纳米金基因芯片技术快速鉴定常见浅部致病真菌

发布时间：2018-03-14 19:31

本文选题：浅部致病真菌　切入点：靶基因序列　出处：《华中科技大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的从标准真菌菌株、临床分离培养的常见浅部致病真菌菌株及阴性对照菌株中提取DNA并扩增出靶基因序列ITS区，针对此区域设计出特异性寡核苷酸探针为后续试验提供基础。方法将标准真菌菌株、临床分离培养的常见浅部真菌菌株及阴性对照菌株参照Biospin真菌基因组DNA提取试剂盒说明操作提取DNA。采用真菌通用引物ITSl（5’端生物素标记）和ITS4对提取的DNA进行PCR反应得到靶基因序列即ITS区并进行电泳检测。运用Array Designer4软件在真菌ITS1或ITS2区设计特异性寡核苷酸探针，依据长度、Tm值、G+C含量、发夹结构、自身二聚体以及重复碱基数等指标筛选出最好的探针，，提交到Genbank进行BLAST特异性分析后进行合成。结果所提取的真菌基因组DNA的PCR产物经琼脂糖凝胶电泳均能扩增出目的条带，扩增产物大小在500-700bp之间。运用探针设计软件设计出了14个特异性寡核苷酸探针，其中红色毛癣菌和紫色毛癣菌共用特异性探针Trirv，犬小孢子菌和铁锈色小孢子菌共用特异性探针Miccf，断发毛癣菌和许兰氏毛癣菌共用特异性探针Trits；其他菌株均有各自的种特异性探针。结论 Biospin真菌基因组DNA提取试剂盒能简便快速的提取真菌DNA；采用通用引物ITSl和ITS4对提取的DNA进行PCR反应能扩增出真菌靶基因序列ITS区，自此区运用Array Designer4软件能设计出待检真菌的特异性寡核苷酸探针。目的为了快速简便、准确直观的鉴定临床常见浅部致病真菌，建立一种采用纳米金基因芯片技术对临床常见浅部致病真菌鉴定的方法。方法将设计出的探针按设计好的顺序排列在醛基化的玻璃片上制备所需的基因芯片。将制备的基因芯片与待检真菌PCR产物杂交，杂交后将纳米金标记的链霉亲和素与芯片温育形成生物素-链酶亲和素-纳米金生物反应放大系统，最后结合银增强染色将检测信号再次放大后直接目测实验结果。结果 3’端氨基化修饰的真菌特异性寡核苷酸探针与醛基化的玻璃片反应后牢固的固定于玻片上，制备出了所需的基因芯片。运用所制备的基因芯片对真菌标准菌株、临床分离的常见浅部致病真菌菌株及阴性对照菌株按上述方法进行了检测。结果显示17株皮肤癣菌和念珠菌的标准菌株对应的特异性探针及阳性对照点可目测到阳性信号，阴性对照及其他探针点无信号,与实际菌株种型相符；2株阴性对照菌株检测结果仅阳性对照点能观察到信号。临床分离的32株皮肤癣菌、33株念珠菌的基因芯片检测结果与临床常规鉴定方法结果一致。结论本实验所制备的纳米金基因芯片能简便快捷、直观准确的鉴定出临床常见的浅部致病真菌。
[Abstract]:Purpose. DNA was extracted from standard fungal strains, common superficial pathogenic fungi strains and negative control strains, and the ITS region of target gene sequence was amplified. Specific oligonucleotide probes were designed for this region to provide the basis for further experiments. Method. Standard fungal strains, Clinical isolation and culture of common superficial fungal strains and negative control strains with reference to the Biospin fungal genomic DNA extraction kit instructions for the extraction of DNA. The fungal universal primer ITSl5'biotinylated) and ITS4 were used to reverse the PCR of extracted DNA. The target gene sequence, ITS region, should be obtained and detected by electrophoresis. Specific oligonucleotide probes were designed using Array Designer4 software in the ITS1 or ITS2 region of fungi. The best probes were screened according to the content of TM, hairpin structure, self dimer and repeat base number. The probe was submitted to Genbank for BLAST specificity analysis and then synthesized. The PCR products of the extracted fungal genomic DNA could be amplified by agarose gel electrophoresis with the size of 500-700bp. Fourteen specific oligonucleotide probes were designed by using probe design software. Among them, Trichophyton rubrum and Trichophyton purplicus shared specific probe Trirvus, Microsporum canis and Microsporum ferruginosa shared specific probe Miccf. Tritsm and Trichophyton schulanensis shared specific probe; other strains had their own specific probes. Specific probe. Conclusion. The genomic DNA extraction kit of Biospin fungi can be used to extract fungal DNA easily and quickly, and the ITS region of fungal target gene sequence can be amplified by PCR reaction of DNA extracted by universal primer ITSl and ITS4. The specific oligonucleotide probes of fungi can be designed by using Array Designer4 software. Purpose. In order to identify the common superficial pathogenic fungi in clinic quickly, conveniently, accurately and intuitively, a method for identification of common superficial pathogenic fungi by using gold nanoparticles was established. Method. The designed probes were arranged in the designed order on the aldehydated glass to prepare the necessary gene chip. The prepared gene chip was hybridized with the PCR product of the fungus to be tested. After hybridization, the streptavidin labeled with gold nanoparticles was incubated with the chip to form a biotin chain enzyme affinity factor nano gold bioreaction amplification system. Finally, the detection signal was amplified again with silver enhanced staining and the results were directly observed. The 3'-terminal amino modified fungal oligonucleotide probe reacted with the aldehydated glass chip and was firmly fixed on the glass slide, and the required gene chip was prepared. The clinical isolates of common superficial pathogenic fungi and negative control strains were tested according to the above method. The results showed that 17 strains of dermatophytes and Candida could be detected by specific probes and positive control points. Negative control and other probe points without signal, The results of gene chip analysis of 32 clinical isolates of dermatophytes and 33 strains of candida were consistent with the results of routine clinical identification. Conclusion. The nano gold gene chip can be used to identify the common superficial pathogenic fungi.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756;R440

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