摘要
建立快速诊断及定型神经毒素对肉毒中毒的治疗、降低死亡率具有十分重要的意义。用一对Ａ型肉毒梭菌特异的寡核苷酸引物，扩增Ａ型肉毒神经毒素基因轻链区域一段４７２ｂｐ的ＤＮＡ片段，对梭状芽胞杆菌属的１０种６８株菌进行了鉴定，并对ＰＣＲ检测Ａ型肉毒神经毒素基因的灵敏度进行了检查。结果表明，所有２０株Ａ型肉毒梭菌ＰＣＲ扩增均为阳性，其余各型肉毒梭菌及其它各种梭菌均为阴性。扩增产物经限制性内切酶分析与预期的酶切片段一致。用溴化乙锭对扩增产物染色，可从３×１０３个细菌中检测出Ａ型肉毒神经毒素基因。用酚－氯仿抽提Ａ型肉毒梭菌全菌体ＤＮＡ进行ＰＣＲ扩增，可从１０ｐｇ的ＤＮＡ中得到清晰的扩增产物。由此可见，该ＰＣＲ扩增系统用于Ａ型肉毒神经毒素基因的检测具有灵敏度高，特异性强等特点，为Ａ型肉毒梭菌的鉴定及肉毒中毒的快速诊断提供了一种新的手段。
Botulism is a neuroparalytic disease caused by the neurotoxin produced from Clostridium botulinum. The rapid diagnosis and typing of botulism is significant for the treatment of botulism and decreasing the mortality.In this study,a polymerase chain reaction (PCR) was developed for detection of neurotoxin gene of Clostridium botulinum type A using a set of oligonucleotide primer which designed from the nucleotide sequence of the light chain of type A neurotoxin gene to amplify a fragment of 472bp.Sixty eight strains belonging to 10 clostridial species were detected by the PCR.The results revealed that all of the 20 strains of Clostridium botulinum type A were positive and the others were all negative in PCR.The fragments amplified from several strains of Clostridium botulinum type A were digested with restriction endonuclease to identify the amplified products,and the digestion patterns were in agreement with the sizes estimated from the sequence data.Clear fragment could be obtained from 3×10 3 cells and as little as 10pg of DNA could be detected with phenol chloroform extracts in this PCR.Therefore,it suggests that the PCR system is specific and sensitive for identification of Clostridium botulinum type A and rapid diagnosis of type A botulism.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1997年第3期176-181,共6页
Chinese Journal of Microbiology and Immunology
基金
甘肃省青年科研基金
关键词
A型
肉毒梭菌
肉毒神经毒素
基因
PCR
检测
Clostridium botulinum type A Neurotoxin Gene Polymerase chain reaction
