大豆及其加工食品转基因成分检测技术研究进展

随着基因工程技术在食用农产品领域的应用发展,形形色色的转基因食品应运而生。目前包括我国在内的许多国家为保护消费者的知情权和选择权,都建立了转基因食品标识管理制度。科学有效的转基因食品检测技术是实现我国转基因产品标识管理的前提。我国食品成分多样且基质复杂,针对初级农产品的转基因成分检测技术并不完全适用于工业加工食品尤其是深加工食品。这对食品监管及技术检测部门提出了新的要求。本文根据技术原理的不同分类介绍了目前几种主要的食品中转基因成分检测技术,除介绍现在已经成熟应用的技术方法外,本文还重点介绍了国内学者新近研发出的几种新技术,为研究人员进一步研发新技术提供重要参考。     

聚合酶链式反应方法检测豆制品中转基因成分的研究概述

大豆作为种植极广的农作物,在人们的生活中占据了重要位置,能加工成多种多样的产品,满足日常需要。 随着生物技术的 快速发展,转基因大豆以其高产量、低成本的优势迅速占领市场。 由于转基因产品的安全性未知,各国对其用途都有严格规定和监 管。该文从我国大豆制品的市场状况、核酸的残留、现行检测标准及实验过程中存在的问题等方面进行了总结和讨论,以期对转基因 产品的市场监管检测体系能够有所裨益。     

Detection of genetically modified organisms in foods by DNA amplification techniques

In this article, the different DNA amplification techniques that are being used for detecting genetically modified organisms (GMOs) in foods are examined. This study intends to provide an updated overview (including works published till June 2002) on the principal applications of such techniques together with their main advantages and drawbacks in GMO detection in foods. Some relevant facts on sampling, DNA isolation, and DNA amplification methods are discussed. Moreover; these analytical protocols are discuissed from a quantitative point of view, including the newest investigations on multiplex detection of GMOs in foods and validation of methods.     

双重数字聚合酶链式反应定量检测转基因马铃薯EH92-527-1品系

目的 建立双重数字聚合酶链式反应(polymerase chain reaction,PCR)法精准定量检测转基因马铃薯EH92-527-1品系的方法。方法 基于马铃薯内源UGPase基因和EH92-527-1品系外源插入片段旁侧序列,设计合成引物和探针,确定反应体系和反应条件,建立转基因马铃薯EH92-527-1品系双重数字PCR定量检测方法。对方法的特异性、定量检测范围、定量检出限、检测准确度进行评估。结果 该方法特异性良好,除转基因马铃薯EH92-527-1品系外,其他物种和品系均无扩增;在线性范围内,内参基因和品系特异性基因拷贝数的相对标准偏差值介于 0.86%～22.90%（＜25%为可接受的范围）,线性决定系数R2＞0.99;品系特异性基因的定量检出限为3拷贝;不同浓度样品EH92- 527-1品系含量测定值与真实值之间的偏差分别为1.54、4.92和-1.31。结论 方法具有良好的重复性和准确度,可以用于进出口产品中转基因马铃薯EH92-527-1成分的定性定量检测。该方法的建立对于转基因马铃薯及其制品的监控监管、安全评价和风险预警具有重要意义。     

数字PCR技术在转基因检测与溯源中的应用

转基因食品的安全性一直备受关注,准确测定转基因成分及其含量十分重要。近年来,数字聚合酶链式反应(digital polymer chain reaction,dPCR)技术因具有高灵敏度、受基质干扰少等优势而被广泛用于转基因食品检测。本研究综述了数字PCR技术的原理、系统各部件的优劣势,梳理了数字PCR技术在转基因检测与溯源技术(标准物质)方面的进展和发展趋势,以期为转基因食品检测领域提供参考依据。     

PCR法定性检测大豆食用油中转基因成分

针对大豆食用油中核酸含量低且被严重破坏、DNA难以提取的问题,对CTAB法进行优化,有效从食用油中提取到可用于PCR扩增反应的DNA模板。同时定性检测出大豆内源基因lectin,外源调控序列启动子CaMV35S、终止子Nos及目的基因CP4-EPSPS序列,成功建立了基于PCR技术在大豆食用油中快速检测转基因成分的方法。     

Nested PCR detection of genetically modified soybean in soybean flour, infant formula and soymilk

Due to the Brazilian market introduction of the genetically modified (GM) crop Roundup Ready™ (RR) soybean, the ability to detect GM crops has become a legal necessity. In order to detect the presence of RR soybean, a polymerase chain reaction (PCR) amplification method was evaluated for the detection of RR in soybean mixtures and commercially available soy flour, infant formula and soymilk powder. To detect the presence of RR soybean, a nested PCR resulted in an amplicon of 169 bp, present for all soybean mixed samples containing 0.01-10% GM soybean and absent for 0% GM soybean. None of the analysed infant formulas showed a positive signal after the nested PCR; four out of six soy flour samples and 15 out of 25 soymilk powder samples were positive for the presence of RR soybean. Results show that the nested PCR method used is adequate to determine the presence of GM soybean in the presented products.     

