本文選題：淺部致病真菌　切入點：靶基因序列　出處：《華中科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 從標(biāo)準(zhǔn)真菌菌株、臨床分離培養(yǎng)的常見淺部致病真菌菌株及陰性對照菌株中提取DNA并擴(kuò)增出靶基因序列ITS區(qū)，針對此區(qū)域設(shè)計出特異性寡核苷酸探針為后續(xù)試驗提供基礎(chǔ)。 方法 將標(biāo)準(zhǔn)真菌菌株、臨床分離培養(yǎng)的常見淺部真菌菌株及陰性對照菌株參照Biospin真菌基因組DNA提取試劑盒說明操作提取DNA。采用真菌通用引物ITSl（5’端生物素標(biāo)記）和ITS4對提取的DNA進(jìn)行PCR反應(yīng)得到靶基因序列即ITS區(qū)并進(jìn)行電泳檢測。運(yùn)用Array Designer4軟件在真菌ITS1或ITS2區(qū)設(shè)計特異性寡核苷酸探針，依據(jù)長度、Tm值、G+C含量、發(fā)夾結(jié)構(gòu)、自身二聚體以及重復(fù)堿基數(shù)等指標(biāo)篩選出最好的探針，，提交到Genbank進(jìn)行BLAST特異性分析后進(jìn)行合成。結(jié)果 所提取的真菌基因組DNA的PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳均能擴(kuò)增出目的條帶，擴(kuò)增產(chǎn)物大小在500-700bp之間。運(yùn)用探針設(shè)計軟件設(shè)計出了14個特異性寡核苷酸探針，其中紅色毛癬菌和紫色毛癬菌共用特異性探針Trirv，犬小孢子菌和鐵銹色小孢子菌共用特異性探針Miccf，斷發(fā)毛癬菌和許蘭氏毛癬菌共用特異性探針Trits；其他菌株均有各自的種特異性探針。 結(jié)論 Biospin真菌基因組DNA提取試劑盒能簡便快速的提取真菌DNA；采用通用引物ITSl和ITS4對提取的DNA進(jìn)行PCR反應(yīng)能擴(kuò)增出真菌靶基因序列ITS區(qū)， 自此區(qū)運(yùn)用Array Designer4軟件能設(shè)計出待檢真菌的特異性寡核苷酸探針。 目的 為了快速簡便、準(zhǔn)確直觀的鑒定臨床常見淺部致病真菌，建立一種采用納米金基因芯片技術(shù)對臨床常見淺部致病真菌鑒定的方法。 方法 將設(shè)計出的探針按設(shè)計好的順序排列在醛基化的玻璃片上制備所需的基因芯片。將制備的基因芯片與待檢真菌PCR產(chǎn)物雜交，雜交后將納米金標(biāo)記的鏈霉親和素與芯片溫育形成生物素-鏈酶親和素-納米金生物反應(yīng)放大系統(tǒng)，最后結(jié)合銀增強(qiáng)染色將檢測信號再次放大后直接目測實驗結(jié)果。結(jié)果 3’端氨基化修飾的真菌特異性寡核苷酸探針與醛基化的玻璃片反應(yīng)后牢固的固定于玻片上，制備出了所需的基因芯片。運(yùn)用所制備的基因芯片對真菌標(biāo)準(zhǔn)菌株、臨床分離的常見淺部致病真菌菌株及陰性對照菌株按上述方法進(jìn)行了檢測。結(jié)果顯示17株皮膚癬菌和念珠菌的標(biāo)準(zhǔn)菌株對應(yīng)的特異性探針及陽性對照點可目測到陽性信號，陰性對照及其他探針點無信號,與實際菌株種型相符；2株陰性對照菌株檢測結(jié)果僅陽性對照點能觀察到信號。臨床分離的32株皮膚癬菌、33株念珠菌的基因芯片檢測結(jié)果與臨床常規(guī)鑒定方法結(jié)果一致。 結(jié)論 本實驗所制備的納米金基因芯片能簡便快捷、直觀準(zhǔn)確的鑒定出臨床常見的淺部致病真菌。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R756;R440

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