霍乱弧菌DPO-PCR特异性检测方法的建立与应用

采用一种新型的引物设计方法——双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）,以霍乱弧菌mdh基因为靶序列,建立了霍乱弧菌DPO-PCR特异性检测方法,分析了DPO引物退火温度不敏感性、检测灵敏度及特异性,并对检测方法进行了初步应用。灵敏度结果显示,DPO-PCR方法对霍乱弧菌的最低检出限为1.07×10^2 CFU/mL。退火温度不敏感性试验中,与常规PCR引物相比,DPO引物在45～65℃退火温度均能够高效扩增出靶基因片段。特异性结果显示,DPO-PCR方法的特异性比常规PCR方法强,不产生任何非特异性扩增。利用建立的霍乱弧菌DPO-PCR检测方法对采集的550份样本进行检测,检出43份霍乱弧菌阳性样本,经行业标准法（SN/T2425-2010）复检,检测结果相同,表明所建立的DPO-PCR检测方法具有良好的实用性,为霍乱弧菌的快速准确检测提供了新途径。

Development and application of DPO-PCR method for specific detection of Vibrio cholerae

In this study, a dual-priming oligonucIeotide（DPO）-based PCR method for detection of Vibrio cholerae was developed using mdh gene of V. cholerae as target gene. Annealing temperature insensitivity,detection sensitivity,specificity of the DPO primers were analyzed and application of the DPO-PCR method was carried out in practical detection. Sensitivity result showed that detection limit of the DPO-PCR method for V. cholerae was 1.07 × 10^2 CFU/mL. In the test of an nealing temperature insensitivity, compared with conventional PCR primers, the DPO primers were able to efficiently amplify target gene in the temperature range from 45~C to 65~C. In the experi ment of specificity,the DPO-PCR showed a higher specificity than conventional PCR, there was no non specific reaction in DPO-PCR. In practical detection,43 V. cholerae positive samples from collected 550 samples were detected by the DPO-PCR method,which was in accordance with the test results according to SN/T 2425-2010, showing a better practicability. DPO-PCR provided a new method for rapid and accurate detection of V. cholerae.