实时荧光定量PCR检测布鲁杆菌方法的应用

Objective To discuss a real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) wether if can be used to detect Brucella. Methods According to the BCSP31 gene sequences specific for Brucella, one pair of primers and one TaqMan probe were designed. A real-time PCR was developed with the BCSP31 fragments cloned into PMD18-T vector. The standard cure was established and the sensitivity, the species specificity and the stability of the assay were evaluated. The clinical blood specimens were detected by QT-PCR and compared with clinical diagnosis. Results The standard curve was established with the standard template and the relationship between the Ct and the DNA copy number was linear(r=0.999). The sensitivity of the real-time PCR was 5 copies/μl. The sensitivity of the common PCR was 5×102 copies/μl. The sensitivity was about 100 times higher than common PCR. Species specificity of this FQ-PCR assay evaluated using genomic DNA from 6 Bmcella strains and 5 non-Brucella strains and strong fluorescence was detected in all Brucella strains. The CV of intra-assay and inter-assay reproducibility were 0.71%,7.23%, reprectively. Twenty-four specimens from clinical brucellosis cases, 19 showed positive, the positive coincident rate was 79%(19/24). The negative results were obtained for all 31 negative control, and the negative coincident rate was 100%(31/31). Two were positive from all 30 specimens clinically suspected. Conclusions Highly specific, sensitive, repeatable and coincidental with clinic, this FQ-PCR is quite useful for rapid detection of tiny DNA of Brucella in various samples and laboratory diagnosis.     

黑龙江省巴彦县布鲁杆菌病流行病学分析

目的了解2003年8月～2005年12月巴彦地区布鲁杆菌病(布病)的流行动态及发展趋势,确定防治对策。方法按全国布病监测方案,对黑龙江省巴彦县人群进行连续监测,并对高发乡镇人群进行了布鲁杆菌素皮试,对阳性者和易感人群做琥红平板试验(RBPT)和试管凝集试验(SAT)。结果2003年8月～2005年12月巴彦县共发生布病79例,年平均发病率为3．9/10万,其中2004年最高,达8．4/10万,并有2名临床医生被感染。结论巴彦县布病疫情呈先升高后降低趋势,原因是病区牛羊大批私自屠宰和外卖,应切实采取有效措施控制疾病蔓延。     

滨州地区生活饮用水细菌污染情况调查分析

为了了解我区生活饮用水细菌污染情况,有针对性的加强管理,防止因生活饮用水引起的肠道传染病的暴发流行。我们/自1989～1998年对全区城镇和农村生活饮用水进行了采样和细菌学指标检测,现将检测情况报告分析如下。1 资料来源及方法1.1 资料来源 本资料来源于滨州地区卫生防疫站档案资料室生活饮用水调查资料及历年的水质、细菌学检测报告。1.2 方法 我区生活饮用水有二种情况,一是地上水,以水库蓄存黄河水和雨水,二是地下水。城镇多数由自来水厂集中供     

实时荧光定量PCR检测布鲁杆菌方法的应用

目的 探讨实时荧光定量PCR(FQ-PCR)用于布鲁杆菌检测的可行性.方法 根据布鲁杆菌BCSP31基因设计合成1对引物和TaqMan探针,以克隆有布鲁杆菌基因片段的PMD18-T质粒作为标准品DNA模板,进行FQ-PCR检测,绘制标准曲线;对所应用的方法分别进行敏感性、特异性及重复性分析;并对临床血液标本进行FQ-PCR,与临床诊断进行比较.结果 用标准品DNA建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999);FQ-PCR检测的灵敏度拷贝数为5个/μl,普通PCR为5×102个/μl,FQ-PCR是普通PCR的100倍;用FQ-PCR检测6株布鲁杆菌菌株及5株非布鲁杆菌菌株,布鲁杆菌均检出较强荧光信号,非布鲁杆菌未检出;5×103个拷贝/μl标准品DNA检测后,Ct值批内变异系数(CV)为0.71%,批间CV为7.23%;用FQ-PCR检测临床确诊阳性血标本24份,检出阳性19份,符合率为79%(19/24),阴性对照血标本31份,全部阴性,阴性符合率为100%(31/31),临床症状可疑标本30份,检出阳性2份.结论 用FQ-PCR方法检测布鲁杆菌具有高度敏感性、特异性及良好的重复性,与临床阳性及阴性标本的符合率较高,可用于快速检测各种样本中的微量布鲁杆菌以及作为布鲁杆菌感染的实验室检测方法.