CRISPR and nanopore technology enable sensitive and specific mpox detection

3 D Monkeypox Infecting Cell Social

A test that combines CRISPR gene editing and nanopore sensing technology could accelerate detection of mpox and other pathogens, according to a paper published in Biosensors and Bioelectronics.

The research, which was also shared at the Biophysical Society Annual Meeting this week, represents a step toward addressing the need for fast tests suitable for use at the point of care (POC). Currently, the World Health Organization (WHO) only recommends lab-based polymerase chain reaction (PCR) testing for mpox, a virus formerly known as monkeypox, but it has shared a target profile for a faster test intended for use in the community. 

A team at Pennsylvania State University applied CRISPR to the challenge of accelerating and simplifying viral detection. The researchers created a genetic sequence combined with a reporter to target mpox. A programmable CRISPR RNA binds to the target and the Cas12a enzyme. Together, CRISPR and Cas12a split the reporter into fragments for analysis by nanopore sensors to enable the detection of the virus.

The scientists used recombinase polymerase amplification (RPA) for pre-amplification, thereby avoiding the need for high amounts of energy and power that make PCR a poor fit for POC testing. The RPA assay finished pre-amplification in 20 minutes and achieved a limit of detection (LOD) of 19 copies in a 50-μL reaction system. Integrating RPA into a CRISPR reaction lowered the LOD.

The LOD is below the level targeted by the WHO in its product profiles. In a statement, Ahasan Ahamed, the Penn graduate student who co-authored the paper, said that the test “takes 32 to 55 minutes to detect the target, depending on viral load.” The WHO targets 40 minutes or less for its desired community test.

The need for a new mpox test has fallen over the past 18 months. The seven-day average of new cases in the US peaked above 450 in August 2022, according to the Centers for Disease Control and Prevention (CDC). Last year, the seven-day average rarely hit 10 cases. The CDC stopped reporting daily case counts last month. While the mpox crisis has eased, the technology is applicable to other pathogens.

“Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care,” the researchers wrote in the paper.

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