微生物酶制剂中转基因微生物的实时荧光PCR检测方法

针对微生物酶制剂中残留转基因微生物的检测问题,根据醇氧化酶-1（AOX1）启动子基因的序列信息设计一对引物及一条MGB探针,建立了转基因微生物的实时荧光PCR筛选检测方法。实验结果表明,该方法灵敏度达到1pg,可以准确判定生产线上不同分离阶段的微生物酶制剂中的转基因微生物残留情况。该方法准确度和灵敏度高,操作方便,可作为微生物酶制剂中转基因微生物分离状况的监测方法,也可为其他转基因微生物检测研究提供借鉴和参考。      

Systematic comparisons of genetically modified organism DNA separation and purification by various functional magnetic nanoparticles

DNA extraction is always an important and key step in bioanalytical fields for target nucleic acid detection. Traditional organic solvent‐based extraction methods are toxic and time‐consuming. In this work, we report a systematic study of magnetic nanoparticles (MNPs) as probes for genomic DNA extraction from genetically modified organism (GMO) plants. Different surface‐functionalised MNPs with controllable diameters have been used to extract the genomic DNA directly from GMO plants for detections. Systematic comparison of the results obtained under different extraction conditions has indicated that carboxyl‐modified MNPs with smaller diameters are more suitable for genomic DNA extractions. The successful qualitative detection of GMO and non‐GMO products based on the MNP extracted DNA is also achieved and discussed in this article. In view of the advantages of magnetic extraction, such as nontoxicity, ease of operation, and rapid and high throughput, this systematic research has demonstrated the great potential of the method and provides theoretical guidance for practical MNP applications.     

转基因大豆及其制品二重荧光定量PCR的建立与验证

研究选用花椰菜花叶病毒35S启动子(P-35S)和根癌农杆菌胭脂碱合成酶基因终止子(T-NOS)为靶标基因,运用多重实时荧光PCR技术对转基因大豆及其制品进行快速筛选检测。实验通过样品核酸提取与质控,多重引物及荧光探针的设计与筛选,反应条件和反应体系的对比优化,摸索出二重荧光定量PCR检测的最佳反应体系。同时通过特异性、重复性、灵敏性和适用性实验验证,确保了该方法在同时检测2个靶标基因时,无荧光信号的相互干扰,不会出现假阴性和假阳性结果。结果表明,方法特异性高,可同时筛查两个靶标基因,扩增效率在90%~110%之间,标准曲线决定系数R²>0.98,确定了最低检测限为2拷贝/μL。开发和建立的二重荧光定量PCR检测技术可以实现一管多检的实际需要,降低试剂成本,缩短检测时间,为大豆及其深加工产品转基因成分的快速检测提供了有效方法,为促进食品进出口提供技术保障。     

转基因大豆DAS44406-6多重荧光定量PCR检测方法的建立

本文针对大豆内源基因Lectin和转基因大豆DAS44406-6品系的5’端插入位点序列,设计特异性引物及探针,建立同时检测转基因大豆DAS44406-6品系和大豆内源基因Lectin的多重荧光定量PCR方法,并运用15种转基因大豆、3种转基因玉米、1种转基因油菜、1种转基因水稻和非转基因大豆对该方法进行特异性评价,并分析该方法的灵敏度和稳定性。结果显示,该方法能准确从20种转基因样品和1种非转基因样品中检出靶目标,检测结果与待检样品信息一致,表明本方法具有良好的特异性;灵敏度高达0.01%;重复性实验表明DAS81419品系4种含量、9次重复反应Ct值的标准偏差介于0.050⁰.222,相对标准偏差介于0.169%⁰.677%,均在可接受范围内。该方法特异性强、灵敏度高、稳定性强,适用于各口岸实验室进行转基因大豆DAS44406-6的快速、准确的检测。      

Quantitative competitive PCR for the detection of genetically modified soybean and maize

 The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques. Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more than 1% RRS were determined by QC-PCR using various amounts of standard DNA. Received: 13 January 1998 / Revised version: 4 March 1998     

实时荧光定性聚合酶链式反应标准方法检测转基因产品的验证及应用

目的验证实验室大豆、玉米和水稻及其制品转基因检测方法,并应用于实际样品检测。方法根据GB 19495.4-2018《转基因产品检测实时荧光定性聚合酶链式反应(PCR)检测方法》要求对无转基因标识的样品进行转基因成分检测。结果方法验证满意。40批次样品(大豆、玉米和水稻及其制品)中发现1批次的转基因成分检出,检出率为2.5%。结论市场中绝大部分未标示转基因成分的大豆、玉米和水稻及其制品确实未检出转基因成分,仅有极少数产品含有转基因成分,但未进行有效标识。     

The impact of non- and genetically modified soybean diets in aorta wall remodeling

