摘要
目的 为监测环境中的霍乱弧菌 ,建立原位PCR(insituPCR ,ISPCR)检测方法。方法纯培养的霍乱弧菌为实验材料 ,霍乱毒素基因 (ctxAB)为靶序列。 4 %多聚甲醛固定细胞 ,1mg/ml溶菌酶消化 2 0min ,PCR反应体系中加入 2 .5 %甘油 ,以荧光素标记的引物为标示物 ,在液相环境下 ,进行靶序列的ISPCR。结果 经蓝光激发 ,荧光显微镜下有扩增产物的细菌发出明亮的绿色荧光 ,阴性对照则无荧光。结论 由于ISPCR既能检测靶序列 ,又保护了细菌形态 ,可以在检测中省去细菌培养的过程 ,因此 。
Objective An in situ PCR-based method for m onitoring the presence of the cholera toxin gene (ctxAB) in Vibrio cholerae was developed. Methods Bacteria cells were fixed in 4% p araformaldehyde and then treated with 1mg/ml lysozyme for 20 min. During ISPCR, 2.5% glycerin was added into the PCR reagents in order to protect the cells to a mplify the ctxAB gene (a long DNA sequence of length 1037bp). Results Cells subjected to ISPCR using FAM-labeled primer as a label, show ed the presence of ctxAB by the epifluorescence microscopy examination. Conclusion A major advantage of ISPCR method is that, while ampl ifying gene copies within the intact bacterial cells, the morphological identity of the cells can still be retained. So even without the cultivation procedure, in situ PCR method can also detect the genes. In fact, it is a new ideal to detect the toxigenic Vibrio cholerae in enviroment quickly and specifically. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第9期751-754,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 (4 0 1760 3 6)
