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.
