交叉引物恒温扩增技术检测创伤弧菌的应用研究

目的 建立一种快速检测创伤弧菌(Vibrio vulnificus)的交叉引物恒温扩增(cross priming amplification,CPA)技术。方法 针对创伤弧菌vvhA基因序列保守区域设计CPA引物和探针,并优化反应体系和反应条件,同时验证方法的灵敏度和特异性。结果 对创伤弧菌的检测具有高度特异性;对创伤弧菌菌液检测的灵敏度达到了1.28×10³ CFU /mL,此法 40～60 min 内即可完成检测。利用CPA法针对本实验室前期研究的CB型、EA型、CAB型、CA型和EB型等亚型创伤弧菌毒株计算特异性和敏感性,5种亚型中除了CB亚型特异性和敏感性为95.65%和100%,其余亚型特异性和敏感性均为100%。结论 本研究建立的CPA法具有较高的特异性和灵敏性,可有效缩短检验时间提高检验效率。

Establishment of a cross priming amplification assay for detection of Vibrio vulnificus

A new method was established based on cross priming amplification (CPA) to detect Vibrio vulnificus. Highly specific primers and probes were synthesized according to the vvhA gene of Vibrio vulnificus, and via optimizing the reaction system and the reaction conditions to establish the optimal reaction system. The amplification products could be the totally enclosed nucleic acid detection device for testing. Specificity and sensitivity of the method were tested. That among all the bacteria stains, only Vibrio vulnificus stain yielded a positive result, indicating that the CPA primers and probes designed were highly specific for target bacteria. The sensitivity of CPA assay for Vibrio vulnificus reached to 1.28×10³ CFU /mL for the pure culture. The test could be completed in 40 to 60 min. The specificity and sensitivity of Vibrio vulnificus strains of CB, EA, CAB, CA and EB were calculated using CPA method for the preliminary study in our laboratory. The specificity and sensitivity of all the subtypes except CB subtypes were 95.65% and 100% (P>0.05). It is indicated that this method showed its wide perspective and practicability by its rapid, simplicity, high specificity and sensitivity on detecting Vibrio vulnificus.