常见角膜致病真菌基因芯片检测方法的实验

Gene microarray for the detection and identification of pathogenic species in fungal keratitis

目的:建立一种根据基因芯片原理,对国内真菌性角膜病常见致病菌种进行快速检测和准确检测的实验方法。方法:实验于2004-11/2006-04在河南省生物工程中心分子生物学实验室和河南省角膜病重点实验室完成。将针对国内临床上常见的角膜致病真菌6属12种,包括腐皮镰孢菌、串珠镰孢菌、梨状镰孢菌、尖孢镰孢菌、烟曲霉菌、黄曲霉菌、土曲霉菌、黑曲霉菌、新月弯孢菌、牵连青霉菌、链格孢霉菌、白色念珠菌,并经修饰的特异性寡核苷酸探针固定在两种材料的片基上。用一对通用引物,对上述真菌的DNA基因片断进行扩增,将带有特殊标记的扩增产物与片基上排列的探针进行杂交,对12株标准菌株和82株临床分离株样本采用双盲法原则进行检测,从而对两种不同片基芯片的特异性和灵敏度进行判断和评估。结果:①在两种不同材料的片基上,均可以高效率结合经过修饰和加尾的寡核苷酸探针,玻片约为35mer,尼龙膜约为400mer,可靠性较好。②通过琼脂糖凝胶电泳观察,12种真菌均产生了530～630bp的聚合酶链反应扩增产物,在相同的条件下对其进行杂交检测,得到了具有各自特征的杂交后显色图谱,玻片为荧光显色,尼龙膜为生物素-亲和素显色,可直接从杂交显色图谱上对不同菌种区分判断。③两种芯片均可与低于琼脂糖凝胶电泳显色浓度一个数量级的聚合酶链反应扩增产物反应,敏感性高于聚合酶链反应检测;在对82株临床分离株样本检测中,79株经聚合酶链反应扩增琼脂糖凝胶电泳均显示扩增产物条带,无非特异性杂交反应发生;玻片灵敏度为92.4%,尼龙膜灵敏度87.3%,χ2检验差异无显着性(P>0.05)。结论:实验建立了以ITS区基因为靶标的寡核苷酸探针芯片检测系统,具有较好的特异性和灵敏度,能够在三四个小时内完成对临床上常见的12种角膜致病真菌的菌种检测。

AIM： To develop a technology for the rapid detection and identification of pathogenic species inducing fungal keratitis in China based on gene chip. METHODS： The experiment was carried out in the Molecular Biology Laboratory, ＂ Bioengineering Center of Henan Province.and Henan.Key Laboratory of Keratonosus from November 2004 to April 2006. Twelve species of fungi causing keratitis including Fusarium solani, Fusarium moniliforme, Fusarium poae, Fusarium oxysporum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Curvularia lunatus, Penicillium implicatum, Altemaria alternate and C.andida albicans ware fixed on two materials chip with modified oligonucleotide probes. The chromosome DNA of these fungi was amplified with a pair of universal primers and the products with specific labels ware applied to the oligonucleotide on the chip for specific hybridization corresponding to each species, and the result was observed with 12 standard strains and 82 clinical isolates based on double-blind principle to assess the specificity and sensitivity of the chips. RESULTS： ①The chips based on two different materials combined with the modified oligonucleotide probes in a high efficiency. The slide was about 35 mer, and the nylon membrane was 400 mer with a good reliability. ②Agarose gel electrophoresis observation showed that about 530-630 bp PCR products of 12 species of fungi were found, and the hybrid color map was obtained with respective features under the same conditions as in the hybridization. The slide was fluorescence, and the nylon membrane was bioepiderm-avidin. The fungi could be identified according to the hybrid color map directly. ③The two kinds of chips could react with PCR product at the concentration lower than that of coloration in agarose gel electrophoresis with a higher sensitivity than PCR detection. In testing of 82 clinical isolates, the amplified product bends ware found in 79 fungi amplified by PCR and agarose gel electrophoresis, but no non-specific hybridization action; the sensitivity of slide was 92.4%, and the nylon membrane was 87.3%; no significant differences ware found in χ^2 test （P 〉 0.05）. CONCLUSION： Oligonucleotide probes detection system with the gene in ITS region as target is established, which has a better specificity and sensitivity, and could detect and identify the 12 clinical pathogenic species causing fungal keratitis in three to four hours.