Wildfeuer A, Schlenk R, Friedrich W
Research and Development, Pfizer/Mack, Illertissen, Germany.
Mycoses. 1996 Sep-Oct;39(9-10):341-6. doi: 10.1111/j.1439-0507.1996.tb00150.x.
The in vitro and in vivo selectivity and sensitivity of a yeast-specific primer system was investigated. A two-step polymerase chain reaction (PCR) was used: the first amplified a 245-bp fragment of the gene for cytochrome P450L1A1 and the second a product of 193 bp. This nested PCR produced an approximately 1000-fold increase in the sensitivity of the test for Candida albicans DNA compared with the first primer pair. The lower level of sensitivity of the test in physiological saline and tissue homogenate was about 10 C. albicans cells ml-1. On the other hand, the sensitivity of the nested PCR method was reduced by a factor of more than 1000 when C. albicans was fixed with 4% formalin. After i.v. injection of different doses of C. albicans into mice, the yeast could be demonstrated in blood and in six different organs. The nested PCR was to some extent more sensitive than culturing for the detection of the yeast in the specimens of organs such as lung, cardiac muscle, liver, kidneys and brain. In contrast, in blood and spleen the culture was superior to the PCR technique used. Nested PCR is thus a useful additional method for the demonstration of yeasts.
研究了一种酵母特异性引物系统的体外和体内选择性及敏感性。采用两步聚合酶链反应(PCR):第一步扩增细胞色素P450L1A1基因的245 bp片段,第二步扩增193 bp的产物。与第一对引物相比,这种巢式PCR使白色念珠菌DNA检测试验的敏感性提高了约1000倍。在生理盐水和组织匀浆中该试验的较低敏感性水平约为每毫升10个白色念珠菌细胞。另一方面,当白色念珠菌用4%福尔马林固定后,巢式PCR方法的敏感性降低了1000倍以上。给小鼠静脉注射不同剂量的白色念珠菌后,可在血液和六个不同器官中检测到该酵母。在检测肺、心肌、肝、肾和脑等器官标本中的酵母时,巢式PCR在一定程度上比培养更敏感。相比之下,在血液和脾脏中,培养优于所使用的PCR技术。因此,巢式PCR是一种用于检测酵母的有用辅助方法。