An integrated rapid detection of Botryosphaeriaceae species in grapevine based on recombinase polymerase amplification, CRISPR/Cas12a, and lateral flow dipstick.

Grapevine Botryosphaeria dieback (GBD), caused by Botryosphaeriaceae species, is an important grapevine trunk disease that poses a threat to grape yield and quality in global viticultural regions. Pathogen diagnosis at the species level using morphological methods is difficult and time-consuming. Therefore, this study aimed to develop a rapid and accurate detection method for the pathogens causing GBD. Recombinase polymerase amplification (RPA) with CRISPR/Cas12a cleavage was combined for detecting pathogens associated with GBD and lateral flow dipsticks (LFD) were employed to monitor the outcomes. Based on the β-tubulin sequences of Botryosphaeriaceae and their related species, specific RPA primers and CRISPR/Cas12a CrRNA were designed and subsequently selected for specifically detecting pathogens associated with GBD. Under optimized reaction conditions and systems, the developed RPA/CRISPR-Cas12a detection system specifically detected Botryosphaeriaceae species within 30 min of RPA and 25 min of CRISPR/Cas12a reactions at 37°C. Specificity tests showed that specific fragments were amplified with the RPA primers in the DNA of six Botryosphaeriaceae species found in China, while none fragments were amplified in the other 22 non-target fungal pathogens species of grapevine. The detection sensitivity of this method was 1 pg μL-1, and which is equal to that of real-time PCR. In summary, our method is simple to perform, produces visual results, does not rely on expensive equipment, and therefore possesses high practical value, providing an efficient and robust detection platform to accelerate the field detection of pathogens associated with GBD.