發展鑑定乳房炎病原菌細菌之寡核苷酸生物晶片

Development of an Oligonucleotide Array for Identification of Bacterial Pathogens of Bovine Mastitis

朱俊育(Chun-Yu Chu)

指導教授 ： 楊季清 ； 張長泉

國立屏東科技大學/農學院/食品科學系所/碩士(2009年)

https://doi.org/10.6346/NPUST.2009.00303

摘要

乳房炎的發生，最常見的是被細菌感染所造成，當病原菌侵入乳腺組織造成炎症的發生。本研究之目的在發展一生物晶片(oligonucleotide array)，期能正確鑑定乳房炎病原菌。這些病原菌包括: Corynebacterium bovis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Mycoplasma bovis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis等。本研究以16S-23S 核糖體核酸基因間隔區(intergenic spacer region)為標的，設計寡核苷酸探針(probe)，點在尼龍膜(nylon membrane)上製成晶片。以PCR放大病原菌菌株之ITS區域後，以晶片雜合反應(hybridization) 鑑定這些菌種。本研究對18種乳房炎病原菌設計了35個特異性探針，將其點製成0.6 cm × 0.5 cm的陣列晶片，經鑑定322個目標菌株(target strains)及154非目標菌株(nontarget strains)後, 晶片之靈敏性(sensitivity)為100%，特異性為96.2%。顯示本研究的晶片能有效鑑定引起乳房炎的病原菌。

關鍵字

乳房炎 ； 生物晶片 ； 16S-23S 核糖體核酸基因間隔區

並列摘要

Cow mastitis is caused by a wide variety of microbial pathogens. In this study, an oligonucleotide array was developed to identify the pathogens causing mastitis. These pathogens include Corynebacterium bovis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Mycoplasma bovis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis. Specific oligonucleotide probes were designed from the 16S-23S rRNA gene intergenic spacer (ITS) region and immobilized on Nylon membrane. The identification method consisted of PCR amplification of the ITS region using bacterial universal primers, followed by hybridization of the digoxigenin-labeled PCR product with oligonucleotide probes on the array. A total of 35 species-specific oligonucleotide probes were immobilized on the array (0.6 cm × 0.5 cm) to identify 18 species of bacteria. After testing a collection of 322 target strains and 154 nontarget strains, the sensitivity and specificity of the array was 100 % and 96.2 %, respectively. The current array has a great potential to rapidly and correctly identify bacteria causing bovine mastitis.

並列關鍵字

mastitis ； microarray ； 16S-23S intergenic spacer region

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