【摘要】 目的探讨PCR技术在快速诊断艾滋病(AIDS)合并播散性马尔尼菲青霉病(PSM)中的应用价值。方法收集21例AIDS合并播散性PSM患者抗真菌治疗前的系列标本:未培养血液、阳性血液培养物和阳性骨髓培养物,以12例健康人血液为阴性对照。提取上述标本的基因组DNA,采用真菌通用引物ITS1和ITS4对基因组DNA进行PCR扩增,扩增产物测序后与GenBank核酸序列数据库进行比对分析。结果 21份阳性血液培养物、21份阳性骨髓培养物及2份未培养血液标本均扩增出目的条带,而12份健康人血液标本未扩增出条带。阳性PCR产物测序结果与广东、日本、泰国、印度尼西亚的马尔尼菲青霉菌(PM)rDNA ITS序列(登录号分别为AB298970、AB298950、L37406、AJ853738)一致性高达97%～100%,证实为PM。PM与新型隐球菌及烟曲霉菌rDNA ITS序列的遗传距离最远,与青霉菌属其他菌种绳状青霉菌和桔青霉菌rDNAITS序列的遗传距离最近。结论 PCR可敏感、特异的检测出培养阳性标本中PM,并能有效缩短PM检出时间,但直接采用患者血液进行PCR检测的阳性率较低,方法有待改进。更多还原

【Abstract】 Objective To explore the application value of PCR technique on rapid diagnosis of disseminated penicillosis marneffei in patients with AIDS.Methods Total of 21 AIDS cases complicated with penicillosis marneffei were enrolled to collect a serial samples including uncultured blood samples,positive blood cultures and positive bone marrow cultures before receiving antifungal therapy.Twelve blood samples from healthy individuals were processed as negative controls.The genome DNA were extracted from these specimens and amplified with fungal consensus primers ITS1 and ITS4.Subsequently,the PCR products were sequenced,then the sequences were aligned with those nucleotide sequences in GenBank.Results A 500-600 bp DNA product was detected in all 21 positive blood cultures and positive bone marrow cultures,and 2 uncultured blood samples,but not in 12 blood samples from healthy individuals.The sequences of these PCR products were highly concordant(97%-100%)with those Penicilium marneffei(PM)isolates originated from Guangdong,Japanese,Thailand and Indonesia(accessions:AB298970,AB298950,L37406,AJ853738),which proved it to be PM.The genetic distances of rDNA ITS sequences between PM and Neoformans cryptococcus or Aspergillus fumigatus were the farthest,while were closest between PM and Penicillium funiculosum or Penicillium citrinum belonging to Penicillium species.Conclusions PM in positive culture samples could be sensitively and specifically detected by PCR,which can effectively shorten the time for PM identification.However,the positive rate of directly detecting PM in uncultured blood by PCR is very low,which needs further improvement.更多还原