AUTHOR=Luo Mei , Meng Fan-Zhu , Tan Qin , Yin Wei-Xiao , Luo Chao-Xi 

TITLE=Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.740177

DOI=10.3389/fpls.2021.740177

ISSN=1664-462X

ABSTRACT=<p>Peach bacterial spot caused by <italic>Xanthomonas arboricola</italic> pv. <italic>pruni</italic> (<italic>Xap</italic>) is a devastating disease worldwide and frequently causes massive economic losses. In recent years, it has become a pandemic outbreak in most peach production areas of China, especially on precocious peaches in the middle reach of the Yangtze River. Rapid, user-friendly detection is extremely important to make the correct diagnosis and develop suitable control strategies. In this study, we described a recombinase polymerase amplification (RPA)/Cas12a-based system that combines RPA and CRISPR/Cas12a for <italic>Xap</italic> identification. A total of three crRNAs were designed to target a highly conserved ABC transporter ATP-binding protein-encoding gene <italic>ftsX</italic> to make specific detection of <italic>Xap</italic>. Results showed that crRNA 2 and crRNA 3 could get consistent detection for <italic>Xap</italic>. To realize the visualization of detection results, we additionally introduced FQ-reporter and FB-reporter. The developed method was highly sensitive and could detect as low as 10<sup>–18</sup> M <italic>Xap</italic> gDNA with a mini-UV torch, corresponding to 1.63 copies/μl or 8.855 fg/μl gDNA of <italic>Xap</italic>, while with lateral flow strips, the sensitivity was 10<sup>–17</sup> M. In addition, this method could specifically detect <italic>Xap</italic> from other closely related bacteria or pathogens associated with peach diseases. Furthermore, this method could make correct identification for <italic>Xap</italic> with crude DNA using NaOH-based extraction (3 min) directly from diseased peach samples. Considering that the developed method could get results within 2 h and could be performed at 37°C (body temperature), it is promising to be applied for <italic>Xap</italic> diagnosis and monitoring in fields.</p>