Objective A droplet digital PCR (ddPCR) method was established for the simultaneous detection of four Verticillium wilt pathogens, which may lay the foundation for timely and accurate quantitative monitoring of pathogen growth dynamics, early diagnosis, and risk assessment. Method Based on the alignment of the internal transcribed spacer (ITS) sequences of four Verticillium wilt pathogens-Verticillium dahliae (Vd, KY039312.1), V. longisporum (Vl, KX058040.1), V. nonalfalfae (Vna, KT362917.1), and V. albo-atrum (Vaa, MH856937.1)-conserved regions were selected for the design of primers and probes. Droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR) were used to screen for the optimal primers, optimize the ddPCR reaction system, and evaluate the specificity and sensitivity of the method. Result The optimal primer/probe set for the established method was Ver5; the optimal annealing temperature was 58 ℃, with primer and probe concentrations of 500 nmol/L and 250 nmol/L, respectively. Specificity testing showed that this method specifically identified the four Verticillium wilt pathogens without cross-amplification for non-target microbes, including 7 fungal and 6 bacterial species. The detection limits for Vd, Vl, Vna, and Vaa were 2.1×10-⁶, 1.6×10-⁶, 6.9×10-⁴, and 3.6×10-⁵ ng/μL, respectively. Detection analysis of 50 cotton and 50 soil samples demonstrated that, compared to qPCR, the ddPCR method showed a significant advantage in detection rate, with sensitivities improved by 46% and 51%, respectively. Conclusion The established ddPCR method for detecting the four Verticillium wilt pathogens demonstrates high specificity, excellent sensitivity, and robust reliability, providing an important technical tool for the accurate detection of Verticillium wilt. This method is advantageous for applications in customs inspection and quarantine, as well as in the monitoring and regulation of plant diseases and pests, thereby enhancing the scientific accuracy and timeliness of disease prevention and control.
