A detection platform for activating Cas12a transcleavage activity without PAM sequence and application thereof in rapid detection of vibrio harveyi

By optimizing the dsDNA target localization and the crRNA spacer region to form a PAM-free CRISPR-Cas12a detection platform, the problems of complex and time-consuming Vibrio harveyi detection operations have been solved, achieving rapid, sensitive, and highly specific detection results, which are suitable for on-site detection in the aquaculture field.

CN122428049APending Publication Date: 2026-07-21HAINAN UNIV
View PDF 0 Cites 0 Cited by

Patent Information

Application Number
CN202511929432.5
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-12-19
Publication Date
2026-07-21

AI Technical Summary

Technical Problem

Existing methods for detecting Vibrio harveyi are complex and time-consuming, and traditional CRISPR-Cas12a detection relies on PAM sequences, which increases detection costs and the risk of sample contamination, failing to meet the needs for rapid on-site screening and early intervention.

Method used

A CRISPR-Cas12a detection platform that does not require PAM sequences was developed. By optimizing the dsDNA target localization and crRNA spacer composition, Cas12a is directly activated, simplifying the detection process and enabling rapid and sensitive detection of Vibrio harveyi using dsDNA.

Benefits of technology

It enables rapid and sensitive detection of Vibrio harveyi, with a detection limit as low as 10 CFU/mL and a dynamic range covering 10³-10⁸ CFU/mL. It has high specificity, good reproducibility, and is suitable for rapid on-site detection. The detection results are consistent with the existing gold standard.

✦ Generated by Eureka AI based on patent content.

Smart Images

  • Figure CN122428049A_ABST
    Figure CN122428049A_ABST
Patent Text Reader

Abstract

The present application relates to the field of microbial molecular diagnosis, and particularly relates to a detection platform capable of activating Cas12a transcleavage activity without PAM sequence and application thereof in rapid detection of waterborne pathogenic bacteria Vibrio harveyi. The present application breaks through the PAM dependence restriction, realizes efficient activation of Cas12a without PAM sequence by optimizing the composition of dsDNA target positioning and crRNA spacer region, and expands the target selection range of the CRISPR-Cas12a detection system without converting dsDNA into ssDNA. The present application simplifies the detection process, eliminates the pretreatment steps such as heat denaturation and enzymolysis, reduces the types of reagents and operation steps, reduces the risk of sample contamination, shortens the total detection time, and is more suitable for on-site rapid detection. The present application has high clinical application value. In the detection of clinical samples of infected Trachinotus ovatus, the detection results of the PFCD platform are completely consistent with those of qPCR (the existing molecular diagnosis "gold standard"), and the infected individuals can be accurately identified, thereby providing reliable technical support for early diagnosis and prevention and control of waterborne diseases.
Need to check novelty before this filing date? Find Prior Art