地衣芽孢杆菌的双位点荧光原位杂交检测

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

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。

Abstract:

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.