论文部分内容阅读
巨大芽孢杆菌是微生物肥料生产中的常用菌种,与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌,传统方法区分两者费时费力,有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物,并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增,以检验其特异性。结果显示,巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物,其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测,其鉴定结果与常规鉴定结果一致。以上结果表明,本文建立的多重PCR方法具有较高的特异性和灵敏度,可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌,在微生物肥料检测方面有良好的实用前景。
Bacillus megaterium is a commonly used microbial strain in the production of microorganisms, and its morphology similar to Bacillus cereus (Bacillus cereus, Bacillus thuringiensis, Bacillus mushroom) is the product of common bacteria, the traditional method It is time-consuming and laborious to distinguish between the two and it is necessary to establish a PCR method for the detection of these two types of Bacillus. In this paper, we designed and screened the specific primers of the two species (groups) respectively using the registered spoOA gene sequences and established the multiplex PCR detection technique. Using this method, the genomic DNAs of 13 24 species of 3 strains of Bacillus megaterium, Bacillus cereus and other Bacillus spores were amplified to examine their specificity. The results showed that Bacillus megaterium and Bacillus cereus genomic DNA produced the only products of different sizes, all other Bacillus were negative. The sensitivity of this multiplex PCR assay was determined to be 105 CFU / mL. At the same time, 10 strains of tested strains and 8 microbial fertilizer products were tested, and the identification results were consistent with the routine identification results. The above results show that the multiplex PCR method established in this paper has high specificity and sensitivity, rapid and accurate identification of Bacillus megaterium and wax-like Bacillus, detection of microbial fertilizer has a good practical prospect.