CPA-核酸试纸条快速检测副溶血性弧菌(Vibrio parahaemolyticus)方法的建立及其在海产品检测中的应用

DEVELOPMENT OF CROSS PRIMING AMPLIFICATION COMBINED WITH NUCLEIC ACID STRIP FOR DETECTION OF VIBRIO PARAHAEMOLYTICUS

本研究将交叉引物恒温扩增技术(cross priming amplification,CPA)与核酸试纸条相结合建立一种副溶血性弧菌(Vibrio parahaemolyticus)快速可视化检测方法。针对副溶血性弧菌特有的不耐热溶血素基因tlh的六个不同区域设计两对特异性引物和一对检测探针,通过优化反应条件确定了最佳反应体系。CPA-核酸试纸条方法对副溶血性弧菌的检测具有较强的特异性,对纯培养物的检测灵敏度达到58 cfu/m L,对污染牡蛎中副溶血性弧菌的检测灵敏度为5.2 cfu/g,比传统的PCR技术灵敏度提高了10倍,且具有较高的稳定性。交叉引物恒温扩增技术与核酸试纸条相结合的方法操作简便、特异性强、灵敏度高且能有效防止污染,可用于现场及基层单位副溶血性弧菌的快速检测。

We developed a new method that combines cross priming amplification （CPA） with nucleic acid test strip for detection of Vibrio parahaemolyticus, and evaluated the specificity and sensitivity of the method. Six primers, including two displacements, two crosses, and two detector primers were designed for recognizing six distinct regions on the sequence of the tlh gene. All bacteria strains including E parahaemolyticus and other reference bacteria strains were amplified by using the primers and probes with Bst DNA polymerase at 63℃. Among all the bacteria stains, only E parahaemolyticus 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 V. parahaemolyticus reached to 58cfu/mL for the pure culture and 5.2cfu/g for the tested contaminated oysters. The test could be completed in 75rain. Therefore, the CPA-nucleic acid test strip detection method is specific, sensitive, rapid, and easy for detecting E parahaemolyticus.