Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification.
Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification.
复制标题
提高环介导等温扩增特异性和灵敏度的两种方法
DOI：
10.3390/molecules20046048
复制
发表时间：
2015-04-07
期刊：
影响因子：
--
通讯作者：
Tomasula PM
中科院分区：
文献类型：
--
作者：
Wang DG;Brewster JD;Paul M;Tomasula PM
环介导等温扩增（LAMP）技术利用四个（或六个）引物靶向基因组的相当小的区段内的六个（或八个）区域进行扩增，其浓度高于传统PCR方法中使用的浓度。使用的高浓度引物导致引物二聚体诱导的非特异性扩增的可能性增加。本研究针对单核细胞增生李斯特菌的prfA基因序列设计了一组LAMP引物，并采用二甲基亚砜（DMSO）和Touchdown LAMP来提高LAMP反应的灵敏度和特异性。结果表明，新设计的引物和添加剂的这种新的LAMP检测方法的检测限为10 fg/反应，这是10倍以上的敏感性比商业等温扩增试剂盒和100倍以上的敏感性比以前报道的LAMP检测。这种高灵敏度的LAMP检测方法已被证明可以检测11株单核细胞增生李斯特菌，而不能检测其他李斯特菌属（包括无害李斯特菌和李斯特菌invanovii），在特异性方面优于商业等温扩增试剂盒和先前报道的LAMP检测方法。
The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.
登录
查看更多内容
影响因子：
4
作者：
Wozniakowski, G.;Niczyporuk, J. S.;Gawel, A.
通讯作者：
Gawel, A.
影响因子：
3.1
作者：
Perera, Namal;Aonuma, Hiroka;Kanuka, Hirotaka
通讯作者：
Kanuka, Hirotaka
影响因子：
3.3
作者：
Nagamine, K;Hase, T;Notomi, T
通讯作者：
Notomi, T
影响因子：
2.4
作者：
Prompamorn, P.;Sithigorngul, P.;Chaivisuthangkura, P.
通讯作者：
Chaivisuthangkura, P.
影响因子：
2.6
作者：
Tang, Meng-Jun;Zhou, Sheng;Gao, Yu-Shi
通讯作者：
Gao, Yu-Shi