牛奶β-乳球蛋白质实时荧光定量PCR检测方法的建立

建立一种快速、特异、灵敏的Taqman实时荧光定量PCR(real-time PCR)方法,用于牛奶主要过敏原β-乳球蛋白质的检测。根据Gen Bank登录的牛β-乳球蛋白质的DNA序列设计,合成一对特异性引物和探针。将扩增产物连接到p MD19-T载体上,制备质粒标准品并测序鉴定,10倍梯度稀释含有β-乳球蛋白质基因的重组质粒,进行实时荧光定量PCR扩增,绘制标准曲线,检测该方法的特异性、稳定性、灵敏性,同时将建立的方法用于10种市售食品的检测。成功建立了β-乳球蛋白质的实时荧光定量PCR检测方法,标准曲线的Ct值与模板浓度在3.18×103~3.18×107copies范围内线性关系良好,R2值为0.997 8;检测灵敏度高(318 copies/μL);特异性强,对羊奶、豆浆DNA均无扩增反应;稳定性好,组内、组间的变异系数均在5%以内。对10种食品牛奶过敏原的检测结果与标签相符。表明所建立的实时荧光定量PCR方法可应用于食品中牛奶过敏原β-乳球蛋白质的检测并可推广到其它过敏原的检测。     

卵转铁蛋白双抗夹心ELISA方法的建立

卵转铁蛋白是鸡蛋中的主要过敏原,针对鸡蛋中主要过敏原卵转铁蛋白建立了双抗夹心ELISA方法,该方法检出限为(15.37±1.20)ng/m L。除白蛋白外,该方法对粘蛋白、溶菌酶、α-乳白蛋白、牛奶总蛋白、β-乳球蛋白、酪蛋白、花生总蛋白、大豆、杏仁和绿豆等致敏蛋白几乎没有交叉反应。板内重复性和板间重复性较好,因此所建立的方法可用于对卵转铁蛋白的检测。     

牛奶过敏原检测方法研究进展

牛奶是八大食物致敏原之一,能引起严重的过敏反应。如何实现食物中牛奶过敏原的快速,准确筛检已成为食品安全领域关注的焦点。牛奶主要过敏原为酪蛋白、α-乳白蛋白、β-乳球蛋白,其检测方法有电泳法、色谱法、酶联免疫实验(ELISA)、传感器、基于质谱的蛋白质组学(LC-MS/MS)、聚合酶链式反应技术(PCR)等。本文就其检测方法的原理和应用进行综述,旨在为食品中牛奶过敏原的检测提供技术方向,以便保障牛奶过敏患者的安全。     

牛奶过敏原制备方法的研究和免疫活性鉴别

目的探索牛奶主要过敏原的制备工艺。方法采用等电点沉淀、凝胶层析和分子筛等技术纯化牛奶中主要过敏原组分;采用SDS-PAGE鉴定蛋白纯度,采用双抗原夹心-ELISA鉴定免疫活性。结果成功获得牛奶中的四种主要过敏原组分:酪蛋白、β乳球蛋白、α乳白蛋白和分子量较高的P1组分,并经过免疫实验证实这四种组分均能与过敏血清产生反应,而其中β乳球蛋白和P1组分反应性较高。结论本研究探索出一种简单、实用的牛奶主要过敏原制备工艺,并证实牛奶中的β乳球蛋白和高分子量蛋白质为主要过敏原。     

牛乳β-乳球蛋白变异体检测方法研究进展

β-乳球蛋白(β-LG)是牛乳中重要的天然活性营养物质,也是牛乳中主要的过敏原之一。β-乳球蛋白约为乳清蛋白总量的50%,是牛乳中主要的乳清蛋白,具有较强的热敏感性和致敏性,在优质乳品工业中起关键作用。其不仅作为牛奶热处理强度的评估指标,还作为食品中牛乳过敏原的标志蛋白,有效识别和检测牛乳β-乳球蛋白非常重要。已报道的牛乳β-乳球蛋白有13种变异体,其中A和B是牛乳β-LG的常见变异体,且含量最高。根据不同的检测原理,文章简述近年来牛乳β-乳球蛋白变异体的三大类检测方法,总结电泳法、色谱法和免疫法的原理、优缺点及部分应用实例,重点介绍液相色谱法和毛细管电泳法两种定量方法,并展望牛乳β-乳球蛋白未来的发展方向。     

食物过敏原水牛乳β-乳球蛋白的交叉反应研究

β-乳球蛋白是乳清中一种主要蛋白质,而且是乳中主要过敏原之一.本研究探讨β-乳球蛋白与其它蛋白的免疫交叉反应性.采用间接ELISA法和免疫印迹的方法,利用β-乳球蛋白和相应的多克隆抗体,检测其交叉反应.结果表明,β-乳球蛋白不仅与水牛乳中其它蛋白有交叉反应,与其他乳源的β-乳球蛋白也有交叉反应.β-乳球蛋白的交叉反应较强,也是导致乳类食物过敏的主要原因之一.     

