植物源性转基因食品PCR衍生技术研究进展

转基因食品尤以植物源性转基因食品的安全性已成为公众关注的焦点。针对植物源性转基因食品甄别与安全性评估,已有多种检测手段得到广泛应用。PCR衍生技术因为具有高特异性、高敏感性等技术优势,为植物源性转基因食品的快速、准确检测提供了有效方法。PCR衍生技术包括多重PCR技术、实时荧光定量PCR技术、多重实时荧光定量PCR技术、多重巢式PCR技术以及多重PCR-DHPLC技术。本文就植物源性转基因食品检测的PCR衍生技术的研究进展作简要综述。     

核酸扩增技术在动物源性食品掺假中的研究进展

核酸扩增技术是一种在体外快速扩增特定DNA片段的分子生物学技术，目前已广泛应用于传染病检测、生物勘测、食品安全检测、临床诊断和公共卫生监测等研究领域。其中，食品安全领域的问题日渐成为人们关注的焦点，尤其是动物源性食品掺假的现象屡禁不止。在过去的科学研究中，动物源性食品掺假的核酸扩增技术发展迅速，取得了很大进展。该文就核酸扩增技术中的凝胶电泳PCR、实时荧光定量PCR、数字液滴PCR、环介导等温扩增、交叉引物扩增、滚环扩增、重组酶聚合酶扩增等技术的原理及在动物源性食品掺假检测中的应用进行综述。讨论了各类核酸扩增技术的关键优势和局限性，简要介绍了现有的挑战和进一步的研究进展，旨在为动物源性食品掺假核酸扩增技术的发展指明方向。     

微滴数字PCR技术在动物源性食品成分鉴定中的应用进展

动物源性食品中的掺假问题是食品加工、流通和餐饮中存在的重要问题。微滴数字PCR(Droplet digital PCR,dd PCR)是绝对定量核酸的一门新的核酸扩增技术,能对动物源性食品成分进行精准定性和定量分析,应用前景广。文章对微滴数字PCR技术的原理、发展、优势以及应用方面进行了综述。     

动物源性食品PCR检测方法的应用研究

动物源性食品的成分检测已成为当前食品质量检测工作的一个重要环节,PCR检测方法在该检测中得到了较为广泛的应用。基于此,本文对PCR在动物源性食品检测中的应用进行了探讨,通过对市售多种肉制品进行PCR检测实验,对PCR检测方法的流程和要点进行详细阐述,以期为今后的相关检测工作提供参考。     

基于DNA条码技术的高通量食品中动物源性成分的快速检测方法

本文应用DNA条码技术,研究通用引物,扩增动物源性加工食品中线粒体基因COI,开发检验快速、准确、低成本和高通量的食品中动物源性成分的快速检测方法.结果表明,该方法有很好的特异性,最低检出限为0.10 ng/μL,可以对未知、已知动物源性成分的食品进行快速、准确的定性、定量检测.     

动物源性食品中抗生素多残留检测的研究进展

研究动物源性食品中抗生素残留的分析检测方法,对保证我国食品安全至关重要。动物源性食品种类繁多、基质效应强,痕量抗生素残留检测的准确性和灵敏度取决于样品前处理效果。该文以动物源性食品中抗生素多残留的样品前处理方法为主线,重点综述近年来新型吸附剂、新前处理方法在动物源性食品中抗生素多残留超灵敏检测方面的研究进展,以期为动物源性食品中抗生素多残留的快速筛查和精准定量提供借鉴思路。     

Taqman探针荧光聚合酶链式反应实时同步鉴定动物源性食品中猪肉、鸡肉源性成分

目的:建立一种能实时同步检测动物源性食品中猪肉源性和鸡肉源性成分的Taqman探针双重荧光聚合酶链式反应(polymerase chain reaction,PCR)方法,应用于动物源性食品的成分掺假快速检验。方法:分别依据猪和鸡的种间保守基因(Cytb)序列设计、合成特异性引物及不同荧光标记(FAM、HEX)的Taqman探针,建立可同步检测动物源性食品中的猪源性和鸡源性成分的Taqman探针双重荧光PCR方法。结果:所建立的Taqman探针双重荧光PCR检测方法特异性强,仅对猪、鸡成分有扩增;灵敏度高,最低检测到猪源、鸡源DNA的含量分别为0.02、0.10 ng;抗干扰性强,当DNA混样中猪源、鸡源性成分含量在2%以上水平时,所建立的混合检测体系均能对DNA混样给出正确判断。结论:所建立的混合检测体系具有高特异性、高灵敏度,能够适用于动物源性食品中猪肉、鸡肉成分的同时快速检测。     

