Portable testing method detects RNA viruses without laboratory equipment

A new protocol detects RNA viruses such as Zika and hepatitis C without traditional medical laboratory equipment. Requiring only a handheld reader and a portable heater, it has the potential to bring rapid testing to low-income regions where low accessibility and availability often hinder time-sensitive diagnoses and treatments.

A team of researchers in the University of Illinois Urbana-Champaign's Grainger College of Engineering has developed a testing protocol for detecting viral RNA directly in whole blood with no laboratory purification. As reported in the journal Science Advances, the protocol uses heat to dry the blood sample before amplifying the preserved viral RNA and detecting it through fluorescence. The entire protocol can be completed on site in a matter of hours, promising a clear advantage in treating diseases such as Zika virus and hepatitis C where early detection is vital to effective treatment.

"Medical infrastructure is currently organized around standard-of-care testing, where samples are collected then transported to a laboratory where time-consuming processing is performed using sophisticated equipment," said the study's lead author Jongwon Lim, former postdoctoral researcher with Illinois Grainger Engineering and current postdoctoral researcher with the Broad Institute of MIT and Harvard. "We want to shift that to point-of-care testing, where the processing is performed where the sample is collected. The main paradigm has been to replicate laboratory approaches at the point of care, but this comes at the cost of reduced sensitivity. Our work reimagines the testing procedure from the ground up as something truly designed to be at the point of care."

This work was a collaboration between the research groups of Illinois Grainger Engineering professors Rashid Bashir of the Department of Bioengineering and Brian Cunningham of the Department of Electrical and Computer Engineering.

Standard-of-care (SOC) testing is based on purification procedures which process whole blood samples to remove substances that interfere with molecular detection. While protocols are accurate and reliable, the purification step is a bottleneck, requiring laboratory-grade equipment such as centrifuges and extraction instruments typically found in centralized laboratories. This can delay diagnosis and treatment particularly in low-income countries where access to medical facilities is limited.

Until now, attempts to address the bottleneck have focused on adapting SOC methods for use outside medical laboratory settings, often by miniaturizing or simplifying laboratory-based sample preparation. However, these approaches still rely on purifying blood samples, which can result in sample loss and compromise detection sensitivity in the field.

The Illinois Grainger engineers decided to take an alternative approach: designing a testing procedure from scratch that is intended for point-of-care use rather than trying to copy SOC procedures. They drew inspiration from the dry blood spot (DBS) technology used to detect HIV and hepatitis B infections.

DBS methods are very simple: you collect one or two drops of blood using a finger prick, then you let it dry. While this simplifies sample collection and storage, the sample still needs to be sent to a centralized laboratory for analysis by specially trained personnel. Our question was whether we could bring the analysis itself to the point of care."

Jongwon Lim, study's lead author 

In prior work, the researchers developed testing protocols for detecting DNA targets called "biphasic reactions." Unlike procedures that mimic SOC testing, the biphasic reaction avoids sample loss associated with conventional purification, helping preserve detection sensitivity even at low target concentrations.

When the team turned to RNA virus, they expected the original process to easily transfer. Instead, they faced a greater challenge because RNA is unstable in whole blood. Enzymes naturally present in blood rapidly break RNA into smaller, undetectable fragments, and heat during sample processing can accelerate its degradation. Lim recalls that he experimented with hundreds of reagent combinations while systematically investigating the mechanisms responsible for RNA degradation and amplification failure.

"We needed to come up with a way to prevent degradation at each step of the procedure," he said. "In trying so many different combinations, we were able to deduce mechanistic explanations for what works and what doesn't. I think this will lead to a huge leap forward in how we detect other diseases caused by RNA viruses such as Ebola and Lassa fever."

In addition to Lim's work in Bashir's Bioengineering research group, the new testing protocol includes a pocket-sized fluorescence device for reading test results. The device was developed in Cunningham's Electrical and Computer Engineering research group, and it was reported earlier this year in the IEEE Sensors Journal. All told, the new protocol only requires a fluorescence device and a portable heater, both of which require low amounts of power and can be easily deployed at the point of care.