本文關(guān)鍵詞： 致病性細(xì)菌 港口航道 基因芯片 檢測　出處：《南方農(nóng)業(yè)學(xué)報》2017年07期 　論文類型：期刊論文

【摘要】：【目的】制備出一套針對港口航道致病性細(xì)菌檢測的基因芯片,為港口航道基因芯片檢測技術(shù)的研究及應(yīng)用打下基礎(chǔ)。【方法】采用常規(guī)方法提取目標(biāo)菌株基因組DNA,以16S r DNA通用引物和gyr B基因保守區(qū)引物進行PCR擴增,使用Allele ID 6.0和Array Designer 4.25對擴增獲得的目的片段進行寡核苷酸探針設(shè)計,經(jīng)PCR篩選驗證,目標(biāo)探針以氨基化修飾后通過芯片點樣儀點制在醛基玻片上;優(yōu)化芯片雜交固定條件,并用于港口航道的水樣檢測,以驗證微陣列基因芯片的檢測效果�！窘Y(jié)果】優(yōu)化后的芯片雜交固定條件為探針點樣濃度10μmol/L、紫外交聯(lián)時間2.0 h、雜交溫度65℃,有效提高了基因芯片檢測的靈敏度,與傳統(tǒng)檢測方法相比可實現(xiàn)快速、高通量、準(zhǔn)確的目標(biāo)。研制的微陣列基因芯片可特異性檢測出港口航道中含有的霍亂弧菌(Vibrio cholerae)、陰溝腸桿菌(Emterobacter cloacae)、溶藻弧菌(V.alginolytivus)、哈氏弧菌(V.harveyi)、副溶血弧菌(V.parahemolyticus)、嗜水氣單胞菌(Aeromonas hydrophila)和創(chuàng)傷弧菌(V.vulnificus)等7株致病性細(xì)菌,且均未出現(xiàn)非特異性雜交�！窘Y(jié)論】針對港口航道致病性細(xì)菌建立的微陣列基因芯片檢測技術(shù)具有特異性強、靈敏度高、快速便捷的特點,可用于港口航道及周邊地區(qū)的海洋環(huán)境監(jiān)測和海產(chǎn)品質(zhì)量安全檢測。
[Abstract]:[objective] to prepare a set of microarray for detection of pathogenicity bacteria in port and waterway. This study laid a foundation for the research and application of gene chip detection in port and waterway. [methods] Genomic DNA of the target strain was extracted by routine method. 16s r DNA universal primers and gyr B gene conserved region primers were used for PCR amplification. Allele ID 6.0 and Array Designer 4.25 were used to design the oligonucleotide probe of the amplified target fragment, which was screened by PCR. The target probe was modified with amino and then was prepared on the aldehyde glass by chip spotting instrument. Optimization of the chip hybridization fixed conditions, and used for port waterway water sample detection. [results] the optimized hybridization conditions were as follows: probe sample concentration 10 渭 mol / L, UV crosslinking time 2.0 h, hybridization temperature 65 鈩，

本文編號：1464866