PCR结合反向斑点杂交法检测侵袭性曲霉感染的初步实验研究

目的评价PCR结合反向斑点杂交法检测动物模型和临床免疫抑制患者的侵袭性曲霉感染的可行性。方法建立侵袭性曲霉感染动物模型,收集动物血清和健康人群、免疫抑制患者的血清,进行PCR-种特异性探针检测。以1对真菌特有的28S rRNA保守序列结构作为真菌通用引物,以临床常见的4个曲霉菌种,即烟曲霉、黄曲霉、黑曲霉、土曲霉的种特异性序列为种特异性探针,与扩增产物进行反向斑点杂交。结果PCR-探针杂交检测动物接种后24h时阳性血清为16/24,48h为18/24,72h为19/24,96h为24/24。阳性率为80%,特异性为95.8%。同时取血行真菌培养,烟曲霉阳性率仅为5%。临床标本显示两例确诊IA患者血清均为阳性,且能鉴定菌种。健康对照人群标本检测显,阴性38例,假阳性2例。结论PCR-探针杂交法较传统血培养法能更快速、灵敏地检测动物模型的侵袭性曲霉感染,并可用于对IA高危患者的监测。

Study on diagnosis of invasive aspergiuosis by PCR and dot blot hybridization

Objective To establish and evaluate a molecular diagnostic method for diagnosis of invasive aspergillosis （IA） in animal modal and immunocompromised patients. Methods Rabbit model of IA was established and sera was collected. Fungal DNA was detected in sera from rabbits, health people and immunocompromised patients by PCR combined with species-specific probe hybridization. Results Sixteen in 24 sera samples showed positive for PCR and dot blot hybridization 24 h after innoculation, 18/24 after 48 h, 19/24 afer 72 h respectively and all showed positive after 96 h. The sensitivity and specificity of the assay were 80% and 95. 8% , with 5% positive rate for blood culture. Thirty eight negative sera and 2 false positive sera were found in health people. Sera of 2 IA patients showed positive results. Conclusions PCR combined with species-specific probe hybridization for serum is more sensi- tive and rapidly than traditional blood culture for early diagnosis of IA in animal model,which may be helpful to monitor the patients at risk for IA.