复合探针荧光定量PCR法检测布鲁氏菌的实验研究

Experimental Research on Brucella Bacteria Detection by Fluorescent Quantitative PCR with Composite Probe

目的:建立用复合探针荧光定量PCR快速检测布鲁氏菌的方法。方法:研究根据BSCP31基因编码31KDa的布鲁氏杆菌表面蛋白的核苷酸序列设计特异引物,通过PCR法的特异性、灵敏度和重复性研究,建立了复合探针荧光定量PCR检测布鲁氏菌的方法,用于布鲁氏菌病的筛选和诊断。结果:结果表明该检测方法的特异性为100%,最低可检出10个拷贝的质粒DNA分子,可对1×101-1×106拷贝范围内的模板进行定量,最低可检测至1×102CFU/ml细菌。该方法的精密度好,阳性质控品和阴性质控品不同时间测定三次及同一时间五次重复实验结果CV值均小于5%。结论:本研究建立的复合探针实时荧光定量PCR检测布鲁氏杆菌的方法,可对布鲁氏病原菌进行快速检测,对布病的筛选和确诊具有重要意义。

Objective： To establish a composite probe fluorescent quantitative PCR assay for Brucella detection. Methods： BSCP31 gene nucleotide sequences specific for 31KDa surface protein of Brucella was used for the PCR primers design. According to the specificity, sensitivity and reproducibility of primers, a composite probe fluorescent quantitative PCR assay for Brucella detection was established for screening and diagnosis of Brucella disease. Results： The specificity of composite probe fluorescent quantitative PCR assay was 100%. The assay can detect the minimum 10 copies of plasmid DNA molecules. Accurate quantization can be achieved with template samples detected within 1 ×10^1-×10^6 copies. The minimum bacteria concentration can be detected by the assay reaching 1 ×10^2CFU / ml. The assay has good precision. The positive controls and negative control materials measured three times at different times or at the same time repeat the experiment five times, from which the CV values were calculated less than 5%. Conclusion： This study established the composite prober fluorescent quantitative PCR assay for Brucella detection, which can rapidly detect Brucella bac- teria and has important meaning on screening and diagnosis of brucellosis.