加工食品中若干动物成分的PCR检测技术应用研究

本研究应用PCR技术检测了12种加工食品中的牛、羊、鸡、猪等动物源性成分，在此基础上还开发了真核生物所共有的18S核糖体DNA（18S rDNA）与食品中牛、羊、鸡、猪等多种动物物种特异性基因片段之间的二重PCR检测方法，还分析了牛、羊源性成分单PCR检测的灵敏度，以及18S rDNA与牛、羊之间的二重PCR检测方法的灵敏度。     

PCR技术检测动物源性食品中沙门菌的应用研究

为快速准确检测动物源性食品中的沙门菌,将建立并优化的沙门菌PCR检测试剂盒应用于动物源性食品的检测,并于国标法进行了比较.对上海地区238份鸡蛋、685份原料牛奶、283份猪肉、314份牛肉和58份虾仁样品的沙门菌检测表明,PCR法的敏感性和特异性均为100%,与国标法的符合率亦为100%,且检测时间仅为2 d,较国标法大为缩短.该方法可用于动物源性食品中沙门菌的检测,并具有快速、特异、敏感等优点。     

近红外光谱分析技术在动物源性食品检测中的应用进展

近年来动物源性食品的消费量日益增大,关于动物源性食品的质量安全也受到人们的高度重视.近红外光谱(NIR)分析技术具有分析速度快、非破坏性、所需样品量小、多组分同时测定,且可对生产过程进行实时监控等优点.本文对NIR分析技术进行了简介,并列举了常用的光谱预处理方法;综述了该技术在动物源性食品检测中的研究进展,包括品质评价和安全评价;并对该技术在动物源性食品检测中的发展趋势进行了阐述.表明NIR分析技术适合评价动物源性食品的品质和安全.     

基于PCR技术的肉类成分溯源鉴定方法研究进展

近几年屡屡曝光的食品安全事故引起了社会的广泛关注,食品安全已经成为社会共同关注的问题,肉类掺假造假现象更是层出不穷,其中用低价鸡肉、鸭肉、猪肉等掺入、冒充牛羊肉成为主要的掺假方式。国内外进行肉类掺假鉴定主要以核酸作为靶标,核酸鉴定也是物种鉴别最常用、最核心的方法,以DNA检测为基础建立起来的DNA条形码、多重PCR、荧光定量PCR、荧光探针等技术也得到空前发展和广泛应用。我国针对动物源性成分检测也制定了相关国家标准和行业标准,但大多现行标准中基于DNA检测建立的PCR技术只能检测单一物种,滞后于技术的发展。目前,基于PCR发展起来的衍生技术凭借其高灵敏度、强特异性和高通量等优势在动物源性成分检测工作中显示出巨大潜力,也是肉类成分鉴定未来的重要方向。本文综述了PCR技术在肉类检测中的研究概况和现行标准的技术概况,以期为肉类成分鉴定研究提供信息。     

Assessing the digestion of a genetically modified tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) R8 DNA in simulated gastric fluid using event-specific real-time PCR

There are numerous studies that have demonstrated the digestion of plant DNA in the gastrointestinal (GI) tracts of various animal species. In this study, we investigate the process of DNA degradation in the GI tract using simulated gastric fluid treatment and quantitative PCR approach. Event-specific SYBR Green and TaqMan real-time PCR methods were developed in the present study for measuring the degradation of GM tomato R8 DNA. The selectivity, sensitivity, accuracy, and precision of real-time PCR detection methods were feasible for detection and quantification purposes. The results of real-time PCR analysis revealed that approximately 99.98% of an endogenous gene was degraded and could not be amplified after 180-min simulated gastric fluid treatment. The rapid acid hydrolysis of tomato fruit DNA in the SGF suggests that only less than 1% of genetically modified tomato fruit DNA could survive through the human digestive system.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

动物源性食品鸭血、猪血DNA提取及多重PCR鉴别研究

目的:研究从鸭血、猪血中提取DNA的快速简便方法并建立多重PCR鉴别方法。方法:用KI提取法从固体块状鸭血、猪血中提取DNA,经PCR扩增检测提取效果。建立多重PCR方法鉴别动物源性食品中的鸭血、猪血成分,并对市售动物源性血制品进行检测。结果:这种方法提取到的DNA纯度较高,凝胶电泳条带整齐,背景清晰;PCR反应能扩增出目的条带。多重PCR能同时扩增出鸭和猪的条带。结论:这种改进的DNA抽提方法能获得高纯度DNA,比传统方法安全、简便、节省试剂,PCR扩增结果很好,应用多重PCR方法能同时检测出血样制品中的鸭、猪成分。     

Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.     

