A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.     

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。     

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.     

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.     

Ochratoxin A reduction in meat sausages using processing methods practiced in households

The aim of this study was to investigate the possibilities of ochratoxin A (OTA) reduction in home-made meat products. Meat sausages (n = 50) produced from raw materials coming from pigs exposed to OTA-contaminated feed, were subject to common heat processes practiced in households (cooking, frying and baking). Concentrations of OTA in pre- and post-processed products were quantified using a validated immunoassay method, enzyme-linked immunosorbent assay, and confirmed using a high-performance liquid chromatography with fluorescence detection. In line with the differences in recipes used and the degree of OTA accumulation in raw materials, OTA concentrations established in Mediterranean and roast sausages were lower than those found in liver and blood sausages. Baking of contaminated sausages at the temperatures of 190–220°C (for 60 min) resulted in significant reduction of OTA levels (75.8%), while 30-min cooking (at 100°C) and frying (at 170°C) proved to be significantly less effective (e.g. yielding OTA reductions of 7.4% and 12.6%, respectively). The results pointed out that despite high OTA stability, heat processes are capable of reducing its concentration in home-made meat products, depending on the processing modality used.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

Utilisation of protein fractions from porcine spleen as technofunctional ingredients in emulsified cooked meat sausages

Spleen is a low commercial value meat by-product from industrial slaughterhouses that is mostly underutilised. In this study, porcine spleens were processed to obtain two protein fractions (i.e. soluble and insoluble extracts in sodium citrate buffer 0.1 m pH 5) and the potential of using them to replace functional ingredients in cooked sausages was assessed. Specifically, a pasteurised insoluble protein extract was used as soy protein replacer in mortadella-type sausages, whereas spray-dried soluble proteins were used instead of sodium caseinate in Frankfurt-type sausages. Both splenic extracts performed well as functional ingredients since no differences in proximate composition, emulsion stability, water-holding capacity or texture were found. The only differences were found in the colour of the sausages containing the soluble protein extract, because of the haem and myoglobin content in this ingredient. The results showed that spleens could be a suitable source of non-allergenic functional proteins for meat products.     

肉制品中牛源性成分多重实时荧光PCR检测 方法的建立及初步应用

目的建立肉制品中牛源性成分的荧光PCR检测方法。方法根据牛特异性线粒体DNA片段,设计合成两对引物,以生、熟牛肉及超市牛肉加工品为材料,建立肉制品中牛源性成分的多重实时荧光PCR检测方法,并用该法与国标法同时对市售的25份肉制品同时进行检测,通过对其他种类的肉源DNA进行扩增验证方法的特异性;对含有不同比例牛肉成分的DNA样本进行检测确定检出限。结果该方法可成功检测出肉制品中的牛源性成分。在25份肉制品检测中,与国标法检测结果一致。该法的特异性为100%,灵敏度检测线为1%。结论本研究成功建立牛源性肉制品的检测方法,该方法快速简便,且具有较高的特异性和灵敏度,可用于市售肉制品中牛源性成分的鉴定。     

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     

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.     

肉制品中鸭源性成分的实时荧光PCR检测

目的：建立基于实时荧光PCR技术的鸭源性成分的快速检测方法。方法：以鸭线粒体DNA序列为目的基因,设计并筛选了鸭源性特异性引物及探针,进行荧光定量PCR扩增,建立鸭源性成分检测方法。通过特异性、灵敏性、及盲样检测试验,对该体系进行验证。结果：该方法能够快速有效的检测鸭源性成分,具有较强的特异性及灵敏性,灵敏度约为0．01％（质量分数）;通过市售盲样肉制品的检测,表明该体系可用于定性检测加工肉制品中的鸭源性成分。结论：该方法特异性强,灵敏度高,可用于对肉制品中鸭源性成分的掺假鉴别。     

多重实时荧光定量PCR检测肴肉中的特定腐败菌

本试验以真空包装水晶肴肉中的3株特定腐败菌屎肠球菌(Enterococcus faecium)、中间耶尔森菌(Yersinia intermedia)和清酒乳杆菌(Lactobacillus sakei)为研究对象,通过克隆3株菌的特异性DNA片段,构建重组质粒作为阳性模板,建立一种多重实时荧光定量PCR方法同时检测这3株特定腐败菌,结果表明:建立的多重实时荧光定量PCR具有较高的特异性和灵敏度,同时还具有很好的稳定性,可用于真空包装水晶肴肉贮藏期间特定腐败菌数量的检测,从而可以间接评价产品的货架期。     

大豆分离蛋白在肉制品加工中的应用

对大豆分离蛋白的保水性、乳化性、吸油性、凝胶性、发泡性等功能特性以及其在肉制品加工中的应用研究进展进行了综述,并根据当前的研究现状对大豆分离蛋白今后在肉制品加工中的应用提出几点展望与建议。     

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     

2015~2017年广州市某食品厂低温肉灌肠生产加工过程微生物污染状况及风险分析

目的 了解低温肉灌肠生产加工过程中主要食源性致病菌的污染状况, 分析生产加工过程存在的微生物污染风险。方法 采集2015～2017年广州市某食品厂低温肉灌肠生产加工过程不同环节样品362份, 按食品安全国家标准规定的检测方法对菌落总数、大肠菌群、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌进行检测, 并对结果进行分析。结果 生产加工过程采集的样品沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌检出率分别为1.10%(4/362)、0.55%(2/362)和25.97%(94/362)。原辅料、中间产品、成品和终产品单核细胞增生李斯特氏菌检出率分别为48.00%、39.13%、20.00%和0.00%, 差异有统计学意义(χ2=22.57, P<0.05)。结论 低温肉灌肠生产加工过程存在微生物污染风险, 以单核细胞增生李斯特氏菌污染风险最高, 二次杀菌可有效降低低温肉灌肠终产品微生物污染。     

实时荧光定量PCR技术在肉及肉制品中的应用

实时荧光定量PCR技术作为一种DNA定量检测的工具,可应用于肉制品种类鉴别和定量分析中。相比于传统感官和理化的鉴别方法,该技术具有灵敏性高、特异性强、操作简便、省时省力等特点。使用Taq Man荧光探针和荧光染料可以直接测定PCR循环后产物的总量,确定扩增DNA片段的种类及数量,从而确定肉品种类及添加量。因此,可以将该技术应用到肉制品成分及安全的检测中。本文介绍了实时荧光定量PCR技术的基本原理,主要综述了该技术在肉品种类鉴定方面的应用,简述其在肉制品细菌污染检测的应用,并对该技术在肉类研究中的应用进行展望。      

一种检测肉品的新型通用单引物多重PCR技术

在市场中,牛、羊肉中掺杂价格便宜的猪肉,以次充好的欺骗行为屡见不鲜,而且近些年来出现疯牛病、禽流感等疾病,肉品的种属鉴定变得至关重要。随着分子生物学的深入研究,多重PCR技术得到了飞快的发展,但是其扩增效率和检测限达不到理想要求。为寻找快速、灵敏、特异的检测生肉品种的方法,在普通多重PCR的基础上发展了一种新的通用单引物多重PCR（CSP-M-PCR）技术,该方法的体系中,所有目的片段共用一样的上游引物进行扩增,检测限可达5μmol/L。此方法不仅弥补了普通多重PCR扩增效率较低、检测限不理想的缺点,还提高了检测的灵敏度、降低了检测的成本。