牛奶过敏原β-乳球蛋白抗体的制备及免疫检测方法的建立

本文将牛奶过敏原β-乳球蛋白抗原分别免疫新西兰大白兔和BALB/c小鼠制备其多克隆抗体和单克隆抗体,并对所获得的抗体进行了效价等特性测定;采用蛋白A亲和层析法将多克隆抗体进行了纯化,采用辛酸-硫酸铵法对单抗细胞株3A7进行了纯化;实验建立了双抗体夹心的ELISA检测方法,并对一些蛋白样品进行了初步的检测。实验结果表明,所制备的抗β-乳球蛋白兔多克隆抗体效价达到了1:6.56×106;Western-blotting鉴定结果显示所制备的多克隆抗体能够与β-乳球蛋白特异性反应;3株单抗的效价均在106以上,纯化后多抗仅与酪蛋白有一定的交叉反应,而纯化后单抗与乳中主要蛋白及其它过敏原蛋白基本没有交叉反应,特异性很好;利用所建立的方法对一些蛋白样品进行检测,准确率100%。     

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。     

牛乳过敏原β-乳球蛋白检测方法的研究进展

目的食物过敏已成为全球性的食品安全问题,欧美等发达国家均要求对食品中的过敏原成分进行标识,其中就包括作为八大过敏性食物之一的牛乳及其乳制品。β-乳球蛋白是牛乳中的主要过敏原,约占乳清蛋白的50%,牛乳总蛋白的10%,并且约有82%的牛乳过敏患者对β-乳球蛋白过敏,因此其可以作为检测食品中是否含牛乳蛋白的一个有效的标志物。建立灵敏、可靠的β-乳球蛋白检测方法,对牛乳过敏原标识及保障牛乳过敏人群的安全消费具有重要意义。本文主要综述了牛乳β-乳球蛋白的高效液相色谱法、超高效液相色谱法、液相色谱-质谱联用法、酶联免疫吸附法、免疫层析法、电化学免疫法、蛋白微阵列法、等离子体共振法等色谱学和免疫学检测方法的研究进展,并对未来发展方向作出展望,以期促进牛乳β-乳球蛋白等过敏原检测方法的研究与开发。     

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(loop-mediated isothermal amplification)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。该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。     

A Simple and Fast Detection Method for Bovine Milk Residues in Foods: A 2‐Site Monoclonal Antibody Immunochromatography Assay

The ingredient declaration on food labels assumes paramount importance in the protection of food‐allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β‐lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β‐lactoglobulin and 49 food samples labeled as not containing milk were negative for β‐lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β‐lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β‐lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as “containing milk” even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β‐lactoglobulin in foods. Practical Application: The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods.     

Identification of Conformational Antigenic Epitopes and Dominant Amino Acids of Buffalo β‐Lactoglobulin

Major allergen β‐lactoglobulin exists in many mammalian types of milk except human breast. Buffalo milk also contains this major allergen but the detailed information on its epitopes is not available. The aim of this work was to map and characterize its conformational antigenic epitopes. Sixty mimotopes of buffalo β‐lactoglobulin were produced by biopanning of phage display peptide library and then 2 mimotopes, specific for sera from rabbit 1 and 2, respectively, were predicted to be conformational epitope candidates by the use of DNAStar and web tool of MIMOX. On the basis of bioinformation analysis, 5 conserved amino acid residues PL‐ENK were identified in 2 conformational epitope sequences and 7 conformational epitopes were derived from 2 mimotopes by molecular modeling. The result showed that these conformational epitopes were located in the 2 regions on buffalo β‐lactoglobulin and composed of 5 hydrophilic and 2 hydrophobic amino acids.     