A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences

Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.     

Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food

DNA-based methods are rapid, cost-effective and broadly applicable approaches for food authentication. Recently, the requirements for food safety and food integrity have increased with improved quality of life. Methodologies regarding food authentication based on DNA analysis are more commonly being used. With the increasing number of vegetarians, searching for markers for blind identification across kingdom species, such as an ingredient of animal origin in vegetarian food, would be valuable and attractive. Using bioinformatic analysis of an existing data source composed of 481 ultraconserved sequences, we selected 6 new candidate DNA segments that exist in most vertebrates but that do not exist in plants. Then, primers were designed for all of the candidate DNA markers, and DNA samples isolated from cow, pig, chicken, duck, soy bean, rice, pepper, wheat, sunflower and colza were amplified using each primer pair. None of the plant DNA samples generated a PCR product, while the DNA samples of animal origin were amplified successfully using 5 of the candidate segment primers; the 6th segment primer failed to amplify the DNA and was discarded. Moreover, a simulation experiment containing a plant product contaminated by an animal component indicated that the candidate DNA markers can be used for the rapid detection of animal adulterants in vegetarian products with a promising 5% detection limit. The identified candidate DNA markers for the blind identification of animal adulteration in vegetarian food may be highly desirable in the vegetarian food market, and these markers may facilitate the study of molecular technology for food authentication.     

Validation of a Diagnostic PCR Method for Routine Analysis of Salmonella spp. in Animal Feed Samples

As a part of a validation study, a comparative study of a PCR method and the standard culture-based method NMKL-71, for detection of Salmonella, was performed according to the validation protocol from the Nordic validation organ for validation of alternative microbiological methods (NordVal) on 250 artificially or naturally contaminated animal feed samples. The PCR method is based on culture enrichment in buffered peptone water followed by PCR using the DNA polymerase Tth and an internal amplification control. No significant difference was found between the two methods. The relative accuracy, relative sensitivity and relative specificity were found to be 96.0, 97.3, and 98.8%, respectively. PCR inhibition was observed for rape seed samples. For the acidified feed samples, more Salmonella-positive samples were found with the PCR method compared to the NMKL method. This study focuses on the growing demand for validated diagnostic PCR methods for routine analysis of animal feed and food samples to assure safety in the food production chain.     

Biochip Technology for the Detection of Animal Species in Meat Products

The verification of declared components in meat products is an essential task of food control agencies worldwide. To date, the ELISA and species-specific polymerase chain reaction (PCR) are two commonly applied analytical tools employed by many authorized food control laboratories. These trusted methods however do not allow the simultaneous detection of all the animal species present in a meat sample. Additionally, detection of undeclared components resulting from inadvertent contamination or deliberate adulteration of the meat products requires additional processing of the samples, resulting in increased expenditure. The use of DNA biochip analysis that allows simultaneous processing of many meat products, while concomitantly generating results for the detection of all animal species present in the meat products is thus highly desirable. In this work, two commercially available animal chip detection systems (CarnoCheck Test Kit and MEAT^species LCD Array) are compared in terms of sensitivity, robustness, reproducibility, and ease of handling. The two animal species differentiation biochip methods compared well in efficiency and could simultaneously detect from eight to 14 animal species in the meat products. Detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Both biochip methods performed well and could be implemented for routine use in any food control agency.     

应用实时荧光PCR技术量化检测羊肉中猪源性和鸡源性成份

目的建立羊肉中猪源性成份和鸡源性成份的定量检测方法。方法以新鲜羊、猪和鸡瘦肉为样本提取DNA分子作为检测模板,针对基因组中单拷贝基因设计特异性的引物和探针,应用荧光实时定量PCR技术对模板DNA进行扩增。通过绘制扩增标准曲线和确定羊、猪和鸡的质量与DNA比值常数,对4种不同掺混比例的混合肉样进行定量分析。结果检测质量百分比的绝对误差可以控制在7%以内,量化结果基本准确。结论对于组织成份单一的样品,可以通过在基因组单拷贝基因上设计特异性的引物,利用PCR技术实现在质量水平上对食品中动物源性成份的量化分析,该技术方法的建立可以为肉类掺假的监管工作提供有力的技术支撑。