两种PCR方法检测食品中花生过敏原Arah1成分

采用套式PCR和荧光实时定量PCR两种方法检测食品中花生主要过敏原Ara h1基因成分,从而推断食品中是否含有花生过敏原成分。根据GenBank提供的花生主要过敏原基因Ara h1 DNA序列(登录号为AF432231)的中一段序列分别设计2对内外特异性引物,扩增目的基因片段来建立套式PCR方法;再用上述内引物扩增目的片段,建立SYBRGreenⅠ荧光实时定量PCR方法,绘出拷贝数-CT标准曲线;并通过这2种PCR方法检测8种食品中含有花生主要过敏原Ara h1基因成分。套式PCR方法具有较高的特异性和灵敏度,SYBR GreenⅠ荧光实时定量PCR方法的标准曲线在3×102至3×108 copies范围内线性关系良好,R2值为0.993 5,检测低限设定为3×103 copies。2种方法检测8种食品样品,结果均与食物过敏原标注内容相符。本研究建立的2种方法具有快速、灵敏等优点,可用于食品中花生主要过敏原基因Ara h1成分的检测。     

食品中花生过敏原及其检测方法的研究进展

花生是常见的过敏原之一,能够引起严重的过敏反应。由于缺乏明确的治疗花生过敏的方法,只能让花生过敏患者尽量避免食入含有花生的食物。但在实际的生产过程中,食品加工往往需要经过复杂的生产工艺,会造成食品之间的交叉污染,部分食品难以准确判断是否含有花生过敏原。因此对于食品中花生过敏原的检测方法的开发就显得尤为重要。本文主要对花生中过敏原的种类及其检测方法的研究进展进行了综述,主要对以下几种方法做了介绍,包括酶联免疫吸附法(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,以及新兴的生物传感器和质谱法,并对检测方法的未来发展趋势进行了展望。     

胶体金免疫层析法检测食品中花生过敏原蛋白成分

建立一种简易快速胶体金免疫层析法(GICA)用于检测食品中花生蛋白成分.采用柠檬酸三钠还原法制备胶体金颗粒、标记抗花生蛋白成分的抗体,制成免疫层析测试条.食品中花生蛋白成分与测试条上金标记抗体结合后沿着硝酸纤维素膜移动,与膜上的固相抗体结合形成肉眼可见的红色线条.自制花生抗原标准品并检测该方法的灵敏度,同时检测19种食品中是否含有花生蛋白成分.结果表明:GICA测试条与花生蛋白抗原呈特异性反应.检测自制的花生抗原标准品的灵敏度可达50ng/mL,用GICA试条检测了19种食品中,其中13种食品标签上标注含有花生过敏原成分的食品检测结果均呈阳性,4种食品标签上标注不含花生过敏原成分的食品检测结果均呈阴性,而2种食品标签上没有标注过敏原成分的食品检测呈阳性.     

时间分辨免疫荧光法测定食物中花生过敏原成分

目的:建立双抗体夹心时间分辨荧光免疫法[Time-resolved Fluoroimmunoassay(TRFIA)]测定食物中花生过敏原蛋白成分,为进出口食品中花生过敏原成分检测和由花生导致的食物过敏性疾病的预防提供技术基础。方法:提取花生蛋白,免疫小鼠制备抗花生总蛋白的多克隆抗体,用该抗体包被酶标板,并用生物素标记该多抗作为捕捉抗体,Eu3+标记的链酶亲和素和以β-二酮体为主的增强液等试剂,建立双抗夹心TRFIA方法,检测该方法的灵敏度,同时用于13种食品中花生过敏原蛋白成分检测。结果:初步地研制出双抗体夹心TRFIA法检测食品中花生过敏原蛋白成分,具有特异性,和其最低检出限为0.1ng/mL,标准曲线在0.1～160ng/mL范围内线性良好;11种食品检测结果与食物过敏原标签标注内容相符,而2种标示不详的食品检测结果均呈现阳性。     

