CRISPR Technologies Transform On-Site Detection of Animal Pathogens

The rapid spread of animal diseases and pathogen evolution have long posed significant threats to the healthy development of animal husbandry. Traditional nucleic acid detection methods such as PCR rely on expensive equipment and professional personnel, making them difficult to meet the needs of on-site scenarios such as farms and rural areas. In recent years, the CRISPR-Cas system, originally developed for gene editing, has been transformed into a novel detection tool due to its ability to precisely recognize nucleic acid sequences-particularly the Class 2 Cas9, Cas12, and Cas13 proteins. When combined with isothermal amplification technology, it enables rapid and sensitive on-site detection. So, how can these technologies overcome the limitations of traditional methods and provide more convenient and efficient solutions for animal pathogen detection?

A review article by Dr. Linlin Zhang's team from the Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, Tianjin Agricultural University, points out that the Class 2 CRISPR-Cas system is the key breakthrough. This type of system operates with only a single Cas protein, featuring a simple structure that is easy to modify. When paired with isothermal amplification technology, it can complete detection in a short time. For example, upon finding the target DNA, the Cas12 system activates its collateral cleavage function, cutting fluorescent probes or labeled molecules on test strips to indicate results through signal changes. The article was published in Frontiers of Agricultural Science and Engineering (DOI: 10.15302/J-FASE-2025659).

For RNA viruses (such as avian influenza virus and duck Tembusu virus), the Cas13 system exhibits outstanding performance. Its SHERLOCK platform combines recombinase polymerase amplification (RPA) technology and transcription to amplify target RNA, followed by precise recognition by Cas13 to trigger fluorescent signals. Studies have shown that detecting H5 subtype avian influenza virus using Cas13 combined with RT-RAA takes only 40 minutes, with a sensitivity of 0.1 virus copy/μL. When paired with lateral flow strips, results can be judged without instruments, facilitating use by grassroots veterinarians in the field. Additionally, Cas13 enables simultaneous detection of multiple pathogens-for instance, identifying both duck hepatitis A virus type 3 and novel duck reovirus-greatly improving diagnostic efficiency.

The core advantage of these technologies lies in their on-site friendliness: while traditional PCR takes several hours, most CRISPR-based methods can be completed within 1 hour. They offer high sensitivity down to single-copy levels (e.g., Cas12 detects porcine reproductive and respiratory syndrome virus in less than 25 minutes, recognizing just 1 virus copy), avoiding false negatives. No expensive equipment is required; operations can be performed with simple heating or at room temperature, and results are visualized (via fluorescence or test strip color changes). For example, when detecting African swine fever, farmers can obtain results within half an hour using portable test strips, enabling timely isolation of infected pigs to prevent herd-wide infection-this is crucial for reducing economic losses and controlling epidemic spread.

However, the article also highlights challenges facing the technology: some methods rely on nucleic acid amplification, posing a risk of cross-contamination; impurities such as blood and feces in animal samples may interfere with detection. Future research directions include developing amplification-free CRISPR detection technologies or optimizing systems to resist sample impurities, ensuring more reliable on-site application.

Overall, the Class 2 CRISPR-Cas system provides a new pathway for on-site detection of animal pathogens. It is expected to become a practical tool for epidemic prevention and control in animal husbandry, facilitating rapid diagnosis in resource-limited areas and safeguarding the healthy development of the industry.