二次PCR用于临床标本真菌感染快速分子诊断的研究

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

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（χ^2=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（χ^2=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。

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%o 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 （χ^2 = 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 （χ^2 = 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.