肺结核继发真菌感染患者分离真菌的基因检测与鉴定的研究

Study on detection and identification of genes for the fungi isolated from pulmonary tuberculosis patients with secondary fungal infection

目的探讨肺结核继发真菌感染患者分离致病性真菌的基因鉴定及其类型。方法采集80例肺结核患者的下呼吸道分泌物标本,接种科玛嘉(Chrom)琼脂平板、置温箱内28℃培养48～72h。分离的真菌分别以常规真菌学方法、PCR扩增核糖体基因转录间隔区(ITS)序列和25SrDNAⅠ型内含子序列进行鉴定。结果分离的99株真菌含78株酵母菌(78.8%)和21株霉菌(21.2%)。78株酵母菌的ITS基因鉴定法与生物学鉴定法的符合率为83.3%;21株霉菌ITS基因鉴定法与生物学鉴定法符合率为71.4%。99株真菌的ITS基因鉴定法与生物学鉴定法总符合率为80.8%。49株白假丝酵母菌经25SrDNAⅠ型内含子基因鉴定分为3型,包括基因A型21株(40.8%)、基因B型14株(28.5%)以及基因C型15株(30.6%),未发现基因D型与基因E型菌株。结论花都区肺结核患者病原性真菌感染常见为酵母菌,其中主要是白假丝酵母菌,基因型为A型。检测ITS序列和25SrDNAⅠ型内含子序列有助于真菌感染的快速基因诊断和提高菌种鉴定的准确率。

Objective To explore the gene test methods and gene type of the pathogenic fungi isolated from pulmonary tuberculosis patients with secondary fungal infection.Methods The lower respiratory secretions collected from 80 patients with pulmonary tuberculosis were inoculated into the CHROM agar medium and cultured at 25℃ for 48～96 h.The cultures of fungi were identified respectively by routine mycological methods and gene test for ITS and 25S rDNA I intron.Results 99 strains of fungi were isolated from the samples,including 78 strains of yeasts（78.8%） and 21 strains of mold（21.2%）.The coincidences between the methods of biology and gene test for ITS were respectively 83.3% for the yeast,71.4% for the mold,and the total coincidence is 80.8% for 99 fungi.With the 25S rDNAⅠintron detection,49 C.albicans were identified as 25 genetype A（40.8%）,14 genetype B（28.5%） and 15 genetype C（30.6%）.Conclusion The common fungi cause secondary infection of the patients with pulmonary tuberculosis are the yeast and in which the C.albicans with genetype A were showed a higher percentage in Huadu area.Determination of ITS and 25S rDNAⅠ intron would be helpful to the rapid gene diagnosis for the fungi infection and improving the accurate rate to the determination of laboratory diagnosis.