Rapid Detection of FAdV-4 by One-tube RPA-CRISPR/Cas12a Assay

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2025 Feb 18

PMID 39963492

Authors

Lei Ma

Xueping Wang

Mingliang Zhang

Mengjie Zhu

Affiliations

Soon will be listed here.

Abstract

Introduction: Fowl adenovirus serotype 4 (FAdV-4) is a highly contagious viral pathogen of global significance that affects various avian species. It primarily infects poultry and wild birds, leading to avian inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS). The development of rapid diagnostic tools for detecting FAdV-4 is crucial for effective disease control and eradication efforts.

Methods: In this study, we developed a recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a assay, specifically targeting the FAdV-4 Hexon gene. RPA and CRISPR/Cas12a reagents were added to the bottom and lid of the test tube at once, allowing the detection process to occur within a single reaction tube. This approach reduced contamination.

Results: The RPA-CRISPR/Cas12a detection method can identify as few as 10 copies of the genome per reaction, demonstrating 100% sensitivity comparable to that of fluorescence PCR (qPCR). This approach exhibits high specificity for FAdV-4, with no cross-reactivity observed with other FAdV serotypes or common avian pathogens. Additionally, the agreement rate between the results of RPA-CRISPR/Cas12a and qPCR for detecting clinical samples is as high as 97.5%.

Discussion: Therefore, the RPA-CRISPR/Cas12a assay presents a promising alternative for the simple, sensitive, and specific identification of FAdV-4.

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