Recombinant DNA in meat additives: Specific detection of Roundup Ready™ soybean by nested PCR

Soybean proteins are widely used by the meat industry as technological coadjutor when producing processed products such as emulsified and ground meat products. Since regulations for the use and labeling of GMOs and derived ingredients are in force in Brazil, a PCR‐based method capable of detecting Roundup Ready^? (RR) soybean was employed for meat additives. Thirty‐two samples of meat additives containing soy proteins were tested for the presence of soybean amplifiable DNA and RR soybean DNA. Twenty‐five samples gave a positive signal for the lectin gene, confirming the presence of soybean amplifiable DNA and 15 samples returned a positive signal for specific RR detection confirming the presence of genetically modified soy. These results demonstrate for the first time the presence of RR soybean in meat additives. This method may be useful for meat industries interested in controlling the presence of RR soybean in additives used for meat products manufacture. Copyright © 2007 Society of Chemical Industry     

转基因大豆Roundup Ready调控元件在食品加工过程中降解变化的研究

35S启动子可能会通过基因的水平转移插入到某一致癌基因上游，活化并导致癌症的发生。为了解转基因植物中调控元件的安全性问题，以转基因大豆Roundup Ready为实验材料，针对Roundup reaqdy转基因大豆中CaMV35S启动子及NOS终止子的序列，设计了不同长度片段的引物，通过对CaMV35S启动子和NOS终止子的PCR扩增，研究了豆腐、豆奶、豆粉3种大豆加工食品中磨浆、煮浆、调配、均质、杀菌、喷雾干燥等关键工艺对Roundup Reaqly大豆中调控元件的影响。结果表明调控原件在食品加工过程中的降解变化与其所处位置有较大关系。扩增长度相近的2个片段，包含大豆基因组。DNA序列的片段受加工过程的影响较小，在3种豆制品的所有加工过程中均能检测到。而只包含CaMV35S启动子序列的片段仅能在原料中检测到，原料经过磨浆后。片段大小降至200bp以下。NOS终止子受食品加工工艺的破坏和影响较小，在被检测食品的每一个加工过程中都能够检测到NOS终止子片段。     

A survey of the use of soy in processed Turkish meat products and detection of genetic modification

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.     

Polymerase chain reaction (PCR) in the quality and safety assurance of food: Detection of soya in processed meat products

A new method for the specific and sensitive detection of soya (Glycine max) in processed meat products has been developed using polymerase chain reaction (PCR) technology. The presence of soya deoxyribonucleic acid (DNA) from several soya protein concentrates was determined with two pairs of specific oligonucleotides yielding a 414-bp (bp=base pair) fragment and an internal 118-bp fragment amplified from the soya lectinLe1 gene. The test detected DNA from textured soya protein concentrates in meat products at a level of 1% and was confirmed by a commercially available enzyme-linked immunosorbent assay (ELISA).     

Detection of soya proteins in heated meat products by "blotting" and "dot blot"

Soy proteins (isolates, concentrates and texturates) as well as meat products containing soya isolate were analysed by SDS-electrophoresis. The separated proteins were blotted on nitrocellulose and stained with a selective immunoperoxidase system with the following sequence: primary (anti-soya) serum, goat anti-rabbit IgG serum and peroxidase-antiperoxidase complex (rabbit allotype). By developing the blot with a peroxidase substrate the antigenic soya fractions were visualised while the meat proteins did not stain. All major (reduced) soya fractions alpha, alpha', beta conglycinin, the acid and basic subunits of glycinin as well as some minor fractions became visible with a commercially available anti-soya serum as primary antiserum. The pattern thus obtained provides a high evidence for the presence of soya protein in meat products. Detection level is about 0.02% of soya protein. During a 24-h incubation at room temp. (before heat processing) of a meat product containing soya product and raw liver a remarkable loss of antigenic material was observed.     

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.     

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。     

Investigation of methods to detect mechanically recovered meat in meat products - IV: Immunology

The possibility of using immunological techniques as a method for the detection of mechanically recovered chicken meat in meat products has been investigated in this preliminary study. Antibodies were raised against a low molecular weight fraction (≤ 30 kDa) of chicken bone marrow proteins and an enzyme-linked immunosorbent assay (ELISA) developed. The system was used to test for the presence of mechanically recovered meat (MRM) in a range of product types, from raw chicken meat through to heat processed samples. The results show that it is possible to raise antibodies to chicken bone marrow proteins which show a strong reactivity with chicken and turkey MRM but show little reaction with extracts of MRM and hand deboned meat of other common meat species. However, blood, skin and soya all affected the accuracy of the ELISA.

This study has demonstrated the potential for the use of an immunological procedure as a rapid test for MRM. The selectivity of the antiserum would, however, have to be increased before this procedure could be considered as a suitable technique for the detection of MRM in meat products.     

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。     

Analysis of commercial soya additives in meat products

The quantitative aspects of the analysis of commercial soya additives in meat products have been investigated using a polyacrylamide gel electrophoresis technique. Extraction of the proteins from the food products was carried out either in 8 M -urea and 1%, 2-mercaptoethanol at 18 to 20 °C for 16 h (Method 1), or 10 M -urea and 4%, 2-mercaptoethanol at 100 °C for 30 min (Method 2). The proteins in the extracts were separated by electrophoresis on gels containing 6% polyacrylamide and either 6 M -urea (Method 1) or 8 M -urea (Method 2) using a 0.06 M -Tris-glycine buffer, pH 8.6. Densitograms of the stained protein bands were used for the quantitative estimation of the soya proteins. Both methods were found to give quantitative results for the analysis of soya protein additives in beefburgers and sausages. Method 1 was also found to give quantitative results for meat pie fillings and canned meat loaf products, which had been autoclaved at 115 °C for 40 min.     

