中国人兽共患病学报2012，Vol.28Issue(12)：1195-1201,7.DOI:10.3969/cjz.j.issn.1002-2694.2012.12.008
微小隐孢子虫LAMP检测方法的探索
Exploration of Loop-mediated Isothermal Amplification (LAMP) for detection of Cryptosporidium parvum
摘要
Abstract
Loop-mediated isothermal amplification (LAMP) by using SYBR Green I as ehromogenie agent was developed and optimized in order to detect C. parvum rapidly. Two loop primers were designed based on the 4 specific primers which recognized the 18S rRNA gene of C. parvum. The process of 95 ℃ thermal denatnration and 80 ℃ enzyme inactivation were cut out and its' most optimal reaction temperature and time were 63 ℃ and 60 min. The best optimum concentrations of Betaine , MgSO4 , dNTP Mixture and BstDNA polymerase were 0 .8 mol/L , 8 mmol/L,0.8 mmol/L and 8 U/25μL , respectively. The most optimal concentrations of inner and outer primers were 1.2 μmol/L and 0 .2 μmol/L , respectively , and the reaction time was shorten to 20 min after using the loop primers . The method could not detect Giardia sp , Entamoeba sp , Eimeria tenella, and Strongyloides sp, showing good specificity . There were several advantages to the method . Besides being simple and fast to operate, it can be used without special equipment . Compared with traditional PCR method , it results in an approximately 100-fold increase in sensitivity , which could be applied in the clinical field for rapid detection of Cryptosporidium in the near future .关键词
微小隐孢子虫/环介导等温扩增/检测/PCRKey words
Cryptosporidium parvum / loop-mediated isothermal amplification (LAMP)/ detection/ PCR分类
农业科技引用本文复制引用
史亚东,董海聚,王荣军,张龙现,宁长申,菅复春..微小隐孢子虫LAMP检测方法的探索[J].中国人兽共患病学报,2012,28(12):1195-1201,7.基金项目
河南省科技厅重点科技攻关项目(No. 92102110138)、国家重大传染病专项-重要寄生虫病检测和监测技术研究(No.2008ZX10004-011)、国家自然科学基金项目(No.31172311)和国家科技重大专项(No.2012ZX10004-220)联合资助 （No. 92102110138）
