摘要
目的:建立布鲁菌单一PCR实验室检测方法,并在模拟样本和染毒动物实验中优化反应条件,为将PCR方法应用于布鲁菌病患者治疗后的追踪随访提供实验室依据。方法:针对编码BP26的基因序列设计布鲁菌属引物,针对插入性序列IS711设计区分布鲁菌羊种、牛种和猪种引物;以M5或M5荧光菌株为实验组、以PBS为对照组制备系列浓度的模拟样本和染毒动物;用热解法从血液样本中提取布鲁菌基因组;用BP26与羊种、牛种和猪种引物对菌液进行PCR扩增,用BP26和羊种引物对模拟样本和染毒动物不同时间点的血液样本进行检测。结果:菌属水平引物BP26和不同种引物在对布鲁氏菌标准菌株、疫苗株基因组的扩增中均得到特异性条带,具有一定的特异性;热解法可提取到模拟样本和染毒小鼠血液样本中的布鲁菌基因组,经PCR扩增后得到特异性条带;染毒小鼠血样PCR检测阳性结果数随时间先上升后下降,同一时间点增加样本量可提高阳性率,热解法在各时间点检测阳性率均高于同期化学试剂法提取结果,PCR检测方法灵敏度高于分离培养方法。结论:采用热解法提取血液样本中的布鲁菌基因组,易于操作;PCR检测方法安全稳定,敏感性高于传统分离培养方法;以核酸为靶标进行检测的特异性较高,可进一步应用于患者治疗后追踪随访研究。
Objective To construct single PCR assay for brucella,and optimize reaction conditions through simulative blood samples and inoculated mice,and provide laboratory evidence for PCR assay application in clinical diagnosis of brucellosis following-up. Methods Primer BP26 on genus level according to BP26 gene and three primers on strain level according to IS711 were designed; simulative samples of serial concentrations were constructed and mice were inoculated with M5 or Pps858-MCS2/M5 as experimental group,while PBS as control; brucella DNA was extracted by boiling method. This PCR assay was used to detect bacteria solution with primers BP26 and three strain primers,simulative samples and inoculated mice were diagnosed with primer BP26 and strain primer Meli. Results Designed primers on genus and strain level showed good specificity through brucella standard strains and vaccine strains; DNA was extracted from simulative samples and blood samples of the inoculated mice by the boiling method and specific bands were obtained; the PCR positive numbers of the inoculated mice increaseed early and desceased later,and increasing sample quantities increased the positive rate; the PCR positive rate of the boiling method was higher than chemical method at the same time point,and the PCR positive rate was more sensitive than traditional culture method. Conclusion The boiling method is convenient to extract DNA from blood. The PCR assay demonstrates higher sensitivity than traditional culture method; this PCR assay is very specific and available to be used for following-up of brucellosis patients.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2009年第6期1172-1176,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅白求恩医学专项基金资助课题(200705392)
吉林省松原市科技发展计划项目资助课题(GY2008004)
