Taqman MGB探针实时荧光定量PCR检测布鲁菌病的研究  

Establishment and application on TaqMan MGB probe real-time PCR for rapid detection of brucellosis

作　　者：刘艳红 王清[2] 

机构地区：[1]山东省淄博市临淄区妇幼保健院皮肤科,山东淄博255400 [2]青岛大学医学院附属医院检验科

出　　处：《现代预防医学》2013年第7期1348-1351,1356,共5页Modern Preventive Medicine

摘　　要：目的建立血液标本布鲁菌DNA荧光定量检测方法,探讨其临床应用价值。方法用PUCm-T载体和PCR纯化产物连接,转染DH5a菌,筛选阳性菌落,提取质粒,制作外标准品;在布鲁菌基因组IS71l序列设计一对引物和Taq-Man MGB探针,严格优化反应物的组成和扩增条件,并对该方法进行评价。结果建立的布鲁菌DNA荧光定量PCR方法最低检出率为400拷贝/ml;批内误差为1.8%～6.5%,批间误差为2.6%～9.1%;在4.0×102～4.0×108拷贝/ml之间与Ct值具有很好的线性(Ct=-1.3911Ln(x)+41.65,r=-0.9958);具有较好的稳定性。在布鲁菌临床监测中发现,157份血液标本,用荧光定量PCR检测与临床检查结果(临床阳性、慢性期患者、症状可疑、阳性畜周围人群与重点职业人群(重点人群)的阳性率和阴性对照的符合率分别为90.3%、40%、6.7%、12.2%和100%。结论建立的荧光定量PCR检测方法能鉴定布鲁菌种属,具有良好的特异性、灵敏度和稳定性。在临床布鲁菌基因诊断方面具有重要意义。OBJECTIVE To standardize a TaqMan MGB probe real-time PCR for screening and detection on Brucella DNA in blood and evaluate its methodology. METHODS TaqMan MGB probe was designed according to the sequence of IS71l gene.The PCR reaction system was optimized strictly.Used clinical and laboratory standards institute （CLSI） evaluation program to evaluate the Brucella DNA quantitative methods’ linear range, sensitivity, specificity and diagnostic accuracy. RESULTS Linear was in the rang 4.0×102-4.0×108 copies / ml, variation within and between groups ranged from1.8% to 6.5% and 2.6% to 9.1% respectively, lower limit was 400 copies / ml of the quantitative method of Brucella DNA.And there was a good linearity with Cr value （Ct = -1.391 1 Ln （x） ＋41.65, r = -0.995 8）. In a test of 157 samples, the positive coincident rate of clinical brucellosis, chronic brucellosis, clinically suspected and risk people was 90.3%, 40%, 6.7%, 12.2% respectively, and the negative coincident rate was 100%. CONCLUSION These results show that the real-time PCR assay is far more sensitive than conventional cultures, which makes this technique a very useful tool for the diagnosis of brucellosis.

关 键 词：TAQMAN MGB探针 荧光定量PCR 布鲁菌 DNA 

分 类 号：R378.5[医药卫生—病原生物学]