食用植物油中动物源性成分PCR检测方法的建立

本文建立了应用PCR技术快速鉴别食用植物油中动物源性成分的方法。以食用植物油中掺入的动物源性基因为靶标,自行设计16S rRNA基因通用引物,同时选用动物物种特异性引物进行PCR扩增,分别得到相应的目的片段。通过三对16S rRNA基因通用引物,建立了从食用植物油中快速检测是否含有动物源性成分的方法;利用所选的动物物种特异性引物,进一步建立了从食用植物油中鉴定猪、牛、羊、鸡、鱼5种动物源性成分的方法,此方法能检测出含有0.1%(m/m)动物源性成分的植物油。     

荧光定量PCR方法检测畜肉食品中猪源性成分

目的 建立一种快速、特异、灵敏的猪源性成分检测方法。方法 本研究以猪线粒体12S rRNA基因序列为靶位点设计引物和探针, 进行荧光定量PCR扩增, 建立猪源性成分检测方法; 以常见畜禽肉包括羊肉、牛肉、鸡肉、鹅肉、鸭肉、兔肉、马肉、鹿肉等参考动物物种作特异性检测; 以50 mg/kg羊肉DNA作为稀释液对猪肉DNA进行梯度稀释, 做灵敏度检测。结果 该方法能够有效对猪源性成分进行快速检测, 具有较强的特异性, 灵敏度较高(可达0.1 μg/kg)并且羊肉成分的存在对猪肉灵敏度检测没有影响。结论 该方法特异性强, 灵敏度高, 可以快速、准确检测畜肉食品中含有的猪源性成分。     

PCR法检测鱼及其制品中的鱼源性成分

目的:建立一种快速、特异、灵敏的鱼源性成分聚合酶链式反应(polymerase chain reaction,PCR)检测方法。方法:根据鱼线粒体基因组12S r RNA中的保守序列设计鱼源性特异性引物,进行PCR扩增,建立鱼源性成分检测方法;对24种鱼及鸡、牛、羊、猪、鸭、虾6种常见的易混于鱼制品的动物源性成分进行特异性检测;将草鱼肉混入其他动物肉中,混合均匀后提取DNA进行PCR扩增,确定肉样水平的检测灵敏度;将草鱼DNA混入其他动物DNA中,以混合后的DNA为模板进行PCR扩增,确定DNA水平的检测灵敏度。结果:该方法能特异性的对鱼源性成分进行快速检测,检测灵敏度达0.5%。结论:该方法能对食品中是否含有鱼源性成分进行初筛,到达快速检测的目的,对防止食品掺假、维护消费者利益、规范市场秩序有重要意义。     

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。     

荧光PCR法检测畜禽肉中的鸡源性成分

为了建立畜禽肉中鸡源性成分荧光定量PCR检测方法,以鸡、鸭、鹅、猪、牛、羊、兔、鸽和鹌鹑等9种动物线粒体DNA 16S rRNA基因序列为靶位点,通过比对分析设计筛选出鸡特异性引物,以9种动物肌肉DNA为模板进行特异性扩增,确立荧光定量PCR扩增条件;同时将鸡DNA模板浓度进行10倍梯度稀释,一直稀释至108倍,检测所建立的荧光PCR方法的灵敏度;并用此方法对市场上样品进行随机抽检。结果显示:所设计的鸡引物仅对鸡肉DNA模板有典型扩增曲线,Ct值为22.11,对其它动物DNA模板无扩增,特异性较强;当鸡DNA模板稀释倍数达到104倍,即DNA浓度为17.5 pg/μL时,仍有典型扩增曲线,Ct值为30.37,方法灵敏度较高;市场流通环节样品抽检结果显示所抽测样品均合格。可见,建立的基于荧光定量PCR和线粒体DNA 16S rRNA基因的畜禽肉中鸡源性成分检测方法,快速而准确,实用性强。     

多重PCR同时检测常见8种食物过敏原

依据大豆Lectin基因,小麦Gliadin基因,花生Arah3基因,腰果Ana o3基因,鱼和虾16S rRNA,牛和鸡线粒体DNA设计特异性引物序列.在单一PCR方法基础上,建立2种4重PCR方法检测8种食物过敏原的技术.该方法检测周期短,具有较好的特异性和灵敏性,可用于对食品中多种食物过敏原的检测和监控.     

