香港海鸥形菌TaqMan-MGB探针实时荧光PCR的建立与应用

Development and application of real-time PCR for Laribacter hongkongensis

目的建立快速、灵敏、特异的香港海鸥形菌TaqMan-MGB探针实时荧光PCR体系,用于食源性致病菌监测和淡水鱼产品贸易往来的快速检测。方法根据GenBank公布的香港海鸥形菌16SrRNA基因高保守序列,设计特异引物对和TaqMan-MGB探针,建立和优化快速检测体系,评价反应体系的特异性、灵敏度、重复性,并在淡水鱼类、急性胃肠炎腹泻患者标本中临床应用,结合细菌分离方法和序列测定分析进行验证。结果建立的香港海鸥形菌TaqMan-MGB探针实时荧光PCR反应体系检测时限短,仅40min就可出结果,特异性好,与沙门氏菌、志贺氏菌、副溶血弧菌等非目标菌无交叉反应,灵敏度高,检测下限达10拷贝/反应,稳定性好,同一浓度阳性质粒进行16次重复检测的Ct值变异系数0.183%。92份淡水鱼及211份医院急性胃肠炎腹泻标本实时荧光PCR与常规细菌分离结果完全一致,均检出了12株香港海鸥形菌,16SrRNA测序结果显示与HKU1株同源性在99.5%以上,进一步验证了实时荧光PCR的特异性。结论本研究建立的香港海鸥形菌TaqMan-MGB探针实时荧光PCR反应体系快速、特异、灵敏,在防止食源性疾病的传播和加快贸易通关速度方面,具有较强的的应用价值。

Objective To develop a TaqMan MGB probe-based fluorescence real-time PCR assay method for rapid detection of Laribacter hongkongensis, and to apply this method for the monitoring of foodborne pathogens in freshwater fishes. Methods Primers and probes specific for the conserved 16S rRNA gene of Laribacter hongkongensis were designed. A TaqMan MGB probe-based fluorescence real-time PCR method was established and optimized. Specificity, sensitivity and stability of the method were assessed. Samples collected from freshwater fishes and gastroenteritis patients were analyzed by real-time PCR. Bacterial isolation and sequence analysis were carried for results confirmation. Results The established real-time PCR assay could be completed within 40 minutes. The detection limit was 10 copies/μl. The specificity was high and did not show cross-reactivity with other bacteria.The stability of the was satisfactory, and the SD value of the CT results was 0.183%. In the testing of 303 clinical specimens, a total of 12 specimens were positive for Laribacter hongkongensis. The result was consistence with the traditional separation method. Compared with the HKU1 strain, the sequence homology of 16S rRNA from the 12 strains of Laribacter hongkongensis was more than 99.5%. Conclusion The TaqMan MGB probe-based fluorescence real-time PCR was sensitive, specific, and useful in the prevention of foodborne diseases.