单核细胞增生李斯特菌在蜂产品中的生存及检测

Listeria Monocytogenes' Survival in Honey and Royal Jelly and Its Detection Methods

目的:为了保障蜂产品免受致病性单核细胞增生李斯特菌的威胁,对蜂产品中单核细胞增生李斯特菌进行检测。方法:采用培养和分子生物学的检测方法研究蜂产品中致病性单核细胞增生李斯特菌的污染状况。将高浓度的病原菌人工污染到蜂蜜和蜂王浆中,室温存放一定时间后,在选择性培养基上增菌培养;以毒力基因hly和16sRNA基因为靶序列,建立双重PCR检测病原菌的方法;以5’、3’端标记FAM、TAMRA的hly基因探针进行荧光定量PCR检测。结果:单增李斯特菌在蜂蜜中的存活时间为5d,而在蜂王浆中不能存活。对未经增菌的人工污染蜂产品采用溶菌酶+蛋白酶K的方法提取DNA,同时采用本实验中建立的双重PCR和荧光定量PCR方法检测,蜂蜜中单增李斯特菌含量均为102CFU/mL。采用这两种方法可在8h内完成蜂蜜中单核细胞增生李斯特菌的快速检测。结论:单增李斯特菌能够在蜂蜜中存活数天,几乎不继续繁殖,而在蜂王浆中不能存活。

Objective： Survival and proliferation of pathogenic microorganism-Listeria monocytogenes in honey and royal jelly need to be characterized to ensure food safety. Methods： Survival and proliferation of Listeria monocytogenes in honey and royal jelly were detected by cell culture, multiplex PCR and real-time PCR. The multiplex PCR was accomplished using the primers from genes of hly and 16 sRNA in matrix as the target. The hly probes were labeled by FAM and TAMRA, which were used successfully to develop a real-time PCR. Honey and royal jelly inoculated by Listeria monocytogenes in high concentration were stored up to 4 W at room temperature. The pathogenic bacteria in the honey and royal jelly were cultured on a selective medium. Survival and proliferation of the pathogenic bacteria was detected by amplification of the HLY and 16 sRNA genes and fluorescence-quantitative PCR, respectively. Results： Listeria monocytogenes in the honey survived for 5 days, but they could not survive in the royal jelly. For the honey and royal jelly with artificial inoculation of Listeria monocytogenes, the detection limit without enrichment is very low（10^2 CFU/mL）. In this study, a simple method for detection of Listeria monocytogenes in honey and royal jelly has been established. It takes only 8 hours to complete the procedure. Conclusions： Listeria monocytogenes can survive for several days and hardly proliferate in honey, but they can＇t survive in royal jelly.