地衣芽孢杆菌的双位点荧光原位杂交检测  被引量:1

Study on Detection of Bacillus licheniformis by Two-site Fluorescence in Situ Hybridization

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作  者:吴茗花 朱小颖 邓锐杰 罗爱民[1] 王西 王鼎 WU Minghua;ZHU Xiaoying;DENG Ruijie;LUO Aimin;WANG Xi;WANG Ding(College of Biomass Science and Engineering,Sichuan University,Chengdu 610065,China;Sichuan Langjiu Distillery Co.Ltd.,Luzhou 646500,China;Sichuan Yifuchun Winery Co.Ltd.,Chengdu 610065,China)

机构地区:[1]四川大学轻工科学与工程学院,四川成都610065 [2]四川郎酒股份有限公司,四川泸州646500 [3]四川宜府春酒厂有限责任公司,四川成都610065

出  处:《现代食品科技》2022年第6期318-326,共9页Modern Food Science and Technology

基  金:川大泸州项目(2020CDLZ-3-SCU);川大泸州战略合作项目(2019CDLZ-02)。

摘  要:为了研究芽孢杆菌荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测的影响因素,该实验以地衣芽孢杆菌(Bacillus licheniformis)为研究对象,分析了不同探针、菌体预处理方式、细胞通透化处理条件及杂交条件对FISH检测结果的影响,并进一步对选出来的探针进行特异性检测。结果表明,在设计的探针中有两个可以特异性识别地衣芽孢杆菌,且使用双探针检测比单探针荧光效果更好,提高1.54倍灰度值;当反应条件为超声分散菌体时间120 s、溶菌酶处理40 min、杂交时间为6 h、甲酰胺浓度为30%时检测结果最佳,其灰度值为41.16、菌体检出率为96.35%;特异性检测表明该方法可以特异性识别地衣芽孢杆菌。该研究建立了一种操作简单的地衣芽孢杆菌的双位点荧光原位杂交检测方法,无需核酸提取及扩增,且具有较强特异性。In order to investigate the affecting factors of fluorescence in situ hybridization(FISH)on Bacillus,this work used Bacillus licheniformis as its research object.It analyzed the effects of different probes,strain pretreatments,strain permeability treatments,and hybridization conditions on the detection results of FISH,and further detected the specificity of the selected probes.The results showed that two of the designed probes could identify Bacillus licheniformis;specifically,the two probes exhibited a better fluorescence effect than a single probe,having 1.54 times higher pixel intensity.The reaction conditions were the following:ultrasonic dispersing time,120 s;lysozyme treatment,40 min;hybridization time,6 h;formamide concentration 30%.Consequently,the detection result was the best,producing a gray value of 41.16 and a detection rate of 96.35%.The specificity detections determined that the said method could specifically recognize Bacillus licheniformis.A relatively simple two-site fluorescence in situ hybridization approach was established to detect Bacillus licheniformis,with strong specificity without nucleic acid extraction and amplification.

关 键 词:荧光原位杂交 地衣芽孢杆菌 双位点 

分 类 号:TS261.11[轻工技术与工程—发酵工程] O657.3[轻工技术与工程—食品科学与工程]

 

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