文章摘要
刘旭,马立才,赵姝,李健,王元,房文红.整合子相关元件多重PCR检测技术建立及其在弧菌中应用[J].上海海洋大学学报,2016,25(6):814-821.
LIU Xu,MA Licai,ZHAO Shu,LI Jian,WANG Yuan,FANG Wenhong.Development of multiplex PCR detection of integron-related elements and its application in Vibrio spp.[J].Journal of Shanghai Ocean University,2016,25(6):814-821.
整合子相关元件多重PCR检测技术建立及其在弧菌中应用
Development of multiplex PCR detection of integron-related elements and its application in Vibrio spp.
投稿时间:2016-03-02  修订日期:2016-06-26
DOI:10.12024/jsou.20160301675
中文关键词: 多重PCR  整合子  SXT  ISCR1  弧菌
英文关键词: multiplex PCR  integron  SXT  ISCR1  Vibrio
基金项目:上海市科技兴农重点攻关项目(沪农科攻字〔2014〕第3-4号);中央级公益性科研院所基本科研业务费专项(东海水产研究所L-2015-1602)
作者单位E-mail
刘旭 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090
上海海洋大学 水产与生命学院, 上海 201306 
 
马立才 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090  
赵姝 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090  
李健 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090
上海海洋大学 水产与生命学院, 上海 201306 
 
王元 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090  
房文红 中国水产科学研究院东海水产研究所 农业部海洋与河口渔业资源及生态重点开放实验室, 上海 200090 fwenhong@163.com 
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中文摘要:
      为了建立一种能同时检测1类整合子、4类超级整合子和1型插入序列共同区的多重PCR检测方法,根据GenBank中1类整合子整合酶基因int1、4类超级整合子整合酶基因intSXT和1型插入序列共同区转座酶基因ISCR1的序列,应用PrimerPlex 2.61设计3对特异性引物,通过改变引物浓度和退火温度对多重PCR体系进行了优化。此后,评价了所建立多重PCR体系的特异性和灵敏度,并应用于222株海水养殖源弧菌中3种整合子相关元件的检测。结果显示,设计的3对引物能分别单独且特异性地扩增出569 bp、430 bp和651 bp的目的条带;当int1、intSXT和ISCR13对引物(10 pmol/μL)的体积依次为0.3 μL、0.6 μL和0.6 μL,退火温度为50℃时,多重PCR体系能特异性地扩增出3个条带清晰、区分度良好的目的条带,方法的灵敏度达到0.02 ng/μL弧菌基因组模板DNA;利用所建立的方法对222株临床分离弧菌进行3种整合子元件检测得到的结果与文献报道的单引物检测结果一致。从上述研究结果可以看出,该多重PCR方法能快速准确地检测Int1、SXT和ISCR1,适用于整合子相关元件流行监测及整合子相关的耐药性研究。
英文摘要:
      In order to establish a multi-PCR detection method for the simultaneous detection of class 1 integron (Int1), class 4 integron (SXT) and insertion sequence common region 1(ISCR1), three pairs of specific primers were designed using PrimerPlex 2.61 based on the gene sequence of int1, intSXT and ISCR1 in GenBank. The multiplex PCR system was established and optimized by changing the primer volume and annealing temperature. The specificity and sensitivity of the optimized multiplex PCR were also evaluated, and then the multiplex PCR was applied to the detection of three kinds of integron-related elements in 222 Vibrio strains of mariculture source. The results showed that the above primers could amplify three bands of 569 bp, 430 bp and 651 bp specifically. The optimized reaction conditions were as follows:the volume of primers 0.3 μL for int1, 0.6 μL for intSXT, and 0.6 μL for ISCR1, and anneal temperature 50℃. Under the conditions, the detection minimum limit was 0.02 ng/μL of genome DNA. The results of detction for the above three integron-related elements in 222 Vibrio strains, using the optimized multiplex PCR, were the same as that of the control assays. In short, this study provides an effective multiplex PCR method for the simultaneous detection of int1, intSXT and ISCR1,which could be applied to studies on integron-associated antimicrobial resistance.
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