胶质类芽胞杆菌PCR快速检测方法

Rapid identification for Paenibacillus mucilaginosus by PCR

摘要:

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

Abstract:

Abstract:［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'-ATGGAGGAAACATGGGGTGA-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．