实时荧光PCR方法检测食品中痕量荞麦成分

目的：建立一种快速、特异、灵敏的荞麦成分检测方法。 方法：针对荞麦内转录间隔区ITS(internal transcribed spacer)和5.8S rRNA基因序列设计一对PCR引物及探针,建立实时荧光PCR检测方法;以同源性(27个荞麦属相关物种)及非同源性(食品中常见的栽培型植物)参考植物物种作特异性检测;以50mg/kg小麦DNA作为稀释液对荞麦DNA进行梯度稀释,做灵敏度检测。 结果：该方法能够有效对荞麦成分进行快速检测,具有较强的特异性,灵敏度较高(可达0.1μg/kg)并且小麦成分的存在对荞麦灵敏度检测没有影响。结论：该方法特异性强,灵敏度高,可以快速、准确检测食品中含有的痕量荞麦成分。     

食品中鲍鱼过敏源基因成分的检测

目的本文建立食品中鲍鱼过敏源基因成分实时荧光PCR检测方法。方法采用蛋白酶K消化法提取鲍鱼肌肉组织中基因组DNA,针对鲍鱼管家基因16S r RNA基因设计特异性引物和探针,确定实时荧光PCR反应体系和反应条件,建立了鲍鱼过敏源基因成分实时荧光PCR快速检测方法。选用鱿鱼、海参等海产品进行特异性试验;采用添加方法制备灵敏度试验样品,分别制备了鲍鱼过敏源基因成分含量分别为100%、10%、1%、0.1%、0.01%、0.001%的样品。结果对非鲍鱼类食品进行检测,结果显示出良好的特异性;灵敏度试验表明,本文建立方法的最低检测下限为0.01%。结论本文建立了特异性好,灵敏度高的鲍鱼过敏源基因成分检测方法。     

实时荧光PCR在鉴别鸡精中鸡源性成分的应用研究

文章利用实时荧光PCR方法,以鸡的cytb基因为目的基因,设计了一套特异性的引物和探针,对鸡精中的鸡源性成分进行定性、定量分析研究.结果表明,该套引物和探针能特异性的检测鸡精中鸡源性成分;荧光PCR检测的灵敏度为0.002％ (W/W),检测时间为3h.此方法具有准确、快速、高效的特点,为食品中鸡源性成分的检测提供了新方法.     

食品中鸡源性成分标准检测方法的比较性研究

为比较食品中鸡源性成分的标准检测方法,本试验对3种标准检验的引物计、方法及灵敏度进行比较。结果显示,实时荧光PCR法引物特异性好、方法简便、灵敏度高。因此得出结论,两种实时荧光PCR法均可作为鸡源性成分检测的补充方法。     

Detection of shrimp-derived components in food by real-time fluorescent PCR

Crustaceans such as shrimp and crabs and their products are important allergens in food, and allergic reactions due to the consumption of shrimp and crabs are frequently reported. However, the chemical properties of shrimp-derived allergens, except for Pen a I, are still unclear. Therefore, it is important to establish a more sensitive and specific method for detecting the composition of foods containing shrimp. In the present study, we developed a real-time fluorescent PCR to identify the specific shrimp-derived components in food. The primers and TaqMan probes for real-time fluorescent PCR were designed based on 16S rRNA genes through comparing a large number of nucleic acid sequences from different species of shrimp that have been published by the National Center for Biotechnology Information. In total, 56 kinds of samples, including different kinds of shrimp, crab, fish, shellfish, and octopus, were subjected to detection by real-time PCR. The results indicated that real-time fluorescent PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish powder containing shrimp powder was treated by heating at 133°C for 30 min. The limit of detection of shrimp-derived components in fish powder was 0.05% (wt/wt).     

实时荧光PCR法测定婴幼儿辅助食品中过敏原鱼类成分

目的建立实时荧光PCR检法测定婴幼儿辅助食品中过敏原鱼类成分。方法通过改进的前处理方法,采用实时荧光PCR法,对45个样品进行检测,分别进行特异性、检出限以及适用性试验。结果用于特异性试验的18个样品中,只有鱼类出现特异性扩增;通过7个不同质量配比的鱼肉样品得出检出限低于0.01%;对市售20批样品进行检测,检测结果与样品标识相符。结论该方法快速、灵敏、准确,适用于婴幼儿辅助食品中过敏原鱼成分的检测。     

可视化膜芯片技术检测常见食源性过敏原成分

目的 建立包括鱼、虾、鸡、鸭、花生、核桃、小麦以及大豆过敏原成分的基因膜芯片技术, 实现对这几种食源性过敏原的同步可视化检测。方法 针对常见食源性过敏物质的成分特异性基因或者过敏原蛋白基因设计特异性的带生物素标记的引物和探针。采用反向斑点杂交(reversedot blot, RDB)结合多重聚合酶链式反应(multiplex polymerase chain reaction, MPCR)技术, 最终通过化学显色直观显示检测结果, 实现对多种食源性过敏原的同步可视化检测。结果 所建立的可视化基因膜芯片方法准确性强, 通过单目标物以及多目标物特异性实验, 显示本方法仅对鱼、虾、花生、核桃、大豆等靶标食源性过敏原有特异性反应, 非靶标物检测均为阴性; 通过制备不同质量浓度比的模拟样品考核方法的灵敏度, 经检测, 方法检测灵敏度可达0.1%(质量分数)。结论 所建立的方法可达到可视化、快速、准确地鉴别食品中鱼、虾、花生、核桃、大豆等常见食源性过敏原。     

