中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (6): 390-392.

• 论著 • 上一篇    下一篇

二次PCR用于临床标本真菌感染快速分子诊断的研究

吕雪莲1,刘泽虎2,梅亚宁3,张晓利1,刘维达1   

  1. 1. 南京 中国医学科学院北京协和医学院皮肤病研究所
    2. 中国医学科学院、中国协和医科大学皮肤病研究所
    3. 南京医科大学第一附属医院临床检验中心
  • 收稿日期:2008-06-11 修回日期:2009-02-19 出版日期:2009-06-15 发布日期:2009-06-03
  • 通讯作者: 吕雪莲
  • 基金资助:

    不符合上述请自行填写具体基金资助项目名称（编号）

Application of two-round PCR in rapid molecular diagnosis of fungus-infected clinical specimens

  • Received:2008-06-11 Revised:2009-02-19 Online:2009-06-15 Published:2009-06-03
  • Contact: LU Xue-lian

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摘要：

目的 探讨二次PCR技术诊断疑似真菌感染临床标本的敏感性。方法 收集临床疑似真菌感染的深部位痰及肺泡灌洗液标本共29份，分别进行真菌直接镜检、真菌培养、真菌通用引物单次与二次PCR扩增rDNA的ITS区，并对真菌检出阳性率和多种真菌菌种检出率进行比较分析。结果 临床疑似病例痰及肺泡灌洗液标本真菌镜检、真菌培养、单次PCR和二次PCR真菌检出阳性率分别为20.7%（6/29）、37.9%（11/29）、17.2%（5/29）和48.3%（14/29）。真菌培养、单次PCR和二次PCR提示二种以上真菌菌种检出的比例分别为6.9%（2/29）、3.4%（1/29）和24.1%（7/29）。二次PCR与单次PCR的真菌检出率差异有统计学意义（χ2 = 6.34，P < 0.05）。在两种以上菌种检出率方面，二次PCR与真菌培养和单次PCR间差异均有统计学意义（χ2 = 4.09，6.30，P值均 < 0.05）。结论 二次PCR技术有助于提高临床标本真菌分子诊断的敏感性。

关键词: 聚合酶链反应;深部真菌感染;分子诊断

Abstract:

Objective To establish a diagnosis method for fungal infection using two-round PCR, and to evaluate its sensitivity in the detection of clinical specimens suspected to be infected with fungi. Methods A total of 29 specimens of clinical sputum and alveolar wash solution were collected from patients with suspicious fungal infection. All specimens underwent direct microscopy with 10% KOH, fungal culture, one-round PCR and two-round PCR. The fungal universal primer targeting ITS regions of rDNA was used in PCR. The detection rate for fungi was compared between these methods. Results The detection rate for fungi was 20.69% by direct microscopy, 37.9% by fungal culture, 17.2% by one-round PCR, and 48.3% by two-round PCR. More than one species of fungus were detected in 6.9% （2/29）, 3.4% （1/29） and 24.1% （7/29） of these specimens by fungal culture, one-round PCR and two-round PCR, respectively. There was a significant difference in the detection rate between two- and one-round PCR （χ2 = 6.34，P < 0.05）. With regard to the detection rate for more than one species of fungus, two-round PCR was significantly higher than one-round PCR and fungal culture （χ2 = 4.09, 6.30，both P < 0.05）. Conclusion Two-round PCR may help to improve the sensitivity of molecular diagnosis of fungus-infected specimens.