巢式聚合酶链式反应检测布鲁氏菌核酸DNA方法的建立

Establishment of a nested polymerase chain reaction assay for detection of Brucella DNA

目的建立一种具有灵敏性高,特异性强的巢式聚合酶链式反应(PCR)方法检测血液标本中布鲁氏菌核酸DNA。方法使用细菌基因组提取试剂盒提取纯菌核酸DNA;使用血液等组织基因组核酸DNA提取试剂盒提取血液标本核酸DNA,对提取的核酸DNA先行常规PCR预扩增,以扩增的PCR产物为模板进行荧光定量PCR第二次扩增(即巢式PCR)。对纯菌提取的核酸DNA进行灵敏度和和特异性测试,构建巢式PCR的Ct值与核酸DNA拷贝数之关系曲线;检测临床血液标本核酸DNA,同时比较常规两种PCR方法检测结果。结果常规PCR检测的灵敏度为512个核酸DNA拷贝数;巢氏PCR检测有效范围为921.6 ng/μl^6.8 fg/μl,对应的Ct值为12.04~37.50,其指数关系为:y=(e-0.695x)×1012;R2=0.9986,巢式PCR扩增效率为2.28×109倍,检测限为2个布鲁氏核酸DNA拷贝数。巢式PCR的灵敏度为91.67%,特异度为93.10%,阳性预测值为91.67%,阴性预测值为93.10%。对一起羊养殖场采集的25份血液标本应用巢式PCR方法检测,结果阳性率为92.00%(23/25);27份健康人群血液标本没有检测出(无Ct值)。结论巢式PCR具有较好的灵敏性和特异性,特别适合于血液标本布鲁氏菌核酸DNA的检测。

Objective To establish a nested polymerase chain reaction(PCR)assay with high sensitivity and specificity to detect Brucella DNA.Methods The bacteria DNA was extracted by using bacterial genome extraction kit.The DNA extraction from blood samples was conducted by using blood and other tissue genomic DNA extraction kit.The extracted DNA was pre-amplified by conventional PCR.The amplified PCR product was used again as the template for the second amplification by fluorescence quantitative PCR(nested PCR).The sensitivity and specificity for bacteria DNA extraction were tested.The relationship curve between Ct value and copy numbers of DNA was constructed.The DNA from blood samples was tested.The results were compared with conventional PCR and conventional fluorescence quantitative PCR.Results The sensitivity of conventional PCR was 512 copies of Brucella DNA.The effective range of genomic DNA was 921.6 ng/μl–6.8 fg/μl for nested PCR.And the corresponding Ct value was 12.04–37.50.The index relationship was y=(e-0.695 x)×1012,R2=0.9986.The amplification efficiency of nested PCR was 2.28×109.The detection limit for nested PCR was 2 copy numbers of Brucella DNA.The sensitivity,specificity,positive predictive value and negative predictive value of nested PCR were 91.67%,93.10%,91.67%and 93.10%,respectively.Twenty five blood samples from a sheep farm were detected by nested PCR.The positive rate was 92.00%(23/25).The detection results of Brucella DNA were negative for 27 blood samples from healthy people(no Ct value).Conclusion The nested PCR has high sensitivity and specificity,which is suitable to use for the detection of Brucella in blood samples.