Hong S T
Department of Parasitology, Seoul National University College of Medicine, Korea.
Korean J Parasitol. 1996 Sep;34(3):191-6. doi: 10.3347/kjp.1996.34.3.191.
Polymerase chain reaction (PCR) is a powerful technique to detect scanty amount of DNA from living organisms. The present study intended to develop specific primers for PCR diagnosis of pneumocystosis and to evaluate diagnostic efficacy by preparation of template DNAs from invasive BAL fluid and also to screen serum or blood as a non-invasive specimen. Albino rats of Wistar or Fischer strains were experimentally infected by Pneumocystis carinii. Extracted DNAs or cell lysates of their blood, bronchoalveolar lavage fluid, and lung homogenate were used as the template DNA. Primers were synthetic oligonucleotides among 16s rDNA sequences. All of the primer combinations gave PCR products, but the primer pair of #24 and #27 gave best quality product of 666 bp. The sensitivity of PCR with lysates of BAL fluid was 57.7% but it increased to 84.6% with extracted DNAs. None of BAL lysate or DNA was positive among 13 microscopically negatives. The serum DNAs were positive only in 2 cases out of 20 morphologically positive rats. DNAs of human, rat, other parasites, yeast, and microorganisms were negative. The findings suggest that the present primers are specific but simple lysate of BAL fluid is not sensitive. PCR may be used as a routine diagnostic method of pneumocystosis if simple and rapid preparation of non-invasive clinical specimens are available.
聚合酶链反应（PCR）是一种用于检测生物体中微量DNA的强大技术。本研究旨在开发用于肺孢子虫病PCR诊断的特异性引物，并通过从侵入性支气管肺泡灌洗（BAL）液制备模板DNA来评估诊断效果，同时筛选血清或血液作为非侵入性标本。用卡氏肺孢子虫对Wistar或Fischer品系的白化大鼠进行实验性感染。将其血液、支气管肺泡灌洗液和肺匀浆的提取DNA或细胞裂解物用作模板DNA。引物是16s rDNA序列中的合成寡核苷酸。所有引物组合均产生了PCR产物，但引物对#24和#27产生了质量最好的666 bp产物。用BAL液裂解物进行PCR的灵敏度为57.7%，但用提取的DNA时灵敏度提高到了84.6%。在13例显微镜检查为阴性的样本中，BAL裂解物或DNA均无阳性。在20例形态学阳性的大鼠中，血清DNA仅2例呈阳性。人、大鼠、其他寄生虫、酵母和微生物的DNA均为阴性。研究结果表明，本研究中的引物具有特异性，但BAL液的简单裂解物不敏感。如果能够简单快速地制备非侵入性临床标本，PCR可作为肺孢子虫病的常规诊断方法。
