布鲁氏菌S2疫苗株实时荧光定量PCR检测体系的建立及应用

Establishment and application of a quantitative real-time PCR detection system for Brucella S2 vaccine strain

目的建立一种布鲁氏菌S2疫苗株的实时荧光定量PCR检测体系。方法依据布鲁氏菌S2疫苗株与其他布鲁氏菌参考菌株全基因组序列的差异,设计引物和探针,建立实时荧光定量PCR检测体系。自中国疾病预防控制中心传染病预防控制所,收集22株布鲁氏菌参考菌株及8种非布鲁氏菌对照菌株的DNA;同时自布病疫苗生产厂采集环境样本,使用血液/组织基因组DNA提取试剂盒提取环境样本细菌DNA。对获取的DNA先行普通PCR预扩增,再以扩增的PCR产物为模板进行实时荧光定量PCR二次扩增(巢式荧光定量PCR),观察是否出现特异荧光曲线及相应的循环数(Ct值),并测试其灵敏性。结果建立的实时荧光定量PCR检测体系,除S2疫苗株外,其他21株布鲁氏菌参考菌株和8种非布鲁氏菌对照菌株均未检出特异荧光曲线(无Ct值)。建立的检测体系,检测布鲁氏菌S2疫苗株DNA的最低限为4.34 fg;采集的14份环境样本,其中3份检测到布鲁氏菌S2疫苗株DNA。结论建立的实时荧光定量PCR检测体系,能够检测到样本中布鲁氏菌S2疫苗株,具有较好的灵敏性和特异性。

Objective To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain.Methods Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella,primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain.The DNA of 22 reference strains of Brucella and 8 non-Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention.At the same time,environmental samples were obtained from the brucellosis vaccine manufacturers,and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit.The obtained DNA was pre-amplified by conventional PCR,and then subjected to quantitative real-time PCR secondary amplification(nested fluorescence quantitative PCR)using the amplified PCR product as a template.The specific fluorescence curve and corresponding number of cycles(Ct value)were observed,and the sensitivity was tested.Results The quantitative real-time PCR detection system established did not detect specific fluorescence curves(without Ct values)for 21 reference strains of Brucella and 8 non-Brucella control strains,except for S2 vaccine strains.The established detection system had a minimum detection limit of 4.34 fg(genomic DNA)for detecting the DNA of Brucella S2 vaccine strain;DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected.Conclusion The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples,with good sensitivity and specificity.