微板核酸杂交检测芽孢杆菌属的方法建立

Development of A Novel Microplate Sandwich Hybridization Assay for Detection of Bacillus

摘要:

以白酒发酵过程中的优势菌枯草芽孢杆菌为模式菌株,针对具有高保守性及特异性的16S rRNA设计一组核酸探针,建立了对芽孢杆菌属进行定性和定量分析的新型的微板核酸杂交检测方法,优化了关键检测条件。结果表明,当S1酶反应温度为42 ℃时,芽孢杆菌属检测探针能分开目标序列仅差一个碱基的近缘属。当捕获探针和信号探针工作浓度均为5 nmol/L,与分析探针杂交温度都为50 ℃,杂交时间分别为60 min和15 min时,检测信号的强弱与细菌数呈良好的线性关系,能实现定量检测,灵敏度高达1.33×102 cells/mL。本方法提供了一个快速定性定量的检测技术,具有很好的应用于白酒发酵过程中芽孢杆菌属实时监测的潜力。

Abstract:

In order to detect bacteria from the complex ecological environment for liquor-making,we developed a novel and efficient assay for identification and quantification of bacteria.We selected Bacillus to be experimental bacterium.Three oligonucleotide probes targeted 16S rRNA of Bacillus were respectively designed.Then the assay based on microplate sandwich hybridization assay with the aid of S1 nuclease treatment(S1-MSH)was developed. Meanwhile,several key operating conditions were respectively optimized.The results showed that the probes targeted Bacillus showed high accuracy for distinguishing homologous species such as Staphylococcus,Acetobacter and Pseudomonas. Besides,the assay could distinguish homologous species with the exclusion of even only one base difference when the reaction temperature of nuclease S1 was 42 ℃.When the concentrations of capture probe and signal probe were 5 nmol/L,the hybridization time were respectively 60 min and 15 min,and hybridization temperatures were 50 ℃,the color intensity was positively linear related to the cellular number,and the detection limit was low to 1.33×102 cells/mL.Therefore,this assay method could be applied for identifying and monitoring Bacillus during liquor fermentation,and the research offers an efficient method for rapid identification and quantification of microbiology in the liquor fermentation process.