基于分子信标探针RT-qPCR快速检测坚果中的霉菌

Rapid detection of mold in nuts based on molecular beacon probe RT-qPCR

霉菌污染是坚果质检不合格的主要原因,实现其快速检测对于坚果行业发展具有重要意义。曲霉属、青霉属和镰刀菌属是坚果霉菌污染的主要污染菌群,根据其内转录间隔区(ITS)基因序列设计引物及3对特异性分子信标探针,建立了一种同时检测坚果中曲霉属、青霉属和镰刀菌属的多重实时荧光定量聚合酶链式反应(qPCR)检测方法。通过对qPCR反应体系进行优化,得到良好的扩增曲线和标准曲线,该方法的检出限分别为:曲霉属2.5×10^(-2)ng/mL(约741 CFU/g),青霉属8.7×10^(-3)ng/mL(约500 CFU/g),镰刀菌属5.6×10^(-3)ng/mL(约454 CFU/g)。本研究为坚果中霉菌的检测提供了一种快速准确的方法,相对于国标检测方法需要耗时7 d,本方法用于检测坚果中的霉菌,用时仅为3 d。

Mold contamination is the main reason for unqualified nuts, and the rapid detection is of great significance to the development of the nut industry. The main mold contaminations of nuts include Aspergillus, Penicillium and Fusarium. According to the internal transcribed spacer(ITS) gene sequences of Aspergillus, Penicillium and Fusarium, primers and three pairs of specific molecular beacon probes were designed to establish a multiplex real-time fluorescence quantitative polymerase chain reaction(qPCR) for the simultaneous detection of these three mold contamination in nuts. The qPCR reaction system was optimized to obtain favourable amplification curve and standard curve. The results showed that the detection limits of Aspergillus, Penicillium and Fusarium were 2.5×10^(-2)ng/mL(about 741 CFU/g), 8.7×10^(-3)ng/mL(about 500 CFU/g) and 5.6×10^(-3)ng/mL(about 454 CFU/g), respectively. Compared with the national standard method which the assay period is 7 days, this proposed method takes only 3 days in detecting mold in nuts.