光滑念珠菌SYBR Green荧光定量PCR检测方法的建立及应用  

Establishment and application of SYBR Green fluorescent quantitative PCR detecting Candida glabrata

作　　者：刘利强 宋阔阔 李慧芳 赵云环 赵诚睿 柳焕章 刘建钗 张鹤平 闫金坤 刘彦威 刘娜 LIU Li-qiang;SONG Kuo-kuo;LI Hui-fang;ZHAO Yun-huan;ZHAO Cheng-rui;LIU Huan-zhang;LIU Jian-chai;ZHANG He-ping;YAN Jin-kun;LIU Yan-wei;LIU Na(College of Life Science and Food Engineering,Hebei University of Engineering, Handan 056021,China;College of Landscape and Ecological Engineering, Hebei University of Engineering, Handan 056021,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,河北邯郸056021 [2]河北工程大学园林与生态工程学院,河北邯郸056021

出　　处：《中国兽医科学》2019年第5期604-610,共7页Chinese Veterinary Science

基　　金：河北省科技支撑项目(17226612D);邯郸市科技计划项目(1622201050-2)

摘　　要：为建立光滑念珠菌快速准确灵敏的检测方法,根据GenBank已发表的光滑念珠菌rDNA内转录间隔区核苷酸序列设计1对特异性引物,应用SYBR Green实时荧光定量PCR方法检测光滑念珠菌,对临床分离的多株不同来源光滑念珠菌病料进行检测,通过临床常见的5种病原真菌对该方法的特异性进行检验,并用人工感染小鼠的血液、肝脏组织样本对所建方法进行检验。结果显示,该方法灵敏度高,光滑念珠菌的最低检出浓度为10 copies/μL,特异性强,与白色念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌、烟曲霉菌等病原菌无交叉反应。该方法操作简单,耗时短,只需1 h即可完成全部检测过程。上述结果表明,SYBR Green荧光定量PCR可应用于光滑念珠菌早期侵袭的快速检测,为光滑念珠菌病的及时正确防治提供了可靠依据。For the purpose of setting up a rapid,accurate and sensitive method to detect Candida glabrata,by means of SYBR Green fluorescent quantitative real-time PCR method,we designed a pair of special primers based on the published ITS sequences of r DNAs of Candida glabrata in Gen Bank.The clinical isolates of Candida glabrata from different sources was tested using the method.The specificity test of the method was performed using 5 clinically important pathogenic fungi.And the method was tested with blood and liver tissue samples from mice infected artificially.In result,the method was high sensitive with the lowest detection as 10 copies/μL of Candida glabrata,was high discriminative without the same reaction in the other pathogenic fungi as Candida albicans,Candida tropicalis,Candida parapsilosis,Candida krusei and Aspergillus fumigatus,respectively,and was less time consumption as long as 1 h for completing the test.In summary,this method can be used to handy and accurate detection for early infection of Candida glabrata,and it offers a reliable basis for timely and correct prevention from Candida glabrata.

关 键 词：光滑念珠菌 SYBR Green荧光定量PCR 快速 

分 类 号：S852.661[农业科学—基础兽医学]