双抗体夹心ELISA法测定食物中花生过敏原蛋白成分

通过建立双抗体夹心ELISA法测定食物中花生过敏原蛋白成分,为进出口食品花生过敏原成分检测和由花生导致的食物过敏性疾病的预防提供技术基础.提取花生总蛋白,免疫小鼠制备抗花生总蛋白的多克隆抗体,用该抗体包被酶标板,并用生物素标记该多抗,从而建立双多抗体夹心ELISA法;自制花生总蛋白标准品并检测该方法的灵敏度,同时检测15种食品中是否含有花生蛋白成分.成功地研制出双抗体夹心ELISA法检测食品中花生过敏原蛋白成分,具有特异性,其最低检出限为8ng/mL,标准曲线在8 ng/mL～125 ng/mL范围内线性良好;13种食品检测结果与食物过敏原标签标注内容相符,而2种标示不详的食品检测结果均呈现阳性.     

超高效液相色谱-电喷雾质谱法检测花生 过敏原Ara h 2

目的建立烘培食品中花生过敏原Ara h 2的液相质谱联用定量检测方法。方法选择花生蛋白中的致敏蛋白Ara h 2作为目标蛋白,筛选出该致敏蛋白的特异肽,人工合成特异肽标准品和特异肽内标,从而建立直接检测花生致敏蛋白Ara h 2的准确定量方法。同时还对全国不同地区的20种花生中致敏蛋白Ara h 2的含量进行检测分析,初步统计得出致敏蛋白Ara h 2和花生蛋白的换算系数,并以Ara h 2作为生物标记物检测10种烘培食品中花生蛋白的残留量。结果花生样品中致敏蛋白Ara h 2的定量限为4.45μg/g,回收率在106.0%~107.8%之间。烘培食品中,定量限可达到6.23μg/g,回收率在107.0%~113.2%之间。结论本方法特异性强、灵敏度高、定量准确,具有良好的应用前景。     

基于焦磷酸测序的花生过敏原基因ara h6检测技术研究

应用焦磷酸测序技术建立了检测食品中花生过敏原基因Ara h6的快速检测方法,该检测方法对花生过敏原成分具有很好的特异性和重现性,测序结果在GenBank中进行同源性比对分析,表明目标序列能够作为鉴定花生过敏原成分很好的特征序列,为食品中的花生过敏原成分快速检测提供良好的技术支撑,保证了食品进出口和食用安全。     

环介导等温扩增技术检测花生过敏原

花生引起的过敏已经越来越受到重视,因此对花生过敏原进行检测也变得越来越重要。目前对花生过敏原的检测大多数采用抗原抗体法或PCR方法,抗原抗体法耗时比较长,而PCR方法需要昂贵的仪器设备。本项目通过建立环介导等温扩增快速检测技术来检测食物中花生过敏原的基因,为食品中花生过敏原成分检测提供方便。该方法快速,简便,灵敏度高,可以很好的应用到现实检测中去,这个方法的建立具有很重大的意义,为食品的安全检测提供了很大的方便。     

加工方式对花生致敏性的影响及其致敏性评价研究进展

花生蛋白是一种理想的食品加工原料,同时也是一种致死率较高的食物过敏原,严重危害人类健康。本文主要综述食品加工方式对花生过敏蛋白致敏性的影响,以及体内、体外致敏性评价方法,为低致敏或无致敏花生制品的开发提供参考和指导。     

时间分辨免疫荧光法测定食物中鸡蛋过敏原蛋白成分

目的:建立双抗体夹心时间分辨免疫荧光法(TRFIA)用于测定食物中鸡蛋过敏原蛋白成分,为进出口食品鸡蛋过敏原成分检测提供技术基础.方法:提取鸡蛋过敏原总蛋白,免疫小鼠获得多克隆抗体,并以此抗体包被酶标板,采用生物素标记抗鸡蛋过敏原总蛋白抗体、Eu3+标记的链酶亲和素和以β-二酮体为主的增强液等试剂,建立双抗夹心TRFIA方法,用于检测10种食品中是否舍有鸡蛋蛋白成分.结果:鸡蛋总蛋白SDS-PAGE显示带大致有10条,其中主要条带有4条,分子量分别为71、55、38、14kDa;成功地研制出双抗体夹心TRFIA法检测食品中鸡蛋过敏原蛋白成分,具有特异性,其最低检出限为0.625ng/mL,标准曲线在12.5～640ng/mL范围内线性良好;8种食品检测结果与食物过敏原标签标注内容相符,而2种标示不详的食品检测结果均呈现阳性.结论:初步建立了检测食品中鸡蛋过敏原蛋白成分的双抗体夹心TRFIA,具有较好的特异性和灵敏度;该方法对于未标注鸡蛋过敏原成分的食品检测具有一定的实际应用价值.     

