Single-Tube CRISPR Test Detects Pathogens and Genetic Variants in 30 Minutes

Rapid, accurate detection of infectious agents and single-nucleotide variants remains difficult to deliver outside centralized laboratories. Although multi-step nucleic acid workflows are sensitive, they often require precise timing and specialized equipment, limiting their use in homes or resource-limited settings. As a result, bridging the gap between rapid antigen tests and laboratory diagnostics remains an important priority for surveillance and genetic screening. New findings now demonstrate a single-tube approach designed to deliver high-sensitivity results in about 30 minutes.

The Hong Kong University of Science and Technology (HKUST) has developed a “one-pot” platform called TEMPO, short for "Thermodynamically Encoded Molecular Programming for One-Pot Diagnostics." Created with researchers at The Chinese University of Hong Kong (CUHK), the system is intended to condense nucleic acid amplification and CRISPR-based detection into a single reaction tube while maintaining laboratory-comparable performance. According to HKUST, the approach aims to simplify highly sensitive testing for settings ranging from clinics to homes.

Image: A research team led by Prof. Hsing I-Ming (center), Professor of the Department of Chemical and Biological Engineering at HKUST, has developed TEMPO, a thermodynamically programmed one-pot CRISPR platform for rapid, point-of-care nucleic acid testing. Mr. Wu Xiaolong (third right) and Ms. Li Yanan (first left), PhD students from the Department of Chemical and Biological Engineering, are the co-first authors of this study. (Photo courtesy of HKUST)

TEMPO uses a pair of thermodynamically engineered DNA primers to enforce the correct reaction sequence. One “pristine” primer efficiently initiates target amplification, while a partner primer carries a deliberate mismatch that delays its engagement until a defined thermodynamic threshold is reached. At that point, the second primer installs a protospacer adjacent motif (PAM) site that activates a programmable CRISPR enzyme, producing a rapid, amplified readout; the behavior was captured in an ordinary differential equation model that predicts threshold-dependent activation.

In evaluations, the platform achieved attomolar (1 aM) sensitivity in a single-step, approximately 30-minute workflow. The team reported full concordance with quantitative polymerase chain reaction on simulated HIV samples and validation on clinical specimens for Influenza A and SARS-CoV-2. A single-step microfluidic chip implementation tested 20 human genomic samples for three clinically relevant single-nucleotide variants, with results fully consistent with DNA sequencing.

The study, titled “Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping,” was published in Nature Communications. According to HKUST, TEMPO has demonstrated applications in influenza, COVID-19, and HIV detection and could also be configured for rapid screening of hereditary conditions. 

The researchers plan to expand target panels, develop an integrated sample-to-answer device within the next year, and explore uses such as at-home testing for sexually transmitted infections, rapid diagnostics for multidrug-resistant bacteria, and genotyping for disorders including thalassemia and conditions covered by newborn screening. Their ultimate goal is an at-home nucleic acid testing platform that offers greater sensitivity and specificity than rapid antigen tests while retaining ease of use.

“Conventional laboratory nucleic acid testing involves multiple steps, including amplifying trace amounts of a pathogen's genetic material, identifying target sequences, and generating a readable signal. A 'one-pot' test integrates all of these processes into a single reaction tube. The greatest challenge lies in ensuring that each step takes place in the correct sequence,” said Prof. Hsing I-Ming, Professor of the Department of Chemical and Biological Engineering at The Hong Kong University of Science and Technology.

“Relying on chemically stable DNA, this process does not require cold chain storage or complex handling. Furthermore, ultra-sensitive nucleic acid results are delivered in a single tube, eliminating the need for bulky instruments. Therefore, TEMPO is well suited to clinics, airports, homes, and remote settings. The advance clears a major hurdle on the road to reliable at-home and point-of-care testing,” added Prof. Hsing.

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