节点文献
环介导等温扩增技术对外源转基因成分检测及其协同验证
【作者】 李荣；
【作者基本信息】 扬州大学 ， 营养与食品卫生学， 2015， 硕士
【摘要】 近些年来转基因食品的安全性问题受到了社会各界的广泛关注,因此国家对转基因食品安全的监管力度也在逐步加强。目前对转基因食品的检测,除了常规的单靶标核酸检测外,还包括对多靶标目的基因进行筛选检测和分析。随着技术的不断发展和进步,转基因食品的检测技术也正向着快速、精确、高通量的检测方向发展。常规的转基因食品检测方法主要是聚合酶链式反应(Polymerase Chain Reaction, PCR)技术、实时荧光定量技术,但这些转基因检测技术都需要精密复杂的温度循环仪器而且检测时间比较长,这样就极大限制了它们在现场或者田间快速检测的推广和应用。但近些年等温核酸扩增技术得到了快速的发展,其中环介导等温扩增技术(Loop-mediated Isothermal Amplification, LAMP)凭着特异性和灵敏度高、简单高效、所需仪器简单等的优势在细菌、病毒、转基因食品等领域得到了广泛的应用。本研究选择了转基因粮油原料中常见的3种外源转基因成分,利用环介导等温扩增方法对其外源转基因成分进行检测。根据这3种常见外源转基因成分((CP4 epsps, Pat, NPTⅡ)的基因序列,设计特异性的环介导等温扩增(LAMP)引物,优化反应体系,并通过在扩增产物中加入SYBR Green Ⅰ (1000×)荧光染料,建立了可视化的环介导等温扩增检测方法。实验结果表明,本研究建立的环介导等温扩增方法对这3.种外源转基因成分(CP4 epsps, Pat, NPT Ⅱ)检测的特异性高,灵敏度强,检测限基本上都能达到5-10拷贝,比传统PCR的检测限高,而且整个LAMP检测过程仅需1小时左右,检测结果可以通过琼脂糖凝胶电泳和染料两种方法来判断。为了验证建立的环介导等温扩增方法的实用性和有效性,我们邀请了国内外10家研究机构进行协同验证。对建立的LAMP检测方法验证之后,将该方法运用于样品的实际检测当中。因此,环介导等温扩增技术的运用不仅实现了快速、高效的实验室内部检测,而且也为现场或田间检测提供了强有力的技术支持。
【Abstract】 The safety of genetically modified food (GM food) in recent years has been the hot topic in the public. Therefore, the genetically modified food safety regulation has becomes more important. For the detection of exogenous transgenic ingredients of genetically modified (GM) food, the detection tend to not only for the conventional single target nucleic acid detection but also for rapid, accurate and high-throughput multiple targets detection analysis at the same time.Conventional GM food detection method is mainly polymerase chain reaction (PCR) technology, real-time fluorescent quantitative PCR technology, but these transgenic detection technology requires sophisticated temperature cycle instrument and spends more time, thus greatly restricts their application of rapid detection on the spot or the field. But the isothermal nucleic acid amplification technique has obtained the rapid development in recent years, the loop-mediated isothermal amplification techniques (LAMP) has been well developed with the advantages of high specificity and sensitivity, simple instruments and operation, high-efficiency in the detection of bacteria, virus and genetically modified foodThis research has developed LAMP detection method for the transgenic exogenous genetically modified ingredients in food, we mainly choose three common genetically modified ingredients among genetically modified food.According to the three common genetically modified ingredients (CP4 epsps, pat ,Npt II) sequence,we designed special LAMP primers, optimized LAMP reaction system, and established visible identification with SYBR Green I (1000 x) fluorescent dye through the loop-mediated isothermal amplification detection method.Experimental results proved that the LAMP detection method for these 3 common exogenous transgenic ingredients in GM food is of high specificity, sensitivity and limit of detection can achieve 5 copies, is higher than the detection limit of traditional PCR, and the LAMP testing just need about one hour. In addition to observed the change of the color by naked eye identification directly, it could also detected through agarose gel electrophoresis.In order to verify the practicability and validity of loop-isothermal amplification method, we had invited ten research institutions to help us validate the LAMP methods. After the validation, these methods have been applied for the practical sample detection. Therefore loop-mediated isothermal amplification technique provides a strong technical support for the rapid, convenient detection field in the future.