Easy determination of the addition of soybean proteins to heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins

The addition of non-meat proteins to processed meat products is limited by regulations. Therefore, this work has investigated the determination of added soybean proteins in commercial heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins. The method consisted of extracting proteins from the meat products in a Tris-HCl buffer (pH 8) and analysing the extract by high-performance liquid chromatography with a linear gradient water-acetonitrile containing 0.05% (v/v) TFA. This method enabled the detection and quantitation of up to 0.08 and 0.28% (w/w), respectively, of soybean proteins (related to 6 g initial product) in these products. Satisfactory precision and recovery data were established. Accuracy was evaluated by a comparison of soybean protein contents determined by the proposed method and the existing AOAC official method based on an enzyme-linked immunosorbent assay (ELISA) from which no statistically significant differences were observed.     

多重实时荧光PCR快速检测转基因大豆及其加工产品

本研究运用多重实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因大豆及其深加工制品进行筛选检测。通过设计大豆内源基因植物凝集素(Lectin)和常用的外源基因花椰菜花叶病毒35S启动子(CaMV35S)、根癌农杆菌胭脂碱合成酶基因终止(nos)的特异性引物和探针,反应条件和反应体系的优化,特异性、重复性和灵敏性的实验比对分析等开发建立了多重荧光定量PCR检测技术。以10%Roundup Ready转基因大豆标准品为材料,建立并优化转基因大豆的定量检测体系,对大豆中的转基因成分进行定量分析。结果表明:该方法重复性好,检测特异性强,扩增效率在90%～110%,标准曲线相关系数R2≥0. 98,确定了最低检测限为每20μL反应2. 4个拷贝。结论:由于使用多重实时荧光PCR技术,可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,为促进农产品和食品进出口提供技术保障。     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

Detection of Residues of Genetically Modified Soybeans in Breaded Fried Turkey Cutlets

The presence of genetically modified organisms (GMOs) in food products is usually ascertained by the polymerase chain reaction (PCR) or nested PCR if sensitivity has to be increased. Since most, if not all, GMO products are of plant origin, the target sequences are the 35S promoter or NOS terminator. The extreme sensitivity of nested PCR can be misleading if the results are not interpreted correctly, since contamination of non-GMO products with residual amounts of GMO may be positive. We report that breaded turkey breast cutlets labeled as containing GMO soybean products were actually prepared from wheat flour that had been contaminated with transgenic soybean.     

Quantitation of soya protein by enzyme linked immunosorbent assay of its characteristic peptide

An enzyme linked immunosorbent assay (ELISA) is described for the quantitative measurement of soya proteins in processed food products. The peptide fragment bearing an antigenicity against anti-glycinin (soya bean 11S globulin) antibody was purified from the trypsin digests of autoclaved glycinin and used as an indicator antigen for soya proteins. ELISA samples were prepared also by a combination of autoclaving and tryptic digestion. Quantitation of soya proteins by ELISA was found to resist interference by other food materials, and the ELISA signal was independent of soya bean cultivar. The ELISA method was evaluated on pork sausages which were prepared in the laboratory to contain 0 to 20·8% soya protein isolate. With commercial soya protein isolate as reference standard, the results showed that the detected amounts were in quantitative agreement with the added amounts of soya protein isolate.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

肉制品中植源性转基因成分多重荧光定量PCR检测方法的建立

为改善产品品质,肉制品加工过程中常常添加植物源性成分,当前转基因农作物的商品化及其在市场上 的广泛流通导致肉制品中被带入植源性转基因成分的风险增加。以转基因植物中常涉及的调控元件CaMV 35S启 动子、NOS终止子以及标记基因NPTⅡ为检测目标,设计相应的引物和Taq man探针,利用载体pRⅠ 101-AN DNA 为模板,通过优化反应体系和反应参数,建立肉制品中植源性转基因成分的单重和多重荧光定量聚合酶链式反应 （polymerase chain reaction,PCR）检测方法。通过比较分析,考察多重荧光定量PCR检测方法的灵敏性、重复性和准 确性,结果表明,多重荧光定量PCR检测方法灵敏度高、重复性好且与单重体系的检测结果具有很好的一致性。     

肉制品中大豆蛋白的应用与检测研究进展

大豆蛋白已经广泛用于各类肉制品加工中。大豆蛋白对肉制品的保水性、质构具有一定的促进作用,但也存在豆腥味、致敏等不利影响。文中对大豆蛋白的功能性及其在肉制品中的应用、检测方法等进行了综述,并提出相关建议。     

PCR法检测大豆加工食品中的转基因成分

通过分子生物学手段,以聚合酶链式反应(PCR)技术为基础,建立了检测大豆加工食品中转基因成分的方法。实验分别采用CTAB法和试剂盒(Kit)法对大豆锅巴、豆浆、豆奶粉、豆腐、豆腐丝等五种大豆加工食品中的DNA进行了提取,用内标基因Lectin对此两种方法的提取效果进行了比较,并以提取的DNA为模板,利用不同的引物分别对目标基因35S和NOS进行了PCR扩增和琼脂糖凝胶电泳检测。结果表明:Kit法的DNA提取效果优于改良CTAB法,上述五种大豆加工食品中均检测出35S启动子和NOS终止子,且均含有转基因成分。