屎肠球菌TaqMan荧光定量PCR检测方法的研究

发布时间：2018-01-12 04:00

本文关键词：屎肠球菌TaqMan荧光定量PCR检测方法的研究　出处：《中国人兽共患病学报》2013年06期 　论文类型：期刊论文

【摘要】：目的建立屎肠球菌的TaqMan荧光定量PCR检测方法。方法根据屎肠球菌的ddl基因设计TaqMan荧光定量PCR特异的引物及探针,在多种常见致病菌及条件致病菌中检测其特异性;将目的基因克隆到pMD18-T载体中建立标准曲线,检测方法的灵敏度和稳定性;使用腹水模拟标本验证方法的应用性。结果在特异性评价中,除了阳性对照外其余菌株均未见特异性扩增曲线。建立屎肠球菌TaqMan荧光定量PCR方法的标准曲线,确定本方法对质粒标准品的检测下限为20copy/管。通过对1.0×107、1.0×105和1.0×1033个浓度质粒标准品的重复检测,确定屎肠球菌TaqMan荧光定量PCR方法的组内变异系数为1.90%～3.91%;组间变异系数为1.52%～1.69%。应用屎肠球菌TaqMan荧光定量PCR方法对含菌量为1.6×100～1.6×108cfu/mL浓度梯度的腹水模拟标本进行检测,其检测灵敏度为1.6×102cfu/mL。结论本研究建立了屎肠球菌TaqMan荧光定量PCR检测方法。
[Abstract]:TaqMan fluorescent quantitative PCR method for detection of Enterococcus faecium. Objective to establish a method according to the primer and probe of DDL gene of Enterococcus faecium TaqMan fluorescence quantitative PCR specific design, the specific detection of common pathogenic bacteria and pathogenic bacteria in a variety of conditions; the target gene was cloned to the pMD18-T vector to establish the standard curve, detection sensitivity and stability; the application of using the simulation verification method. Ascites specimens results in specific evaluation, in addition to the positive control strains had no specific amplification curve. A standard curve of Enterococcus faecium TaqMan fluorescence quantitative PCR method, the method of determining the standard plasmid detection limit of 20copy/ tube. The repeated detection of 1 x 107,1.0 x 105 and 1 * 1033 concentration of standard plasmid, identified Enterococcus faecium TaqMan fluorescence quantitative PCR method within group variation coefficient is 1.90% ~ 3.91%; group variation. The number is 1.52% ~ 1.69%. application of Enterococcus faecium TaqMan fluorescence quantitative PCR method of bacteria quantity is 1.6 * 100 ~ 1.6 * 108cfu/mL concentration gradient of ascites simulation specimens were detected, the detection sensitivity is 1.6 * 102cfu/mL. conclusion: This study established the Enterococcus faecium TaqMan fluorescence quantitative PCR detection method.

【作者单位】：中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室;
【基金】：国家科技重大专项(No.2011ZX10004-001,No.2012ZX10004-501) 卫生科研行业专项(No.201302006)联合资助~~
【分类号】：R378.1
【正文快照】： 屎肠球菌是人类胃肠道常见的细菌之一,广泛存在于土壤、水、动物(鸟类、昆虫)中。虽然很少引起正常人的感染性疾病,但是屎肠球菌是院内感染的常见病原菌。最近十年,在美国、欧洲和南美多个国家中由屎肠球菌引起感染的比例逐年升高[1-2]。由于屎肠球菌对多种一线抗生素包括青霉

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