Detection of exogenous gene doping of IGF‐I by a real‐time quantitative PCR assay

Zhang Jin‐Ju; Xu Jing‐Feng; Shen Yong‐Wei; Ma Shi‐Jiao; Zhang Ting‐Ting; Meng Qing‐Lin; Lan Wen‐Jun; Zhang Chun; Liu Xiao‐Mei

摘要

Abstract > Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin‐like growth factor I (IGF‐I) exogenous gene for gene doping detection. First, the synthetic IGF‐I gene was subcloned to recombinant adeno‐associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF‐I‐GFP vectors. Second, in an animal model, rAAV2/IGF‐I‐GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real‐time quantitative PCR was employed to detect the exogenous gene doping of IGF‐I. In view of the characteristics of endogenous IGF‐I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF‐I gene to distinguish it from the exogenous IGF‐I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false‐positive results through comparison of their threshold cycle ( Ct ) values. Thus, an accurate exogenous IGF‐I gene detection approach was developed in this study. </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract XMLNS =“http://www.wiley.com/namespaces/wiley”type =“main”> <title类型=“main”>抽象</ title> >自产品以来可以轻松隐藏基因掺杂类似于内源性蛋白质，使其有效的检测非常具有挑战性。在这项研究中，我们选择了胰岛素样生长因子I（IGF-I）外源基因进行基因掺杂检测。首先，将合成的IGF-I基因亚克隆至重组腺相关病毒（RAAV）质粒以产生重组rAAV2 / IGF-I-GFP载体。其次，在动物模型中，将RAAV2 / IGF-I-GFP载体注入小鼠的大腿肌肉组织中，然后在不同时间点对肌肉和血液进行取样进行总DNA分离。最后，使用实时定量PCR检测IGF-1的外源基因掺杂。鉴于内源IGF-I基因序列的特征，在IGF-I基因的外显子2和3的结处设计了Taqman探针，以将其与外源IGF-I基因区分开。此外，通过比较其阈值循环（ CT-I>）值，在PCR中使用内部参考控制质粒及其探针以排除假阳性结果。因此，在本研究中开发了一种准确的外源IGF-I基因检测方法。 </ p> </ abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15016/'>《Biotechnology and Applied Biochemistry》</a> <b style="margin: 0 2px;">|</b><span>2017年第4期</span><b style="margin: 0 2px;">|</b><span>共6页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Zhang Jin‐Ju&option=202" target="_blank" rel="nofollow">Zhang Jin‐Ju;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Xu Jing‐Feng&option=202" target="_blank" rel="nofollow">Xu Jing‐Feng;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Shen Yong‐Wei&option=202" target="_blank" rel="nofollow">Shen Yong‐Wei;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Ma Shi‐Jiao&option=202" target="_blank" rel="nofollow">Ma Shi‐Jiao;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Zhang Ting‐Ting&option=202" target="_blank" rel="nofollow">Zhang Ting‐Ting;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Meng Qing‐Lin&option=202" target="_blank" rel="nofollow">Meng Qing‐Lin;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Lan Wen‐Jun&option=202" target="_blank" rel="nofollow">Lan Wen‐Jun;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Zhang Chun&option=202" target="_blank" rel="nofollow">Zhang Chun;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Liu Xiao‐Mei&option=202" target="_blank" rel="nofollow">Liu Xiao‐Mei;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>College of Physical EducationSoochow UniversitySuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> <p>CAS Key Lab of Bio‐Medical DiagnosticsSuzhou Institute of Biomedical Engineering and Technology Chinese Academy of SciencesSuzhou People's Republic of China;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/180.html" title="生物工程学（生物技术）">生物工程学（生物技术）;</a><a href="https://www.zhangqiaokeyan.com/clc/177.html" title="生物化学">生物化学;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=gene doping&option=203" rel="nofollow">gene doping;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=IGF‐I&option=203" rel="nofollow">IGF‐I;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=gene therapy&option=203" rel="nofollow">gene therapy;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=recombinant adeno‐associated virus (rAAV)&option=203" rel="nofollow">recombinant adeno‐associated virus (rAAV);</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=internal reference control (IRC)&option=203" rel="nofollow">internal reference control (IRC);</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=real‐time quantitative PCR&option=203" rel="nofollow">real‐time quantitative PCR;</a> </p> <div class="translation"> 机译：基因掺杂;IGF-I;基因治疗;重组腺相关病毒（RAAV）;内部参考控制（IRC）;实时定量PCR; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> <h3 class="all_title" id="enpatent66">相似文献</h3> <div class="similaritytab clearfix"> <ul> <li class="active" >外文文献</li> <li >中文文献</li> <li >专利</li> </ul> </div> <div class="similarity_details"> <ul > <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/academic-journal-cn_agricultural-science-technology_thesis/020122758254.html">利用SYBR Green实时定量PCR法检测转基因植物外源基因的拷贝数</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=裘劼人&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">裘劼人,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=许颖&option=202" target="_blank" rel="nofollow" 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target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,喻富根</a> <span> <a href="/journal-cn-7281/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 安徽农业科学 </a> </span> <span> . 