Development of a rapid, accurate, and field deployable LAMP-CRISPR-Cas12a integrated assay for Xylella fastidiosa detection and surveillance

Abstract

AbstractXylella fastidiosa is an aggressive plant pathogenic bacterium of significant quarantine concern. Accurate and reliable detection tools are essential to minimise the risk of the pathogen’s spread and for outbreak control, as limited post-infection management strategies are possible. Here, we report the development of a specific and potentially field-deployable assay combining a pre-existing Loop-Mediated Isothermal Amplification (LAMP) assay and a Cas12a-based DNA Endonuclease-Targeted (DETECTR) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) trans reporter for X. fastidiosa detection. The LAMP-CRISPR-Cas12a integrated assay detected the amplified target region of the X. fastidiosa specific rimM gene at the low femto-molar range within 10 min of initiation. The assay detected varied X. fastidiosa sub-species in a range of naturally infected and economically relevant host material, with no non-target amplification recorded. The results show integration of LAMP with CRISPR-based detection is a specific, sensitive and a potentially field-adaptable strategy for the detection of X. fastidiosa and has the potential for further operationally focused improvements.