Bialek Ralf, Kern Jan, Herrmann Tanja, Tijerina Rolando, Ceceñas Luis, Reischl Udo, González Gloria M
Institute for Tropical Medicine, University Hospital Tübingen. Institute of Medical Microbiology, University of Regensburg, Germany.
J Clin Microbiol. 2004 Feb;42(2):778-83. doi: 10.1128/JCM.42.2.778-783.2004.
A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.
设计了一种用于检测波萨达斯球孢子菌DNA的传统巢式PCR和实时荧光定量LightCycler PCR检测方法，并在120株临床菌株中进行了测试。这些菌株是从墨西哥新莱昂州蒙特雷10年内的114名患者中分离出来的，该地已知是球孢子菌病的流行地区。编码特异性抗原2/富含脯氨酸抗原（Ag2/PRA）的基因用作靶标。所有菌株均被正确鉴定，而发癣菌科相关成员的DNA仍为阴性。通过LightCycler进行熔解曲线分析以及对第一次PCR的526 bp产物进行测序，结果显示与现在已知为波萨达斯球孢子菌的Silveira菌株的GenBank序列（登录号AF013256）有100%的同一性，或者在第1228位有一个单碱基沉默突变。对两个含微卫星的位点（GAC和621）进行长度测定，将所有120株分离株鉴定为波萨达斯球孢子菌。通过传统巢式PCR从三个显微镜下观察到有球形体的石蜡包埋组织样本中扩增出了特异性DNA，而20份对其他双相真菌呈阳性的人体组织样本仍为阴性。此外，使用10株菌株评估了一种改良的市售DNA提取方法每个步骤的安全性。结果表明该方法至少有三个步骤能充分杀死关节孢子。这种安全的方法适用于培养物和临床标本。
