摘要
目的通过对核糖体18S rRNA基因内部转录间隔区（internal transcribed spacer,ITS）序列的PCR鉴定,建立快速、准确地鉴定真菌性角膜炎致病真菌种属的方法。方法采用眼科常见5种致病菌标准菌株;镜检确诊真菌阳性的角膜炎临床标本29例,其中真菌培养阳性15例,真菌培养阴性14例。所有标本均采用液氮研磨结合小玻璃珠振荡法破碎真菌细胞壁,提取真菌基因组DNA,并以此为模板PCR扩增其18S rRNA基因ITS保守区序列,进行基因测序后在基因库中查询致病菌的种属,与镜检结果比对。结果禾谷镰刀菌、尖孢镰刀菌、燕麦镰刀菌、烟曲霉、白色念珠菌等5种标准菌株培养物和29例临床真菌样本（包括15例真菌阳性培养物和14例真菌培养阴性的角膜病变组织样本）,各5mg样品中提取的DNA样品的浓度在1.7~22.5mg·mL-1。PCR扩增后5种标准菌株均有目的条带,15例真菌阳性培养物也均有目的条带,14例真菌培养阴性的角膜病变组织样本有2例有目的条带,其余12例没有目的条带。标准菌株的PCR鉴定阳性率100%,镜检确诊真菌阳性的临床样本PCR阳性率58.6%,高于真菌培养法鉴定的阳性率51.7%。PCR产物进行基因测序后在基因库中查出了致病菌的种属,检索结果与镜检结果完全一致。结论通过核糖体18S rRNA基因ITS序列PCR鉴定真菌性角膜炎致病菌的方法特异性强,灵敏度较传统的培养加镜检法更高。若能改进从角膜病变组织中提取真菌基因组DNA的方法,其灵敏度还能进一步提高。
Objective To identify the genus and species of fungal keratitis pathogens by PCR quickly and accurately through internal transcribed spacer（ITS）sequences in the ribosomal 18S rRNA gene.Methods Five standard fungal strains which were the most popular in fungal keratitis were used,and 29 clinical specimens including 15 cases of positive fungal cultures and 14 cases of negative fungal cultures which were all confirmed under microscope in advance were also used.All the specimens were ground in liquid nitrogen and vortex with little glass beads in extract buffer to crush into micro particles.Their genomic DNA were extracted,which were used as template to amplify the ITS conserved sequences in the 18S rRNA genes by PCR.The ITS sequences were sequenced and the genus and species were checked in the GenBank,the results were compared with the ones under microscope.Results The concentration of DNA in each 5 mg sample from the five standard fungal strains（including fusarium graminearum,fusarium oxysporum,fusarium avenaceum,aspergillus fumigatus,blastomyces albicans）and 29 clinical specimens were from 1.7 ng·μL-1 to 22.5 ng·μL-1.The aimed lines were appeared after PCR amplification in the retinal samples of all five standard fungal strains,15 cases of positive fungal cultures and 2 cases of negative fungal cultures.The positive rate of PCR identification on the standard fungal strains was 100%,on the 29 clinical specimens which were all confirmed under microscope was 58.6%,which was higher than the positive rate of fungal culture method 51.7%.The PCR products were sequenced and the pathogenic genus and species were identified in the GenBank,the results and the ones under microscope were completely consistent.Conclusions The method to identify the fungal keratitis pathogens through ITS sequences in the ribosomal 18S rRNA gene is specific,and the sensitivity is higher than the traditional method.The method of extracting the fungal genomic DNA from diseased corneal tissue is still need to be improved to increase the sensitivity of the method.
出处
《眼科新进展》
CAS
北大核心
2010年第11期1017-1020,共4页
Recent Advances in Ophthalmology
基金
西安市科技局医疗卫生研究基金资助[编号:SF09023(3)]~~
