A Ypt1-targeted TaqMan real-time fluorescence quantitative PCR method for detection of Phytophthora cryptogea P. on Gerbera jamesonii

Abstract:

[Background] Phytophthora cryptogea P. is a quarantine pathogen causing serious symptoms in a variety of plants in the global range. [Objective] To establish a specific, sensitive, and quantitative assay for the detection of P. cryptogea. [Methods] Specific primers and probes for real-time fluorescence quantitative PCR were designed and screened according to the conserved sequence of Ypt1 in P. cryptogea. The reaction system and procedure were optimized in terms of primer concentration, probe concentration, and annealing temperature, and the standard curve and detection system were established under the optimal reaction conditions. The specificity, sensitivity, and repeatability of the established detection system were evaluated. [Results] Considering C_q, RFU, and cost, the optimal PCR conditions were determined as the primer concentration of 20 μmol/L, the probe concentration of 10 μmol/L, and annealing at 60 ℃. The linear equation of the established standard curve was y=−3.323x+40.767, with R²=0.998 and E=99.9%, which indicated the established system had good correlation and high amplification efficiency. No amplification curve appeared for the other eight common species of oomycetes and fungi, which indicated that the designed primers and probe had strong specificity. The established method showed the minimum limit of detection being only 1 copy and the minimum limit of stable detection being 10 copies. Compared with conventional PCR, the method established in this study increased the detection sensitivity by 10−100 times. The intra-batch and inter-batch tests with the established method showed the standard deviation (SD) of C_q ranging from 0.04 to 0.13 and the coefficient of variation (CV) ranging from 0.20% to 0.58%, indicating that the method had good repeatability, stability, and reliability. [Conclusion] We established a Ypt1-targeted TaqMan real-time fluorescence quantitative PCR method for the detection of P. cryptogea. This method can efficiently and specifically identify P. cryptogea infecting Gerbera jamesonii, serving the rapid and efficient detection of this pathogen at port clearance and in the field, particularly for the detection and identification of trace pathogens in core materials such as breeder’s seed and original species. It is essential for the early diagnosis and effective prevention and control of diseases, demonstrating a broad application prospect.

Key words:Gerbera jamesonii;Phytophthora cryptogea P.;Ypt1;real-time fluorescence quantitative PCR;TaqMan probe