Costa C, Vidaud D, Olivi M, Bart-Delabesse E, Vidaud M, Bretagne S
Laboratoire de Parasitologie-Mycologie, Hôpital Henri Mondor-APHP and Université Paris XII, 51 avenue du Général DeLattre de Tassigny, 94010, Cedex, Créteil, France.
J Microbiol Methods. 2001 Apr;44(3):263-9. doi: 10.1016/s0167-7012(01)00212-3.
Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.
已经开发了几种聚合酶链反应（PCR）检测方法，用于检测侵袭性曲霉病患者血液中的烟曲霉DNA。然而，尚未确定最佳的血液检测部分，并且用作DNA扩增靶标的多拷贝基因也未得到表征。首先，我们基于TaqMan技术开发了一种针对单拷贝基因的实时PCR检测方法。为了比较血清、白细胞沉淀和血浆作为血液检测部分的有效性，我们向全血中加入烟曲霉DNA，并对这些部分进行类似处理。白细胞沉淀和血清之间的差异不显著。相比之下，血浆的产量比血清低10倍。然后，我们比较了立即处理或在室温下放置24小时后的血清，发现24小时后的产量较低。其次，还开发了一种针对线粒体基因的实时PCR检测方法。估计每个单拷贝基因的线粒体基因拷贝数在9到10个之间。因此，我们建议使用尽快储存和冷冻的血清来检测循环中的烟曲霉DNA以进行诊断。此外，对线粒体多拷贝基因进行了表征，以便比较不同患者的结果。
