New CRISPR-based diagnostic test detects viruses in honeybees

Leslie Dan Faculty of Pharmacy team aims to develop technology that makes accurate diagnostic tools more convenient and accessible

New technology being developed at U of T’s Leslie Dan Faculty of Pharmacy has potential to improve detection of devastating viruses in honeybee colonies without the time and expense of sending samples to labs.

In a recent publication in Nature Communications, Margot Karlikow, a former post-doctoral fellow in the Pardee lab at the Leslie Dan Faculty of Pharmacy, described a newly developed diagnostic tool that combines the accuracy of lab-based systems with the convenience and practicality of rapid tests and used the system to detect harmful viruses that infect honeybees.

"It’s tremendous what they carry on their small wings.”

“As much as we have to take care of the human health, we also have to take care of ecosystem health, and that involves bees,” says Karlikow. “We rely on bees for a lot of things in our life – food and agriculture, the economy, and environment. It’s tremendous what they carry on their small wings.”

Honeybees are important pollinators and essential for many crops to grow. In fact, Agriculture and Agri-Food Canada estimates that honeybees contribute $4 billion to 5 billion to the Canadian economy.

Diagnostic tools have often had to trade convenience and affordability for accuracy. Lab-based diagnostic tools are highly accurate but inconvenient and costly, while rapid tests are practical and inexpensive but typically not as accurate.

Keith Pardee, associate professor at U of T’s Leslie Dan Faculty of Pharmacy, leads a research team that is developing new diagnostic and therapeutic manufacturing tools that are portable, affordable and provide results comparable to lab-based testing.

“Our lab is focused on de-centralizing health care tools so that patients in low-resource settings have access to clinical-grade diagnostics and drugs that can be logistically challenging to distribute,” says Pardee. “This new diagnostic technology addresses some key practical limitations with current CRISPR-based diagnostics – our new approach makes testing more robust to deployment outside of traditional health care settings and enables the detection of more than one target pathogen at a time.” 

In conventional CRISPR-based diagnostic tools, the Cas enzyme looks for viral genetic signatures in a patient sample, and when it detects this signature, it produces a signal such as fluorescence or a colour change, that can be read. However, for the enzyme to detect the genetic signature, the signature needs to be close to a short genetic sequence called a PAM – if the signature is not near a PAM, the enzyme can’t detect it, which limits CRISPR’s diagnostic applications.  

Karlikow developed a new system that does not rely on these PAM sequences. Instead, the system itself inserts a specific sequence anywhere in the genetic material that researchers choose, a process called DNA barcoding, and the CRISPR enzyme looks for the barcode instead of the PAM sequence.

“The idea with our approach is that by getting rid of the need for the PAM sequence, we would then be able detect any genetic sequence of interest or to diagnose virtually anything,” explains Karlikow.

In addition, the researchers have designed the system so that the test can produce different types of readouts – such as colorimetry, fluorescence or glucometers – that might be better suited for different types of settings outside of the laboratory.

New diagnostic tool could detect honeybee viruses as accurately as lab-based technologies

Pardee’s research team has worked to develop diagnostic tools for diseases including COVID-19, Zika, and Ebola, but these tools aren’t only needed for human disease. When Karlikow was given the opportunity to test the next diagnostic technology in honeybees, she jumped at the chance.

Karlikow used the barcoding and diagnostic system to look for two different types of honeybee viruses and found that the new system was able to detect the viruses with nearly 95% accuracy compared to conventional lab-based diagnostics.

“This research is really about whether we can do as well as lab-based tools in a different setting,” says Karlikow. “It’s very easy to be very accurate when you’re in a perfect environment. The question is, can you be as good as with the technology that can be taken out of the perfect environment? The new system demonstrates that we can.”

Since completing her work with Pardee’s lab, Karlikow has launched a start-up company called En Carta Diagnostics, with Pardee as strategic advisor, to develop molecular diagnostic systems using techniques similar to Pardee’s lab, first focusing on Lyme disease. Meanwhile, Pardee’s team will continue developing the new technology into a usable diagnostic tool.

“The recent report in Nature Communications is an exciting proof-of-concept project that provides a fresh take on how CRISPR can be used to create a diagnostic sensor,” says Pardee. “Our next steps will focus on optimizing the assay for speed and sensitivity, as well as incorporating it into fluidics to make it simple to use.”