不同PCR方法检测布鲁菌阳性样本的敏感性分析

目的利用布鲁菌分离株及对应的患者血液样本验证不同引物扩增的特异性及其敏感性,为将PCR方法应用于人布鲁菌病临床诊断提供参考。方法对血液样本进行传统布鲁菌分离培养,根据表型对分离株进行种型鉴定;用玻片凝集试验(PAT)和试管凝集试验(SAT)两种血清学方法检测培养阳性的血液模板;应用不同布鲁菌属引物B4/B5、BP26以及羊、牛和猪种引物对布鲁菌分离株进行鉴定,同时对其血液模板进行PCR检测。结果从急性发热患者血液分离到24株布鲁菌,经表型鉴定均为羊种。对分离株进行PCR鉴定,布鲁菌属引物B4/B5、BP26及羊种引物在分离株中均扩增到目的条带。应用不同引物PCR方法对血液模板检测的阳性率依次为B4/B5(79.17%),羊种(66.67%)和BP26(25.00%);B4/B5和羊种引物扩增同为阳性的符合率为41.67%,B4/B5或羊种引物扩增为阳性的符合率为91.67%。结论在布鲁菌分离培养阳性的血液样本中,应用单一引物PCR进行人布鲁菌病诊断的敏感性较低,将布鲁菌属B4/B5和地方流行种引物结合可提高PCR检测方法的敏感性和特异性。

Sensitivity analysis on different PCR assays for Brucella detection

To test the sensitivity and specificity of different PCR primers with Brucella strains and cultured-positive blood samples, the conventional culture method and strain genotyping for Brucella were conducted. Both Plate Agglutination Test （PAT） and Standard Agglutination Test （SAT） were also used for identification. Brucella isolates and brucellosis patients’ blood samples were identify with PCR primers B4/B5, BP26, B. Melitensis, B. Abortus, and B. Suis, respectively. And the 24 isolates from the blood of patients with fever in acute phase were genotyped as B. Melitensis. Target bands were obtained in all identifications of these isolates by PCR with primers B4/B5, BP26, and B. Melitensis, respectively. The positive coincidence rates of blood samples were 79.17% （B4/B5）, 66.67%（B. Melitensis） and 25.00%（BP26）, respectively. The positive coincidence rate of B4/B5 and B. Melitensis was 41.67%, and the positive coincidence rate of B4/B5 or B. Melitensis was 91.67%. Results suggested that single primer PCR showed low sensitivity in culture-positive blood samples. However, the combination of B4/B5 and local epidemic strain primer might improve the sensitivity and specificity of PCR assay.