基于双启动引物PCR方法检测溶藻弧菌的研究  

Research on Dual-priming Primers-based PCR Method for the Detection of Vibrio alginolyticus

作　　者：李丹丹[1] 徐义刚 邱索平 王昱[4] 高会江[5] 高慎阳[6] 

机构地区：[1]海南出入境检验检疫局检验检疫技术中心,海口570311 [2]黑龙江出入境检验检疫局检验检疫技术中心,哈尔滨150001 [3]从化出入境检验检疫局,从化510900 [4]重庆出入境检验检疫局检验检疫技术中心,重庆404100 [5]中国农业科学院北京畜牧兽医研究所牛遗传育种研究室,北京100193 [6]辽宁医学院畜牧兽医学院,锦州121001

出　　处：《食品工业》2016年第8期277-280,共4页The Food Industry

基　　金：海南省社会发展科技专项(2015SF29);国家质检总局科技项目(2013IK031;2013IK051;2015IK089);重庆市科技计划项目(cstc2014yykf A80017);海南省应用技术研究与开发专项项目(ZDXM20130025);广东检验检疫局科技计划项目(2013GDK04;2015 GDK53)

摘　　要：为建立检测溶藻弧菌（VA）的快速检测方法,试验以VA Collagenase基因为靶基因设计了一对双启动寡核苷酸引物（DPO）,建立了DPO-PCR快速检测VA的方法。结果显示,退火温度范围在48℃-68℃内都能有效地扩增出目的基因,表明该检测方法对退火温度不敏感;该DPO引物仅与VA的扩增反应呈阳性,而与其他菌株无非特异性扩增反应,表明该方法特异性强;该方法的灵敏度为1.14×10^2 CFU/m L。利用该检测方法对采集的550份样品进行检测,共计检出4份VA阳性样品,与商检行业标准（SN/T 1870—2007）检测结果一致,显示了良好的实用性。该DPOPCR方法设计简单、特异性强,具有良好的实用性。To establish a rapid assay for Vibrio alginolyticus（VA） detection,a dual-priming oligonucleotide（DPO）-based PCR method was developed using the DPO primers targeting collagenase gene of VA.The DPO-PCR method had a detection limit of 1.14×10^-2 CFU/m L for VA,which was able to efficiently amplify target gene in the annealing temperature range from 48 ℃ to 68 ℃,showing insensitivity to annealing temperature.In addition,the DPO-PCR method had high specificity due to special structure of DPO primers,thus no non-specific amplifications were produced in reaction.Furthermore,a total 4 positive samples for VA were detected from 550 clinical samples by the DPO-PCR method,which was in accordance with the testing result by SN/T 1870—2007 standard detection protocol.Therefore,the DPO-PCR method provided a novel rapid and sensitive detection method for VA infection.

关 键 词：溶藻弧菌 Collagenase基因 DPO-PCR 

分 类 号：TS207.4[轻工技术与工程—食品科学]