速冻水饺细菌总数FISH检测技术的优化

摘要

探索荧光原位杂交(FISH)技术检测速冻水饺中细菌总数的可行性及特异性,优化样品的快速、准确和便捷的检测体系.采用TAMRA标记的EUB338探针与大肠杆菌进行FISH实验条件的优化,并利用优化的FISH技术对4种速冻水饺中的8个样品进行检测和计数.应用FISH技术检测速冻水饺中细菌总数,理想的样品预处理实验条件为:热固定2h,4％多聚甲醛固定20 min,系列乙醇脱水5 min,杂交条件为杂交温度为46℃,杂交液中去离子甲酰胺浓度为20％,杂交时间3h,洗脱液中NaCl浓度为225 mmol/L；检测的8个样品中,有2例细菌总数超标.FISH可以特异性的检出速冻水饺中的细菌总数,简便,检测周期短,灵敏度高,为保障食品安全提供更多的理论支持.%This paper was focused on the evaluation of the feasibility and specificity of fluorescence in situ hybridization ( FISH) method in the detection of the total bacteria in Fast-frozen Dumplings, and the construction of a fast, accurate and convenient detection system.TAMRA-labeled EUB338 probe was used to optimize the FISH detective conditions, and eight kinds of Fast-frozen Dumplings samples were detected by FISH to test its effect.After the optimization, the ideal Fast-frozen Dumplings sample preparation conditions were obtained as follows: 2h thermal fix-ing-time, 20min of 4% poly-formaldehyde fixing-time, and 5min of ethanol dehydration time.The hybridization conditions were as follows: hybridization at 46℃ in 20% formamide for 3h, with the dilution concentration of 225mmol/ L NaCl.The testing results suggested the total amounts of bacteria from 2 samples out of 8 were exceeding the Food-standard.FISH can be effective for the detection of the total number of bacteria in the Fast-frozen Dumplings, which will provide more theoretical support for the food safety detection.