食品中牛奶过敏原成分LAMP检测方法的建立

目的 建立食品过敏原牛奶成分LAMP检测方法,并与实时荧光PCR(real-time PCR)检测方法比对。方法 针对牛线粒体细胞色素b(cyt-b)基因设计LAMP引物并建立反应体系,在特异性和灵敏度方面与real-time PCR检测方法比对。结果 本研究建立的LAMP方法检测9份不同品牌的牛奶和羊奶及其加工制品,没有出现交叉反应,具有良好的特异性。通过添加试验方法的检测灵敏度为0.5 %,与real-time PCR方法检测灵敏度相当。检测了69份实际样品,检测结果与real-time PCR检测结果一致。结论 本研究建立的食品过敏原牛奶成分LAMP检测方法简单经济,检测结果可靠,可有效缩短检测时间,适用于过敏原牛奶成分的检测,具有良好的应用前景。     

Simultaneous detection of allergenic fish,cephalopods and shellfish in food by multiplex ligation-dependent probe amplification

People suffering from food allergy rely on correct food labelling as the ingestion of minimal amounts of the respective allergen can trigger severe allergenic reactions. Probes for the detection of DNA from allergenic fish, shellfish and cephalopod species in food using multiplex ligation-dependent probe amplification were developed. The specificity and the sensitivity of the detection system were investigated. The limit of detection was 20 mg kg^?1 for scallop, fish and bivalve species and 100 mg kg^?1 for cephalopod, gastropod and crustacean species using self-prepared sushi spiked with the analytes in different concentration levels. The analysis of 10 commercial food samples demonstrates the applicability of the developed method and its suitability for food quality control. Therefore, the method can be used to monitor the compliance with labelling rules regarding food allergens.     

食品过敏原牡蛎成分PCR检测方法的初步研究

目的:建立基于PCR技术的过敏原牡蛎成分快速检测方法,结合溶解曲线及特异的Tm值与Ct值,用于不同产品的牡蛎成分检测。方法:根据NCBI上的牡蛎线粒体序列,通过DNA Star等软件设计引物,分别采用普通PCR和SYBR Green I实时荧光PCR的方法建立食品过敏原牡蛎成分的检测方法,对十种牡蛎阳性样品和二十余种阴性样品进行实验,并且对几种牡蛎相关食品进行检测。结果:研究建立的检测方法可以检测出含牡蛎成分0.1%的样品,其中荧光PCR的灵敏度达到0.01 ng/μL,特征峰的Tm温度为80.08 ℃,能够检测出牡蛎相关食品中的牡蛎成分。结论:该方法是一种操作安全方便、成本较低、特异、灵敏的实时荧光PCR方法,对于收集到的食品相关产品以及保健品中牡蛎粉的检出率为100%,该方法可广泛应用于食品中牡蛎成分的快速鉴定。     

Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients sesame,almond, lupine and Brazil nut

The inter-laboratory (=ring-trial) validation of 4 food allergen quantification methods using real-time PCR is described. Three single real-time PCR methods for the specific detection and quantification of sesame, almond and Brazil nut were used. Additionally, a multiplex real-time PCR method combining the detection of sesame, almond, Brazil nut and lupine was tested in parallel. Matrix based calibrants (rice cookies) spiked (=incurred) with defined amounts of sesame, almond, lupine and Brazil nut were applied for quantitative evaluation. Cookies based upon wheat and rice flour as well as sauce hollandaise powder each incurred with these allergenic ingredients in the range of 10–123 milligram per kilogram were used as ring-trial samples. The lowest spike level of 10 mg/kg could reproducibly be detected by 6 of 7 PCR systems. In quantitative evaluation of the results, reproducibility standard deviations of approximately 50 % and below were obtained. In addition, the effect of the food matrix on allergen quantification was examined. The range of “recoveries” over all matrices and methods was from 43 to 109 %.     

Quantitative analysis of shrimp allergen in food matrices using a protein chip based on sandwich immunoassay

Food allergy is increasingly becoming a serious concern these days. With packaged foods becoming the norm of the day, food allergy cases out of accidental consumption are becoming rampant, thereby generating great risks for the subjects involved and prompting food authorities in different countries to formulate new regulations about displaying food allergen data on food labels. Detection of food allergens is conventionally carried out by ELISA or PCR tests. These techniques are limited in that they can only detect one or few allergens at one time. Therefore, in the present study a novel sandwich protein chip assay was developed for quantitation of shrimp allergens in food matrixes. The shrimp allergen model used 3D aldehyde slides as the solid carrier, rabbit antisera as the capture reagent, and biotin-labeled monoclonal antibody as the detector reagent. Resulting antigen–antibody complexes were visualized in the presence of commercial strepavidin labeled with Cy3 to produce fluorescence for quantification. With the LOD of the protein chip being 0.054 mg tropomyosin/kg, the protein chip can quantify down to 0.096 mg tropomyosin/kg. The protein chip was not found to be sensitive to other kinds of foods but cross-reacted to some extent with allergens of some other crustaceans. The recoveries ranged from 69.2 to 99.9%, while the intra- and inter-assay coefficients of variation were <13% and <19%, respectively. It seems that the new assay is reliable enough to detect shrimp allergens in food and food products and help minimize the instances of shrimp allergy. It is also possible to use the protein chip for simultaneous detection of other food allergens.