斑点热群立克次体实时荧光定量PCR检测方法的 建立及应用

摘要:目的　探讨实时荧光定量PCR快速检测斑点热群立克次体标本的应用价值。方法　根据斑点热群立克次体外膜蛋白A（OmpA）基因保守序列设计TaqMan特异性探针和引物，进行实时荧光定量PCR方法学评估，并同时运用该法和普通PCR法对对海南省2012年不明原因发热病人157份血标本进行斑点热群立克次体的检测及结果分析。结果　本研究建立的定量标准曲线循环阈值（Ct值）和模板拷贝数呈良好的线性关系（R2值为0.998）。该法能检出斑点热群立克次体阳性标准品—西伯利亚立克次体（R.sibirica）最小浓度为102 copies/μl，灵敏度比普通PCR高100倍。用该法检测斑点热群立克次体DNA，结果为阳性，检测金黄色葡萄球菌、铜绿假单胞菌、致泻性大肠埃希菌和副溶血弧菌等其他病原细菌DNA结果均为阴性。采用实时荧光定量PCR法对实验室库存的发热病人血标本DNA进行回顾性检测，结果示阳性2例，检出率为1.2%（2/157），而普通PCR检测结果均为阴性。结论　实时荧光定量PCR法具有快速、特异和灵敏的优点，在处理突发公共卫生事件中可发挥快速检测的优势，在斑点热诊断中具有重要的临床意义。

Establishment and application of real-time fluorescence quantitative PCR in spotted fever group rickettsiae detection

Abstract: Objective This study was aimed to explore the application of rapid detection of spotted fever group rickettsiae by real-time fluorescence quantitative PCR. Methods Based on the outer membrane protein A (OmpA) gene conservative sequence of spotted fever group rickettsiae, specific primers and probes were designed and applied for real-time fluorescence quantitative PCR evaluation. Additionally, this method and the conventional PCR were used to detect and analyze 157 blood specimens from patients with fever of unknown cause in Hainan province in 2012. Results This study showed that the cycle threshold (Ct) of quantitative standard curve and template copy number had a good linear relationship (R2=0.998). The minimum concentration detected was 102 copies/ul, and the sensitivity was 100 times higher than the conventional PCR. The result of the detection of spotted fever group rickettsiae using this method was positive, while the detection of other pathogenic bacteria DNA was negative. Using the real-time fluorescence quantitative PCR for the detection of the DNA from fever patients' blood samples, the results were positive in two cases, with a positive rate of 1.2% (2/157), while the ordinary PCR test results were negative. Conclusion Real-time fluorescence quantitative PCR analysis had the advantages of rapid, specific and sensitive. It can play a role in rapid detection dealing with public health emergencies, possessing important clinical significance in the diagnosis of spotted fever.