CAJ | 학술논문

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（rDNA）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103 CFU/ml，即104 CFU/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。
목적：장다중실시형광정량PCR기술여라자주부집기술련용，건립랄초양품중상견3류（곡매속、청매속、렴도균속）산독성진균적쾌속정량검측방법。방법근거진균핵당체RNA（rDNA）기인서렬선택진균엄보인물급속특이성탐침，련용라자주부집기술，건립다중실시형광정량PCR체계，병평개소건방법적령민도、특이성、중복성화일치성。결과라자주-다중실시형광정량PCR방법령민도고，여본연구우화후적보통실시형광정량PCR상비제고료10배，시원보도적100배，직접검측랄초양품중3류산독성진균적검출한균가체103 CFU/ml，즉104 CFU/g，차특이성량호（여비목표균무교차반응）、중복성량호（CV<1.5%），해방법모의검측랄초양품중3류산독성진균적계수결과여표준배양법적계수결과상비균무통계학차이（P>0.05），실험과정부수7 h（표준배양감정법모시7~14 d）。결론구건적라자주-다중실시형광정량PCR방법능대대축단산독성진균적검측시간，병성공응용우모의랄초양품중상견3류산독성진균적정량검측，치득진일보연구화추엄。
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 qPCR). Methods The primers and genus-specific probes were designed based on the rDNA sequences to develop a multiple real-time PCR using non-modified 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 104 CFU/g, demonstrating a 10-fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple qPCR 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 qPCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.