A study of rapid diagnosis for invasive pulmonary aspergillosis by molecular biological technique

我们建立了一种巢式PCR方法，用于检测曲霉病动物模型和侵袭性肺曲霉病患者血清中曲霉菌属特异性DNA。1）巢式PCR：用14株临床重要真菌和10株细菌进行PCR检测，根据18 S可变区V7和V9序列设计了两套寡核苷酸引物，烟曲霉的rRNA基因。单PCR和巢式PCR均能从5株曲霉菌中扩增出特异性片段，而从其他微生物中均未扩增出。用巢式PCR方法检测目标DNA，仅需50 fg的烟曲霉菌DNA。2）动物模型血清中曲霉菌特异性DNA的检测。目的：研究实验性曲霉病小鼠和大鼠血清中曲霉菌特异性DNA的检测。用巢式PCR方法在71%的感染小鼠血清中检测到特异性片段，其中43%的血清中检测到半乳甘露聚糖抗原。在大鼠模型中，我们也显示了巢式PCR比半乳甘露聚糖抗原血症测定和（1*3）β-D-葡聚糖测定更高的灵敏度。结果：20例病理诊断为侵袭性肺曲霉菌病的患者中，14例（70%）巢式PCR阳性，12例（60%）半乳甘露聚糖抗原阳性。

We developed a nested PCR method for detecting DNA specific for Aspergillus species in serum samples of animal medels of aspergillosis and patients with invasive pulmonary aspergillosis.1)Nested PCR : Fourteen strains of clinically important fungi and 10 strains of bacteria were used for examination of specificity of the PCR.Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 and V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A.fumigatus.2)Detection of DNA specific for Aspergillus species in serum of animal models. : We investigated the detection of DNA specific for Aspergillus species in serum of murine and rats with experimental aspergillosis. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice, while galactomannan antigen was detected in 43% of them. In rats model, we have also shown enhanced sensitivity of the nested PCR over galactomannan antigenemia assay and (1*3) beta-D-glucan assay.Detection of Aspergillus DNA in clinical samples. : The nested PCR was positive for 14 of 20 patients (70%) with a histological diagnosis of invasive pulmonary aspergillosis, while galactomannan antigen was positive 12 (60%).In conclusion, the high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for immunocompromised patients with invasive pulmonary aspergillosis.