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【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 one of the most widely used functional microorganisms for microbial fertilizer. Screening and identifying its specific primers and establishing a rapid detection method for this strain is very important for the detection and evaluation of microbial fertilizer products . 【Method】 A non-coding sequence of Bacillus giomycete was screened as a specific primer (orf06701-F: 5’-ATGGAGGAAACATGGGGTGA-3 ’/ orf06701-R: 5’-TCAGGAATGAAGGCCCCCTT-3’ Reaction conditions / system optimization, specificity and sensitivity detection, established rapid detection of Bacillus Geobacillus. 【Result】 The results showed that this primer could only specifically amplify 333bp conserved sequence when it was used as a template, and its sensitivity was limited to 400-1000 cells per microliter reaction system. The other strains of Bacillus and Bacillus strains as a template can not be extended when the sequence of the primer has good specificity; the same time, the method was successfully identified from the soil isolated gliobacillus, and The specific fragment sequence homology was 100%. 【Conclusion】 This study provides technical support for the detection and ecological evaluation of Bacillus giobacillus in microbial fertilizer.