2011</span><span>,第021期</span> </span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/academic-journal-cn_journal-zhejiang-sci-tech-university-social-sciences-edition_thesis/0201248261441.html">SYBR Green实时定量PCR检测外源基因拷贝数</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=庄强&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 庄强</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=钱程&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,钱程</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=刘立&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,刘立</a> <span> <a href="/journal-cn-4538/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 浙江理工大学学报 </a> </span> <span> . 2010</span><span>,第001期</span> </span> </div> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/academic-journal-cn_medical-laboratory-science-clinics_thesis/0201225464453.html">实时定量PCR与常规PCR检测乙型肝炎病毒感染的比较及临床意义</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=刘长生&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 刘长生</a> <span> <a href="/journal-cn-5835/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 医学检验与临床 </a> </span> <span> . 2008</span><span>,第004期</span> </span> </div> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/academic-conference-cn_meeting-43316_thesis/020222755062.html">牛副流感病毒3型(BPIV3)荧光实时定量PCR检测方法的建立及在牛源样本检测中的应用</a> <b>[C]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=王吉&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 王吉</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Wang Ji&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Wang Ji</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Fu Rui&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Fu Rui</a> <span> <a href="/conference-cn-43316/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 第十三届中国实验动物科学年会 </a> <span> <span> . 2017</span> </span> </div> </li> <li> <div> <b>6. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020312383518.html">利用实时定量PCR技术检测柑橘外源基因的拷贝数</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=文莉&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 文莉</a> <span> . 2011</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06120103023452.html">一种检测牛IGF-1基因的特异性引物、其荧光定量PCR检测试剂盒及其检测方法和应用</a> <b>[P]</b> . <span> 中国专利： CN104313124A </span> <span> . 2015-01-28</span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06120106659561.html">一种造血嵌合体实时定量PCR检测方法的PCR反应体系</a> <b>[P]</b> . <span> 中国专利： CN102443632A </span> <span> . 2012-05-09</span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130416670572.html">Automatic realtime PCR system for the various analysis of biological sample, method for Automatic nucleic acid purification and realtime quantification of gene amplification, method for automatic viable cell count of pathogenic bacteria using realtime quantitative PCR, method for automatically getting antigene density using quantitative immunity PCR</a> <b>[P]</b> . <span> 外国专利：  KR101420094B1 </span> <span> . 2014-07-17</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：用于对生物样品进行各种分析的自动实时PCR系统，自动核酸纯化和基因扩增的实时定量方法，使用实时定量PCR进行病原菌自动活细胞计数的方法，使用定量免疫PCR自动获得抗原密度的方法 </span> </p> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/patent-detail/06130422055878.html">AUTOMATIC REALTIME PCR SYSTEM FOR THE VARIOUS ANALYSIS OF BIOLOGICAL SAMPLE, METHOD FOR AUTOMATIC NUCLEIC ACID PURIFICATION AND REALTIME QUANTIFICATION OF GENE AMPLIFICATION, METHOD FOR AUTOMATIC VIABLE CELL COUNT OF PATHOGENIC BACTERIA USING REALTIME QUANTITATIVE PCR, METHOD FOR AUTOMATICALLY GETTING ANTIGENE DENSITY USING QUANTITATIVE IMMUNITY PCR</a> <b>[P]</b> . <span> 外国专利：  KR20120044197A </span> <span> . 2012-05-07</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：用于各种生物样品分析的自动实时PCR系统，自动核酸纯化和基因扩增的实时定量方法，利用实时定量PCR自动检测病原菌的活细胞数量的方法，用于定量自动定量抗原性的方法 </span> </p> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/patent-detail/06130408591183.html">Real-time PCR assays for quantitative detection of Viral hemorrhagic septicemia virus</a> <b>[P]</b> . <span> 外国专利：  KR101744181B1 </span> <span> . 2017-06-07</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：实时PCR测定法定量检测病毒性败血病病毒 </span> </p> </li> </ul> </div> </div> </div> <div class="theme cardcommon" style="overflow: auto;display:none"> <h3 class="all_title" id="enpatent55">相关主题</h3> <ul id="subject"> </ul> </div> </div> </div> </div> <div class="right rightcon"> <div class="details_img cardcommon clearfix" style="margin-bottom: 10px;display:none;" > </div> </div> </div> <div id="thesis_get_original1" class="downloadBth" style="bottom: 19px;z-index: 999;" onclick="ywcd('0704021881154','4',7,2,1,'',this,24)" class="delivery" prompt="010401" title="通过人工服务将文献原文发送至邮箱" >获取原文</div> 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