摘要
目的建立可视化肺炎支原体环介导等温扩增(LAMP)检测体系,并对其敏感性和特异性进行验证,以期开发出适合各级医院和现场高通量检测的肺炎支原体试剂盒。方法克隆肺炎支原体P1基因质粒,并以此为模板建立LAMP体系,优化肺炎支原体LAMP体系的各组分和反应参数,添加反应指示剂羟基萘酚蓝,用已知浓度的肺炎支原体P1质粒检测体系敏感性;以相近种类支原体和细菌检测体系特异性。结果建立的可视化肺炎支原体LAMP体系能特异性检测肺炎支原体,检测限为2×102copies/m L。结论建立的可视化肺炎支原体LAMP检测体系可应用于人肺炎支原体检测,价格低廉,无需贵重仪器设备,操作简便,适合各级医院和现场高通量肺炎支原体检测。
Objective To develop a loop-mediated isothermal amplification（ LAMP） assay for detect Mycoplasma pneumoniae（ Mp）,and verify its sensitivity and specialty for preparing the test kit which can be used in the hospitals at different levels for high-throughput detection. Methods The plasmid of Mycoplasma pneumoniae P1 gene was cloned and used as template to develop LAMP assay. The components and reaction parameters were optimized. Hydroxynaphthol blue（ HNB） was added into the reaction system as color indicator. The detection limit and specificity of the LAMP assay were verified by P1 plasmid with given concentration and similar mycoplasma and bacterium respectively. Results The visual LAMP assay was developed. The developed LAMP assay in this study could be used in Mycoplasma pneumoniae detection,which was inexpensive,simple and easy-operated wihout requiment for special equipment,hence it could be used in hospitals at different levels for Mycoplasma pneumoniae detection of high-throughput samples.
出处
《临床检验杂志》
CAS
CSCD
2016年第2期85-89,共5页
Chinese Journal of Clinical Laboratory Science
基金
金华市科学技术局课题(2014-3-034)
关键词
肺炎支原体
环介导等温扩增
可视化
羟基萘酚蓝
Mycoplasma pneumoniae（Mp）
loop-mediated isothermal amplification（LAMP）
visual
hydroxynaphthol blue
