摘要
目的 建立一种用于同时检测多种支原体污染的环介导等温扩增(loop-mediated isothermal amplification,LAMP)法,并进行验证。方法 通过SnapGene软件的多序列比对工具比对13种支原体16S rRNA基因的保守序列,根据保守序列设计2条内引物FIP和BIP、2条外引物F3和B3、环引物LOOP(LF/LB);建立LAMP体系:Bst DNA聚合酶大片段(8 U/μL)、10×ThermoPoly反应缓冲液[20 mmol/L Tris-HCl,10 mmol/L KCl,10 mmol/L(H4)2SO4,2 mmol/L MgSO4,0.1%Triton X-100,pH 8.8]、MgSO4(100 mmol/L)、dNTP Mix、FIP/BIP Primers、F3/B3 Primers、LOOP Primers、无核酸酶水、模板DNA(13种搭载支原体16S rRNA的质粒)。产物进行1%琼脂糖凝胶电泳鉴定。验证方法可行性、特异性、可重复性,确定方法检测限。结果 筛选的2组LAMP引物,组合使用可同时检测出11种支原体序列;在其他非特异模板检测中未出现非特异扩增,引物组特异性良好;建立的方法灵敏度好,最低检测限为30.1 copies/μL,且重复性好,未见假阳性和假阴性扩增。结论 LAMP技术可用于检测细胞培养中多种支原体污染的情况,特异性好、耗时短,可作为细胞培养中快速检测支原体污染的技术平台之一。
Objective To develop and verify a loop-mediated isothermal amplification(LAMP)method for simultaneous detection of multiple mycoplasma contamination.Methods The conserved sequences of 16S rRNA genes of 13 mycoplasma species were aligned by the multiple sequence alignment tool of SnapGene software.Two internal primers FIP and BIP,two external primers F3 and B3 and loop primer LOOP(LF/LB)were designed according to the conserved sequences.The LAMP system was developed as follows:Bst DNA polymerase large segment(8 U/μL),10×ThermoPoly reaction buffer[20 mmol/L Tris-HCl,10 mmol/L KCl,10 mmol/L(H_(4))_(2)SO_(4,)2 mmol/L MgSO_(4) and 0.1%Triton X-100,pH 8.8],MgSO_(4)(100 mmol/L),dNTP Mix,FIP/BIP Primers,F3/B3 Primers,LOOP Primers,nuclease-free water and template DNA(13 plasmids carrying mycoplasma 16S rRNA).The products were identified by 1%agarose gel electrophoresis.The feasibility,specificity and repeatability of the method were verified,and the detection limit was determined.Results A total of 11 mycoplasma sequences were detected simultaneously by using two sets of LAMP primers screened.There was no nonspecific amplification in other nonspecific template detection,and the primer group had good specificity.The developed method showed good reproducibility and good sensitivity with the minimum detection limit of 30.1 copies/μL,and no false positive or false negative amplification.Conclusion LAMP technology can be used to detect a variety of mycoplasma contamination in cell culture,with good specificity and short time consumption,which can be used as one of the technical platforms for rapid detection of mycoplasma contamination in cell culture.
作者
柯晓梅
吴宇曼
孔伟圣
陈智妍
刘帅
吴华佐
朱霞燕
张健
KE Xiaomei;WU Yuman;KONG Weisheng;CHEN Zhiyan;LIU Shuai;WU Huazuo;ZHU Xiayan;ZHANG Jian(Department of Bioengineering,Zhuhai Campus of Zunyi Medical University,Zhuhai 519041,Guangdong Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第8期980-986,共7页
Chinese Journal of Biologicals
基金
贵州省科技支撑计划(黔科合支撑[2019]2758号)
贵州省基础研究计划(黔科合基础-ZK[2022]一般620)。
关键词
环介导等温扩增
支原体
16S
rRNA
Loop-mediated isothermal amplification(LAMP)
Mycoplasma
16S rRNA