The aim of this study was to evaluate the influence of nongenetically modified soybean (non-GMS) and genetically modified soybean (GMS) meal on growth and cardiometabolic parameters in rats. Thirty male Wistar rats were divided into 3 groups (n= 10): non-GMS, GMS, and control group (CG). All animals received water and an isocaloric diet ad libitum for 455 d. Blood was drawn by cardiac puncture, and serum was separated for subsequent biochemical analyses (total cholesterol, triacylglycerols, insulin, glucose, and testosterone). The aorta was quickly harvested and fixed; the body fat mass was removed and weighed. Non-GMS and GMS had a growth index (GI) similar to CG but with a lower body weight (P < 0.05) and a lower amount of body fat mass (P < 0.05). Total cholesterol, triacylglycerol, glucose concentrations, and aortic tunics were reduced (P < 0.05) in non-GMS and GMS compared to CG. Non-GMS and GMS are able to reduced serum cholesterol, triacylglycerols, glucose, and aortic remodeling in aged rats. No differences were observed between non-GMS and GMS in all parameters.     

Detection of genetically modified soybean and its product tou-kan by polymerase chain reaction with dual pairs of DNA primers

Since genetically modified (GM) crops and foods began to appear in market, the detection of these GM foods has become an important issue. Efficient and reliable methods are urgently needed to analyze the GM content of a large quantity of foods. However, most foods are processed through heating or cooking, and their proteins are denatured. Therefore, DNA rather than protein is the major target for detecting GM foods. In this report, polymerase chain reaction (PCR) was performed with dual sets of DNA primers, which co-amplified the soybean-specific lectin gene and the transgenic petunia transit peptide sequence (CTP) in the 5′ end of the 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene within the same reaction. PCR with these two sets of primers reacted specifically with lectin gene and CTP, and can detect soybean with GM content less than 1%. PCR detecting both lectin gene and CTP was also applied to a soybean-derived product tou-kan, a traditional oriental food. Results indicated that although DNA was partially degraded in tou-kan, this PCR method was still able to detect the presence of EPSPS gene. However, when the DNA within tou-kan was destroyed badly, neither lectin nor EPSPS genes were detected by the PCR suggested here. Finally, an examination procedure for the Roundup Ready soybean was suggested according to the results of PCR with dual pairs of DNA primers. The proposed PCR method has proved to be a reliable and efficient method for detecting the GM content of the processed foods from Roundup Ready soybean. An erratum to this article can be found at     

2012~2015年深圳市售转基因大豆及其产品的监测结果分析

目的 了解2012~2015年深圳市售转基因大豆及其制品的市场占有率、标识情况及发展态势。方法 在深圳各大连锁超市随机抽取大豆及其制品, 采用试剂盒法提取样品DNA, 采用实时荧光PCR的方法扩增大豆内源基因Lectin, 并扩增外源基因CaMV35S启动子和NOS终止子对样品进行定性筛查, 分析不同类别、不同年份样品的阳性率差异及变化趋势。结果 4年监测大豆及豆制品共283份, 检出阳性样品19份,总体阳性率为6.71%; 大豆样品的转基因阳性率为0; 不同年份的豆制品转基因阳性率之间存在显著性差异且有逐年上升的趋势; 所有检出的阳性样品均未按照规定标识。结论 目前深圳市场转基因大豆及其制品(大豆油除外)的市场占有率还很低, 但豆制品转基因阳性率有逐年上升的趋势。政府应该加强转基因产品的标识管理, 并加强转基因农产品的监管。     

多重PCR方法对大豆转基因食品的定性检测

为了同时检测转基因食品中所含的多个目标基因序列,本文采用多重PCR分析技术对转基因大豆食品进行了检测。为了排除扩增结果的假阴性,大豆外源凝集素基因和肌动蛋白基因被作为内部对照以评价所有PCR反应的效率。当转基因大豆含量仅为0.15%时,仍然可以对转基因食品进行可靠的鉴定,从而表明该方法的高度敏感性。该文所述的多重PCR系统是一种简单、准确并且敏感的检测方法,只需要进行一个反应就可以检测多个目标基因序列,因而可以用来对转基因原材料及加工成品进行高精度、高灵敏的检测。     

An overview of genetically modified crop governance,issues and challenges in Malaysia

The application of agricultural biotechnology attracts the interest of many stakeholders. Genetically modified (GM) crops, for example, have been rapidly increasing in production for the last 20 years. Despite their known benefits, GM crops also pose many concerns not only to human and animal health but also to the environment. Malaysia, in general, allows the use of GM technology applications but it has to come with precautionary and safety measures consistent with the international obligations and domestic legal frameworks. This paper provides an overview of GM crop technology from international and national context and explores the governance and issues surrounding this technology application in Malaysia. Basically, GM research activities in Malaysia are still at an early stage of research and development and most of the GM crops approved for release are limited for food, feed and processing purposes. Even though Malaysia has not planted any GM crops commercially, actions toward such a direction seem promising. Several issues concerning GM crops as discussed in this paper will become more complex as the number of GM crops and varieties commercialised globally increase and Malaysia starts to plant GM crops. © 2017 Society of Chemical Industry