基于Ypt1靶标的非洲菊隐地疫霉TaqMan探针实时荧光定量PCR检测

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

【背景】隐地疫霉(Phytophthora cryptogea P.)是一种可危害多种植物且危害症状较为严重的检疫性病原卵菌,在世界范围内危害严重。【目的】建立一种特异性强、灵敏度高且能定量分析P.cryptogea携带情况的高效检测方法。【方法】根据隐地疫霉Ypt1基因的保守序列设计并筛选实时荧光定量PCR的特异性引物和探针,在优化引物浓度、探针浓度和退火温度的基础上获得最优反应体系和条件,利用最优反应条件构建标准曲线及检测体系,并验证检测体系的特异性、灵敏性和重复性。【结果】综合考虑Cq值、最终荧光信号值(relative fluorescence units,RFU)和经济角度,优化后的隐地疫霉荧光定量PCR最佳退火温度为60℃,上、下游引物浓度均为20μmol/L,探针浓度为10μmol/L;所建立的标准曲线为y=−3.323x+40.767,相关系数(R^(2))为0.998,扩增效率(E)为99.9%,相关性好、扩增效率高;对其他8种常见卵菌及真菌均无扩增曲线,表明所设计的引物探针组合特异性强;灵敏度检测结果显示最低检出限为1 copy,最低稳定检出限为10 copies,较常规PCR技术的灵敏度提高了10−100倍;同浓度批组内和批组间重复性试验Cq值标准差为0.04−0.13,变异系数(coefficient of variation,CV)为0.20%−0.58%,重复性及稳定性好、可靠性高。【结论】基于Ypt1靶标建立了非洲菊隐地疫霉TaqMan实时荧光定量PCR检测技术体系,可高效特异地鉴别出疫霉属中广泛侵染非洲菊的隐地疫霉病菌,对于口岸通关和田间隐地疫霉的快速高效检测,尤其是原原种、原种等核心材料的痕量病原菌的检测鉴定和病害早期精准诊断,以及有效防控意义重大,应用前景广阔。

[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 Cq,RFU,and cost,the optimal PCR conditions were determined as the primer concentration of 20μmol/L,the probe concentration of 10μmol/L,and annealing at 60℃.The linear equation of the established standard curve was y=−3.323x+40.767,with R^(2)=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 Cq 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.