多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

Multiplex-PCR Approach to Identify Bacillus megaterium and Bacillus cereus Group Applied in Microbial Fertilizers

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 strains are commonly used in microbial fertilizer （MF）. MF products are often contaminated by other B. cereus group members, which have similar phenotype such as Bacillus cereus, B. thuringiensis, B. mycoide. For quality control and safety of MF, a rapid and accurate method is needed to distinguish the strains of Bacillus megaterium from B. cereus group. Based on specific nucleotide sequences of the spoOA genes, 2 pairs of species-specific primers were designed and a multiplex-PCR （mPCR） was developed for this purpose. When the optimized mPCR was used to detect the DNAs of 24 reference strains from three genera of Bacillus, Paenibacillus, and Brevibacillus, all B. megaterium strains showed single fragment of 443 bp and Bacillus cereus group showed a fragment of 411 bp. However, no any amplified product was from the other bacteria. The sensitivity of mPCR was 10^5 CFU/mL. The mPCR results of 10 isolates of B. megaterium/B, cereus group and 8 products of MF coincided with the biochemical assay. Taken together, our newly developed mPCR assay was species-specific and effective in application. It can be used to detect and identify the strains of B. megaterium and B. cereus group from microbial fertilizer products.