中国食品学报2016，Vol.16Issue(5)：230-237,8.DOI:10.16429/j.1009-7848.2016.05.032
环介导等温扩增技术检测醋化醋杆菌
Detection of Acetobacter aceti Using Loop-mediated Isothermal Amplification Technology
摘要
Abstract
In order to develop a method for the rapid and specific detection of acetic acid bacteria,the Acetobacter actei was took as the model strain,the 16S-23S rRNA nucleotide sequence was selected for primer design and Loopmediated isothermal amplification (LAMP) technology was used to test Acetobacter actei for the first time.The specificity of the primers was detected and the reaction system and reaction condition was optimized.Then the detecting methods for the products were compared.Results indicated LAMP primers had strong specificity,the 25 μL optimized reaction system contained 6 mmol/L MgCl2,1.4 mmol/L dNTPs,0.8 μmol/L inner primers,0.2 μmol/L outer primers,l ×Bst DNA polymerase thermal buffer,8U Bst DNA polymerase,0.8 mmol/L betaine,2 μL DNA template,and sterilized distilled water.The reaction achieved the positive results in 30 minutes at 62 ℃ in the optimized reaction system,the detection sensitivity was up to 3.63×101 CFU/mL which was examined by agarose gel electrophoresis.The white magnesium pyrophosphate precipitate visual inspection sensitivity was up to 3.63×102 CFU/mL,and the naked eye detection limit by color change was achieved by adding lO000× 0.1 μL SYBR Green Ⅰ dye after reaction to the same detection sensitivity as agarose gel electrophoresis detection and the detection time was also shortened.关键词
醋酸菌/醋化醋杆菌/LAMP/灵敏度/产物检测Key words
Acetic acid bacteria/Acetobacter aceti/LAMP/sensitivity/product detection引用本文复制引用
周巍,付晓华,章晶晶,杨岚,王红,张岩,张志胜..环介导等温扩增技术检测醋化醋杆菌[J].中国食品学报,2016,16(5):230-237,8.基金项目
河北省质量技术监督局科技计划项目(100108) （100108）
