空肠弯曲菌TaqMan-MGB双重探针实时荧光定量PCR快速检测

Rapid Detection of Campylobacter Jejuni with TaqMan-MGB Dual Probe Real-time Fluorescence Quantitative PCR

目的开发一种特异、灵敏的TaqMan MGB双重探针实时荧光定量PCR方法,用于空肠弯曲菌的快速定量检测。方法针对空肠弯曲菌鞭毛蛋白A和马尿酸酶基因设计特异性引物和探针,建立一种新型TaqMan-MGB双重探针实时荧光定量PCR检测空肠弯曲菌方法,对该方法的定量检测线性范围、特异度、灵敏度、重复性、稳定性进行评价,应用该方法对临床标本中的空肠弯曲菌进行检测,同时用细菌培养、普通PCR、基因克隆和测序鉴定。结果建立的空肠弯曲菌TaqMan MGB双重探针实时荧光定量PCR检测方法专属性强,能准确检出空肠弯曲菌,而与其他细菌无交叉反应,特异度为100%。该技术灵敏度高,能精确定量检测空肠弯曲菌DNA线性范围达10个数量级,最低检测限为4个菌落形成单位。重复性和稳定性良好,组内和组间相对标准偏差均小于1%。应用该方法成功从78例临床标本中定量检出28例空肠弯曲菌阳性标本,用普通PCR和基因克隆测序分析确认,细菌培养方法仅获得6株存活的空肠弯曲菌。结论 TaqMan MGB双重探针实时荧光定量PCR具有快速简便、可靠稳定、特异灵敏的优点,可用于临床标本中空肠弯曲菌定量检测,值得推广应用。

Objective To develop a specific and sensitive TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay in order to rapidly detect and quantity Campylobabter jejuni. Methods Specific primers and probes were designed to conserved regions of flagellin A(flaA) and hippuricase(hipO) genes of Campylobabter jejuni. A novel TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay was developed. The linear range, sensitivity, specificity, reproducibility and stability of the assay were evaluated. The assay was applied to detect Campylobabter jejuni in clinical specimens, and bacterial culture, conventional PCR, gene cloning and sequencing were concurrently performed. Results It was shown that the explored TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay could accurately detect Campylobabter jejuni with 100% specificity, and had no cross reaction with other bacteria. The technology was demonstrated to be highly sensitive, allowing a precise Campylobabter jejuni DNA quantitation over a range of 10 orders of magnitude, and the limit of detection of the assay was determined to be 4 colony-forming units of Campylobabter jejuni. The reproducibility and stability of this assay was excellent with the relative standard deviation within and between groups of less than 1 percent. The assay was successfully applied to quantifiable detection of Campylobabter jejuni genomic load in 78 clinical specimens, and confirmed using conventional PCR and gene cloning and sequence analysis, in which 28 specimens were positive, while only 6 specimens were positive for Campylobabter jejuni obtained by bacterial culture. Conclusion TaqMan MGB dual probe real-time fluorescence quantitative PCR has the advantages of rapid, simple, reliable, stable, specific and sensitive, which can be used for quantitative detection of Campylobacter jejuni in clinical specimens, and is worthy of popularization and application.