基于vvhA基因检测创伤弧菌实时荧光PCR方法的建立

doi:10.13652/j.issn.1003-5788.2016.09.007

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基于vvhA基因检测创伤弧菌实时荧光PCR方法的建立
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                        10.13652/j.issn.1003-5788.2016.09.007
                    
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作者单位:(1.海南出入境检验检疫局检验检疫技术中心，海南 海口 570311;2. 东北农业大学动医学院，黑龙江 哈尔滨 150001;3. 从化出入境检验检疫局，广东 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，辽宁 锦州 121001)
作者简介:李丹丹，女，海南出入境检验检疫局高级兽医师，博士。
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基金项目:海南省社会发展科技专项（编号：2015SF29）；国家质检总局科技项目（编号：2013IK031，2013IK051，2015IK089）；重庆市科技计划项目（编号：cstc2014yykfA80017）；海南省应用技术研究与开发专项项目（编号：ZDXM20130025）；广东检验检疫局科技计划项目（编号：2013GDK04，2015GDK53)

Establishment of a quantitative real-time PCR for detecting vvhA gene in Vibrio vulnificus

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(1. Animal Quarantine Lab, Inspection & Quarantine Technology Center of Hainan Entry-Exit Inspection & Quarantine Bureau, Haikou, Hainan 570311, China; 2. College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjinag 150001, China; 3. Conghua Entry-Exit Inspection & Quarantine Bureau, Conghua, Guangdong 510900, China; 4. Animal Quarantine Lab, Inspection & Quarantine Technology Center of Chongqing Entry-Exit Inspection & Quarantine Bureau, Chongqing 404100, China; 5. Laboratory of Bovine Genetics and Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 6. Department of Animal Husbandry & Veterinary Medicine, Liaoning Medical University, Jinzhou, Liaoning 121001, China)

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摘要:

为建立创伤弧菌（VV）的快速检测方法，根据vvhA基因序列设计合成引物和探针，建立实时荧光PCR方法。结果显示：所建立的方法能特异扩增出VV标准阳性菌株，而对其它14种菌株没有扩增；该方法的灵敏度为15 CFU/mL；稳定性和重复性试验结果表明，同一样品重复检测4次Ct 值(循环阈值)的变异系数均小于2%；利用该检测方法对采集的156份样品进行检测，共计检出2份VV阳性样品，与行标法（SN/T 1870—2007）检测结果一致。该检测方法灵敏度高、特异性强，具有良好的实用性。

Abstract:

To establish a rapid assay for Vibrio vulnificus (V. vulnificus) detection, a quantitative real-time PCR (qRT-PCR) method was developed targeting vvhA gene of V. vulnificus . The results showed that the tests for 15 strains of different bacteria by using this method, only the ones of V. vulnificus were positive, indicating high specificity of qRT-PCR. Moreover, the sensitivity of this specific method was 15 CFU/mL. Further stability and reproducibility test results showed that the coefficient of variation of Ct values of four duplicates were less than 2%. In addition, a total 2 positive samples for V. vulnificus were detected from 156 clinical samples by this method, and the results were in accordance with those by SN/T 1870-2007 standard detections. Therefore, the qRT-PCR method provides a novel rapid and sensitive detection method for V. vulnificus infection.

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李丹丹,徐义刚,邱索平,等.基于vvhA基因检测创伤弧菌实时荧光PCR方法的建立[J].食品与机械,2016,32(9):31-33,70.
LIDandan, XUYigang, QIUSuoping, et al. Establishment of a quantitative real-time PCR for detecting vvhA gene in Vibrio vulnificus[J]. Food & Machinery,2016,32(9):31-33,70.

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收稿日期:2016-01-06
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在线发布日期: 2023-03-09
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