Tian Luan,Lu Wang,Jiyu Zhao,Hui Luan,Yueling Zhang,Chunlai Wang,Paul R Langford,Siguo Liu,Wanjiang Zhang,Gang Li

Front Microbiol.2022 Sep 9;13:928307.doi: 10.3389/fmicb.2022.928307. eCollection 2022.

Abstract

Actinobacillus pleuropneumoniae  is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for the control of epidemic diseases. In this study, we formulated an  A. pleuropneumoniae  species-specific  apxIVA -based CRISPR/Cas12a-assisted rapid detection platform (Card) that combines recombinase polymerase amplification (RPA) of target DNA and subsequent Cas12a ssDNase activation. Card has a detection limit of 10 CFUs of  A. pleuropneumoniae , and there is no cross-reactivity with other common swine pathogens. The detection process can be completed in 1 h, and there was 100% agreement between the conventional  apxIVA -based PCR and Card in detecting  A. pleuropneumoniae  in lung samples. Microplate fluorescence readout enables high-throughput use in diagnostic laboratories, and naked eye and lateral flow test readouts enable use at the point of care. We conclude that Card is a versatile, rapid, accurate molecular diagnostic platform suitable for use in both laboratory and low-resource settings.

Keywords: Actinobacillus pleuropneumoniae; CRISPR; Cas12a; crRNA; diagnostics.