 10 copies/ml (in CARMEN v1 - 86% and 77.8%, respectively).

The authors created an additional mCARMEN panel that allows distinguishing 6 variants of SARS-CoV-2, including delta and omicron, by 26 mutations in the S-protein. (This requires 26 pairs of sgRNAs.) A check on 2,088 patient samples showed that the classification of variants almost perfectly (97.9% of matches) corresponds to the results of NGS sequencing. The Broad Institute used a new technology to monitor omicron in Massachusetts.

Finally, the authors adapted the model for the quantitative determination of viruses, which required two different CRISPR effectors — the Cas13 and Cas12 proteins and reporter molecules specific to each of them. (Cas13 has increased sensitivity compared to Cas12, so Cas12 provides detection of large amounts of material, and Cas13 — smaller amounts.) In this form, the technology allows you to measure the number of copies of SARS-CoV-2 and influenza A viruses in samples. 

The ideal diagnosis, the developers note, is the simultaneous processing of hundreds of patient samples, with the detection of several viruses in each sample, differentiation of viral variants and determination of viral load. Such tests do not currently exist, but mCARMEN could be a step in this direction.

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