多粘芽孢杆菌芽孢荧光原位杂交检测技术研究及其在有机肥发酵中的应用

Detection of Paenibacillus polymyxa spores by fluorescent in situ hybridization (FISH) and its application in organic compost fermentation

采用化学和萌发两种预处理方法,对多粘芽孢杆菌SQR21芽孢进行荧光原位杂交(FISH),结合扫描电镜比较不同预处理芽孢表面结构和形态变化。结果表明,两种预处理均可在2 h内完成SQR21芽孢的荧光原位杂交,且萌发预处理比化学预处理杂交效率高,荧光信号强。扫描电镜结果显示,萌发预处理芽孢无裂解,而化学预处理芽孢有不同程度的裂解。将萌发预处理荧光原位杂交用于检测猪粪有机肥中二次发酵的SQR21芽孢,发现在37℃,含水量50%的条件下,2 h后芽孢开始生长,4 h后营养体细胞出现,12 h后营养体细胞有显著优势。本研究建立的芽孢荧光原位杂交检测技术,比平板计数更能准确反映多粘芽孢杆菌芽孢的数量和生长动态,在生物有机肥中芽孢杆菌的检测方面有良好的应用前景。

This paper studies on the detection of Paenibacillus polymyxa SQR21 spores by fluorescent in situ hybridization（FISH） after treatments of chemical substance and germinant pretreatment.Pretreatment effect and surface structure was analyzed by scanning electron microscopy.The results show that pretreated P.polymyxa spores can be detected by FISH in less than 2 hour after pretreatments,and germinant pretreatment has higher hybridization efficiency and stronger signal than chemical substance pretreatment.The germinant pretreatment spores were intact,but the chemical pretreatment spores were lysed at different degree.FISH with germinant pretreatment was used to detect SQR21 spores fermented in pig slurry compost at the condition of water content 50% under 37℃.The spores were germinated and enlarged after 2 hours,vegetable cells became visible after 4 hours,and vegetable cells were dominant after 12 hours.Compared with plate counting method,FISH with germinant pretreatment reflected the number and the change of SQR21 spores in organic compost more accurately,and it will be applied in detection of Bacillus.sp in bio-organic fertilizer.