实时荧光PCR法检测食物中榛子过敏原成分

榛子是最常见的食品过敏原之一,选择针对榛子不同基因的引物和探针,进行验证、比对和条件优化,建立榛子过敏原的实时荧光PCR检测方法。经实验比较,针对榛子热休克蛋白设计合成的引物和探针具有相对较高的灵敏性,检测限可达0.11ng DNA,而且和常见的食物种类无交叉反应,适于榛子过敏原的检测。该方法的建立,对于加强食品过敏原的标识管理,保护消费者利益、促进进出口贸易具有重要意义。     

奶粉中阪崎肠杆菌PCR和荧光PCR检测方法的研究

建立了奶粉中可致婴幼儿高死亡率的阪崎肠杆菌的PCR和荧光PCR检测方法。利用细菌16S和23SrDNA的保守区设计通用引物,对6株阪崎肠杆菌16S-23SrDNA间区序列(ITS)进行扩增和测序,在比对阪崎肠杆菌ITS序列的基础上,设计了11条PCR和荧光PCR检测引物,组合成30对PCR引物,并筛选出一对种特异性引物,建立了奶粉中阪崎肠杆菌PCR和荧光PCR检测方法。用10株阪崎肠杆菌,18株近源菌株验证实验表明,本文所建立的PCR和荧光PCR方法特异性强;加菌实验表明,奶粉样品中阪崎肠杆菌检测低限为(2.2～5.4)CFU/100g,灵敏度高;新建的PCR和荧光PCR方法与FDABAM(美国食品及药品管理局微生物分析手册)方法比对实验表明,三种方法的检测结果完全一致。由于PCR和荧光PCR检测方法快速、可靠,因此可替代传统检验方法。     

Combination of immunomagnetic separation with real-time PCR for rapid detection of Salmonella in milk, ground beef, and alfalfa sprouts

A rapid, specific, and sensitive method for detecting Salmonella spp. in pasteurized milk, ground beef, and alfalfa sprouts was developed. The method combined immunomagnetic separation with a real-time PCR assay based on the double-stranded DNA binding dye SYBR Green I. The primers used produced a product with a melting temperature of 87+/-0.5 degrees C during the PCR assay by amplifying a 284-bp sequence from the invasive gene (invA) of Salmonella. The method was successful in detecting 20 Salmonella strains, but the expected PCR product was not formed by any of 11 other bacterial strains. To test this combined method for the monitoring of Salmonella, Salmonella enterica serotype Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered peptone water, cells were removed by immunomagnetic separation and DNA extracted using the High Pure PCR template preparation kit. The DNA produced was used as a template in the real-time PCR assay. When spiked pasteurized milk, ground beef, and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of 1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13 h. These results indicate that the combination of immunomagnetic separation and real-time PCR assay was a highly specific and sensitive method for the rapid detection of Salmonella.     

食源性单增李斯特菌的实时定量PCR检测

根据GenBank数据库单增李斯特菌的O基因序列(AF253320)设计引物,建立单增李斯特菌实时荧光定量PCR检测方法。标准曲线的相关系数为0.996,检出底限约8个细菌/反应,而常规PCR检出底限约60个细菌/反应。比较常规PCR和实时荧光定量PCR对人工污染样品检测,发现实时定量PCR也具有较高的灵敏度;对40份牛奶和40份火腿样品进行检测,阳性率分别为12.5%和10%,与传统细菌分离检测结果完全相符。因此,实时定量PCR检测单增李斯特菌具有快速、灵敏的优点,适宜于食品中单增李斯特菌污染的调查及监测。     

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time <2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.     

乳品中致病大肠杆菌荧光双重PCR技术建立

建立乳及乳制品中致病性大肠杆菌(EPEC)的快检方法。以EPEC特异的微绒毛粘连基因(eae gene)和茵毛束形成编码基因(bfp gene)为模板,设计两对引物,荧光探针对EPEC进行特异性扩增。该方法快速,特异性好,对EPEC的最低检出量为1 cfu/μL(纯菌),检出时间为3 h,同一样品重复检测3次ct值的变异系数均小于5%。建立可快速特异检测乳及乳制品中EPEC的双重荧光PCR方法。     

多重PCR快速检测婴幼儿奶粉中的病原菌

为了建立同时检测婴幼儿奶粉中阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌的多重PCR方法,根据阪崎肠杆菌ompA基因、金黄色葡萄球菌nuc基因、蜡样芽孢杆菌hblA基因分别设计3对引物进行多重PCR扩增,并对反应条件进行优化。结果多重PCR扩增出长度为514、156、235bp的特异性目的条带。不增菌的情况下,多重PCR同时检测3种病原菌的灵敏度是103cfu/mL,3种病原菌在奶粉中的检出限是104cfu/g。建立的多重PCR反应准确、快速、高效,为同时检测婴幼儿奶粉中的阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌提供了新方法。