Brancart Françoise, Rodriguez-Villalobos Hector, Fonteyne Pierre-Alain, Peres-Bota Daliana, Liesnard Corinne
Laboratory of Microbiology, Erasme Hospital-ULB, 808 Route de Lennik, 1070 Brussels, Belgium.
J Microbiol Methods. 2005 Jun;61(3):381-7. doi: 10.1016/j.mimet.2005.01.001.
We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.
我们基于靶向β-微管蛋白编码基因的引物和探针，开发了一种用于检测和定量支气管肺泡灌洗（BAL）标本中耶氏肺孢子菌的定量实时PCR检测方法。该检测方法能够检测每毫升含有靶序列的标准质粒中的50个DNA拷贝。在5个对数（10）值范围内，批内和批间变异系数分别为0.46%-4.27%和0.05%-2.00%。对57份人、病毒、细菌和真菌DNA样本进行扩增，结果均为阴性。对53份送至实验室诊断肺孢子菌病的BAL样本，采用实时PCR以及吉姆萨染色和直接免疫荧光的直接显微镜检查（DME）进行前瞻性研究。所有PCR阴性样本经显微镜检查均为阴性。在24份（45%）PCR阳性的BAL样本中，8份经显微镜检查为阳性（35%）。显微镜检查阳性样本的靶基因拷贝数为每毫升4.4×10³至2.8×10⁶，显微镜检查阴性样本的靶基因拷贝数为每毫升8至9.2×10³。总之，我们开发了一种快速、灵敏且特异的实时PCR方法，用于诊断和定量BAL样本中的耶氏肺孢子菌。
