纯培养微生物荧光原位杂交技术检测的影响因素探究

Exploration of affecting factors of fluorescence in situ hybridization detection on pure culture microorganism

为了研究纯培养微生物荧光原位杂交技术(FISH)检测的影响因素,该实验以大肠杆菌(Escherichia coli)及植物乳杆菌(Lactobacillus planetarium)为研究对象,研究不同种属、不同生长期菌株、菌体预处理方式及杂交条件对基于探针EUB338的FISH检测结果的影响。结果表明,菌体种类及菌体生长阶段均较明显影响FISH计数的准确性;确定超声分散菌体时间60 s(100 W、每30 s间歇)、溶菌酶处理60 min可较大幅度提高FISH对菌体的检出率;杂交时间和洗脱液中NaCl浓度对FISH检测结果影响小,杂交温度、杂交液中甲酰胺浓度对大肠杆菌影响小而对植物乳杆菌有一定影响。

In order to investigate the affecting factors of fluorescence in situ hybridization(FISH)on pure culture microorganism,the effect of bacteria species,different strain growth period,strain pretreatment methods and hybridization conditions on the FISH determination results based on probe EUB338 was investigated using Escherichia coli and Lactobacillus planetarium as the research objects.The results showed that the species and growth period significantly affected the accuracy of FISH count.It was determined that ultrasonic wave time 60 s(100 W and intermittent intervals every 30 s)and lysozyme treated time 60 min could significantly improve the detection rate.The effect of hybridization time and NaCl concentration had little effect on FISH determination.Hybridization temperature and formamide in washing buffer had little effect on E.coli and some effect on L.planetarium.