胶质类芽胞杆菌PCR快速检测方法  

Rapid identification for Paenibacillus mucilaginosus by PCR

作　　者：王璇[1,2] 马鸣超[2] 关大伟[2] 姜昕[2] 李力[2] 丁延芹[1] 李俊[2] 

机构地区：[1]山东农业大学生命科学学院,泰安271018 [2]中国农业科学院农业资源与农业区划研究所,北京100081

出　　处：《微生物学报》2011年第11期1485-1493,共9页Acta Microbiologica Sinica

基　　金：863计划资助项目-根际固氮微生物功能基因组(2010AA10A203);中央级公益性科研院所科研业务专项资助项目(2011-12);现代农业产业技术体系建设专项资助项目(nycytx-004)~~

摘　　要：【目的】胶质类芽胞杆菌(Paenibacillus mucilaginosus)是微生物肥料广泛应用的功能菌种之一,筛选并鉴定其特异性引物,建立该菌种快速检测方法,对微生物肥料产品检测和评价至关重要。【方法】本文筛选了胶质类芽胞杆菌基因间的一段非编码序列作为特异性引物(orf06701-F:5'-ATGGAGGAAACATGGGGTGA-3'/orf06701-R:5'-TCAGGAATGAAGGCCCCCTT-3'),通过PCR反应条件/体系的优化、特异性及灵敏度检测,建立了胶质类芽胞杆菌快速检测方法。【结果】研究表明,该引物仅能在以胶质类芽胞杆菌为模板时特异性扩增333bp保守序列,其检出灵敏限度为每微升反应体系400-1000个细胞,以实验选取的类芽胞杆菌属其他菌株和芽胞杆菌属菌株为模板时均不能扩出该序列,该引物具有良好的特异性;同时,用该方法成功地鉴定了从土壤中分离得到的胶质类芽胞杆菌,并且得到的特异性片段序列同源性达到100%。【结论】本研究方法为微生物肥料中胶质类芽胞杆菌的检测及生态评价提供技术支撑。[Objective] Paenibacillus mucilaginosus is widely used as microbial fertilizers,therefore,rapid identification of this species by Polymerase Chain Reaction（PCR） is necessary for detection and ecological evaluation of microbial fertilizer.[Methods] A species-specific primer pair of P.mucilaginosus,orf06701-F（5′-ATGGAGGAAACATGG-GGTGA-3′）/orf06701-R（5′-TCAGGAATGAAGGCCCCCTT-3′） was designed.Optimization of PCR,specificity and sensitivity determination were followed and the rapid identification was established.[Results] A single band with 333 bp was consistently amplified from all the strains of P.mucilaginosus tested and negative results were obtained from all the reference strains,such as other strains of Paenibacillus and Bacillus.The PCR detection limitation was 400-1000 cells per assay,indicating the sensitivity of the rapid culture-PCR method.To verify the rapid identification,soil was sampled and cultured.Five of 11 soil isolates were rapidly identified as P.mucilaginosus by the method,and the similarity of 333bp sequences were 100%.[Conclusion] The methods can be used in the rapid identification of P.mucilaginosus and the results will provide technical supports for the detection and ecological evaluation of microbial fertilizer.

关 键 词：胶质类芽胞杆菌 PCR检测 特异性引物 

分 类 号：S154.3[农业科学—土壤学]