Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae
Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae
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DOI：
10.1128/jcm.43.4.1581-1586.2005
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发表时间：
2005-04-01
影响因子：
9.4
通讯作者：
Maeno, M
中科院分区：
文献类型：
--
作者：
Seki, M;Yamashita, Y;Maeno, M
很难将肺炎链球菌与基因相似的微型链球菌和口腔链球菌区分开来，这两种细菌都是人类口腔的共生菌。研究了一种新的核酸扩增技术--环介导的等温扩增(LAMP)技术，该技术在63℃等温条件下扩增DNA，具有高特异性、高效率和快速的特点，并对其在肺炎链球菌检测中的适用性进行了研究。设计了针对lytA基因的肺炎链球菌特异性LAMP引物。用10种链球菌和7种非链球菌验证了该引物的特异性。在60min内，该方法可以检测到10个或更多拷贝的纯化肺炎链球菌DNA，灵敏度是常规PCR的1000倍。对另外21株含有毒力因子编码基因(lytA或plY)的临床分离株(口腔链球菌3株、米氏链球菌17株和链球菌1种)进行了鉴别。LAMP方法对肺炎链球菌临床分离株的检测较常规聚合酶链式反应具有更高的选择性。因此，LAMP是诊断肺炎链球菌感染的一种敏感而可靠的方法。
It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.