Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV)

Sep 20, 2025Journal of virological methods

Rapid and easy visual test using CRISPR to detect Tilapia Lake Virus

AI simplified

CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗

Abstract

The detection limit for Tilapia Lake Virus (TiLV) was found to be 9.10 copies per reaction for recombinant plasmid standards.

A new method was developed for detecting TiLV using the CRISPR-Cas12a system.
The assay demonstrated high sensitivity with a detection limit of 91.82 fg/μL for TiLV RNA.
There was no cross-reactivity with other bacterial and viral pathogens in fish.
The method showed 100% concordance with standard fluorescence testing, indicating accuracy.
This approach combines reverse transcription recombinase aided amplification with CRISPR/Cas12a for rapid on-site diagnostics.

AI simplified

In this study，we developed a new, highly efficient, and sequence-specific method for detecting Tilapia Lake Virus (TiLV) based on the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 12a (Cas12a) system. TiLV is a highly contagious virus that has caused significant damage to the global aquaculture industry. Specific primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporters were designed to detect TiLV genome segment 3, with the ssDNA reporters modified at the 5' and 3' ends with fluorophore and quencher groups, respectively. The assay showed no cross-reactivity with other bacterial and viral pathogens in fish. The detection limit was 9.10 copies per reaction for recombinant plasmid standards and 91.82 fg/μL for TiLV RNA, demonstrating high sensitivity. The reverse transcription recombinase aided amplification (RT-RAA) coupled CRISPR/Cas12a method showed 100 % concordance with the standard fluorescence method, indicating its accuracy and suitability for clinical testing. This study innovatively combined the RT-RAA technique with the CRISPR/Cas12a reaction system, offering a new diagnostic method for TiLV that is fast, portable, highly specific, and sensitive. This enables on-site rapid screening for TiLV, ensuring aquaculture safety and the secure circulation of aquatic animal products.

Full Text

Full text is available at the source.

View full text (XML) ↗View full text ↗

Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV)

Abstract

Full Text

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the crispr gene editing brief

Abstract

Full Text

Related papers

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the crispr gene editing brief