辣椒中常见产毒性真菌的裸磁珠-多重实时荧光定量PCR检测方法

Non-modified magnetic beads coupled with multiple real-time PCR for detection and quantification of mycotoxigenic fungi in paprika samples

目的将多重实时荧光定量PCR技术与裸磁珠富集技术联用,建立辣椒样品中常见3类（曲霉属、青霉属、镰刀菌属）产毒性真菌的快速定量检测方法。方法根据真菌核糖体RNA（r DNA）基因序列选择真菌广谱引物及属特异性探针,联用裸磁珠富集技术,建立多重实时荧光定量PCR体系,并评价所建方法的灵敏度、特异性、重复性和一致性。结果裸磁珠-多重实时荧光定量PCR方法灵敏度高,与本研究优化后的普通实时荧光定量PCR相比提高了10倍,是原报道的100倍,直接检测辣椒样品中3类产毒性真菌的检出限均可达103CFU/ml,即104CFU/g,且特异性良好（与非目标菌无交叉反应）、重复性良好（CV〈1.5%）,该方法模拟检测辣椒样品中3类产毒性真菌的计数结果与标准培养法的计数结果相比均无统计学差异（P〉0.05）,实验过程仅需7 h（标准培养鉴定法耗时7~14 d）。结论构建的裸磁珠-多重实时荧光定量PCR方法能大大缩短产毒性真菌的检测时间,并成功应用于模拟辣椒样品中常见3类产毒性真菌的定量检测,值得进一步研究和推广。

Objective To establish a method for detecting 3 common toxigenic molds（Aspergillus, Penicillium, and Fusarium）based on non- modified magnetic beads coupled with multiple real- time PCR（NMB- multiple q PCR）. Methods The primers and genus- specific probes were designed based on the r DNA sequences to develop a multiple real- time PCR using nonmodified magnetic bead to enrichment of fungal spores. The sensitivity, specificity and repeatability of this assay were evaluated. Results The detection limit of this assay for spiked samples was 104CFU/g, demonstrating a 10- fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple q PCR assay also showed good specificity and reproducibility and yielded comparable results with those by traditional colony counting method for spiked samples（P〈0.05）.Conclusion NMB-multiple q PCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.