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SMART Team Improves Virus Detection Speed With Quantitative CRISPR-Based Isothermal Amplification

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NEW YORK – A team at the Singapore-MIT Alliance for Research and Technology has created a new method for faster detection of viruses and bacteria.

The approach was developed by the alliance's Critical Analytics for Manufacturing Personalized-Medicine (CAMP) Interdisciplinary Research Group and has so far been tested on SARS-CoV-2 synthetic DNA and RNA and Epstein-Barr virus in cultured B cells and patient serum.

Xiaolin Wu, a postdoctoral associate at CAMP, said the team combined CRISPR and PCR technology to provide a quicker quantitative result, with less variability, for viral loads in a patient sample. The Rapid Digital Crispr Approach, or RADICA, integrates CRISPR-based isothermal amplification and digital PCR-based partitioning methods.

While the RADICA method was developed to detect contamination in cell therapy products and monitor cell therapy manufacturing processes, early in the pandemic a commercial collaborator recommended the team try using it to detect COVID-19 and quantify virus loads, Wu said.

In a proof-of-concept paper published online in the journal Biomaterials last month, the researchers noted that "quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in [one hour] but suffer from inter-site variability." In addition, "absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application." The RADICA method is intended to bypass both of these issues.

In the RADICA method, a sample such as a nasal swab is collected, and the RNA or DNA are extracted. The 15 microliter CRISPR-based reaction mix is independently partitioned into thousands of microwells on a high-density chip, with some wells that contain the target and some that don't. The RNA or DNA is amplified and identified by the Cas12a protein, which turns the target signal into a fluorescent signal. The microwells that have the target DNA or RNA are lit up and are counted to determine a patient's viral load.

The method has a detection limit of .89 copies per microliter, Wu said, and the turnaround time is 40 to 60 minutes. The sensitivity and accuracy were comparable to digital PCR, the researchers wrote in the Biomaterials paper. It is "highly specific to the target nucleic acids, without cross-reactivity to other similar targets, and is insensitive to human background DNA," they added.

The RADICA method works similar to qPCR and costs about the same as digital PCR but doesn't need thermocycling to run. The only necessity is a water bath since the reaction occurs at a constant temperature, Wu said, making it potentially useful in rural areas or places without high-tech central laboratories. "It can be further applied to some areas with limited resources or instable electricity," she said. "The requirements for this method are much less than the PCR-based method."

Catherine Freije, a postdoctoral scientist at the Rockefeller University who previously worked at the Broad Institute developing CRISPR-based diagnostics, echoed Wu, saying that "the power of isothermal amplification and CRISPR-based detection is that these methods only require incubation at a single temperature."

In addition, "the use of CRISPR enhances the method's specificity, which can be a challenge for isothermal amplification methods used alone, unless the amplification method is thoroughly optimized for the target DNA or RNA of interest."

Hanry Yu, the co-lead principal investigator at CAMP and a professor at the National University of Singapore, noted that the test "shouldn't be difficult" to do with a simple water bath and a chip reader, and mentioned that an undisclosed partner wants to deploy the method in Africa.

He added that as the COVID-19 pandemic begins turning endemic and people get vaccinated, there is a need for more quantitative assays to detect superspreaders of the virus. "People care about … virus load more than whether there's an infection or not," he said. There's more precision in a quantitative assay and that can help direct efforts to reduce spread or stop an outbreak. Knowing the exact amount of virus in a sample can also help clinicians determine the course of treatment.

According to Freije, "One of the challenges with CRISPR-based detection is having a quantitative versus a qualitative readout," which the RADICA method addressed by "using partitioning within a specified range of concentrations."

She noted, though, that because RADICA relies on partitioning the reactions, "it still requires commercial chips and a machine for dropletization, which is likely to be more expensive than a simple heating device." The SMART team used an instrument from JN Medsys for its proof of concept, Wu said.

Another potential problem Freije brought up is that "traditional PCR reagents have been manufactured at scale for many years, so it may be more challenging to get large amounts of Cas12 at a comparable price to PCR reagents." However, "as CRISPR-based diagnostics become more popular, this may no longer be a challenge," she said.

There's still a lot to be done before the RADICA method can be implemented clinically, Wu said, and a lot of data must be generated to determine how stable and robust it is. The team has an undisclosed commercialization partner that will help generate that data, she added. She declined to provide additional details about the future work.

Freije said she saw a need for validation of the method on other droplet PCR machines, as well as ensuring the sensitivity and analysis are "robust to changes in equipment" before it could be implemented in other labs. Labs would also need the equipment to perform nucleic acid extraction from patient samples, she said.

Tim Lu, a principal investigator for CAMP and a professor at the Massachusetts Institute of Technology, said the method would be easy to adapt to other viruses besides SARS-CoV-2 and Epstein-Barr virus. "Once the protocol is set up, adapting it to new viruses is actually pretty straightforward," he said.

Wu added that the team is "definitely interested in other areas for the technology."

For cell therapeutic products, the current method of detecting contaminants or inhibitors is culture-based, which usually takes a long time and doesn't provide a quantitative result, Lu said, adding performing contamination testing is a significant bottleneck for biomanufacturing companies. Cell therapy products have a "quite short" shelf life, so a rapid method is needed to perform contamination testing, Wu added.

The quantitative readout and speed are two things "that are missing from many of the existing assays that are out there," Lu said. "The applications are quite broad."

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