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

TITLE=A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.928307

DOI=10.3389/fmicb.2022.928307

ISSN=1664-302X

ABSTRACT=<p><italic>Actinobacillus pleuropneumoniae</italic> 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 <italic>A. pleuropneumoniae</italic> species-specific <italic>apxIVA</italic>-based <underline>C</underline>RISPR/Cas12a-<underline>a</underline>ssisted <underline>r</underline>apid <underline>d</underline>etection 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 <italic>A. pleuropneumoniae</italic>, 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 <italic>apxIVA</italic>-based PCR and Card in detecting <italic>A. pleuropneumoniae</italic> 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.</p>