Peanut Allergy,Peanut Allergens,and Methods for the Detection of Peanut Contamination in Food Products

ABSTRACT: Attention to peanut allergy has been rising rapidly for the last 5 y, because it accounts for the majority of severe food‐related anaphylaxis, it tends to appear early in life, and it usually is not resolved. Low milligram amounts of peanut allergens can induce severe allergic reactions in highly sensitized individuals, and no cure is available for peanut allergy. This review presents updated information on peanut allergy, peanut allergens (Ara h1 to h8), and available methods for detecting peanuts in foods. These methods are based on the detection of either peanut proteins or a specific DNA fragment of peanut allergens. A summary of published methods for detecting peanut in foods is given with a comparison of assay formats, target analyte, and assay sensitivity. Moreover, a summary of the current availability of commercial peanut allergen kits is presented with information about assay format, target analyte, sensitivity, testing time, company/kit name, and AOAC validation.     

花生过敏原检测方法研究进展

花生是重要食物过敏原之一,能引起严重的过敏反应。目前尚没有花生过敏的特效疗法,严格避免食入含花生的食物是花生过敏患者的最佳选择,所以食物中花生的检测就显得尤为重要。本文对花生主要过敏原及其过敏反应研究进展进行简要介绍,重点介绍花生过敏原的主要检测方法的新发展,包括传统的酶联免疫吸附实验(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,ELISA和免疫印迹法都是应用较为普遍的检验方法,PCR方法应用较少,但它能从DNA/RNA方面检测花生过敏原的存在;新兴检测方法主要包括生物传感器和质谱法,这两种方法在花生检测方面具有可观的前景。未来的这些检测方法将朝着快速、灵敏、简便的方向发展。     

花生主要致敏物质及其脱敏方法研究进展

花生作为影响部分人群生活质量的主要过敏食物之一,其中含有的过敏原会引起人体强烈的过敏反应,严重时甚至会导致人的死亡。花生过敏反应因其潜在的危险性、长期性以及不断增加的发病率而日益受到各国的广泛关注。研究花生致敏机理及脱敏方法已成为热点,探索有效脱敏方法,对保证花生食品的安全性具有重要的现实意义。本文对花生致敏机制,过敏原致敏组分分析和脱敏方法进行阐述。     

IDENTIFICATION AND QUANTIFICATION OF MAJOR PEANUT ALLERGENS (IN CHINA) WITH IGE-BINDING PROPERTY

Peanut allergens have not been studied in China. This study aimed to investigate (1) whether there are differences in the relative amounts of major peanut allergens between the Chinese peanut varieties and the American, and (2) the effect of cooking methods on peanut allergenicity. The allergenic property of raw peanuts and peanut preparations was assessed by immunoblotting and enzyme-linked immunosorbent assay. The relative contents of the major peanut allergens were quantified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. For results, Ara h 1 and Ara h 3 were major peanut allergens in China. The amounts of Ara h 1 and Ara h 3 in peanut varieties differ significantly and were both lower than the American varieties. The immunoglobulin E (IgE)-binding ability of different processed peanuts to IgE was not significantly different. Therefore, peanut varieties may induce different amounts of allergens. The relative lower contents of Ara h 1 and Ara h 3 may lead to the lower prevalence of peanut allergy.

PRACTICAL APPLICATIONS

Because of its nutritional and rheological properties, peanut is used in a wide range of different foods, and peanut allergy represents an important health problem. It is essential to identify the compounds of peanut allergens and to study their characteristics in order to explore approaches for the therapies and to breed the nonallergenic peanut seed.     

花生致敏性评估方法及食品加工对其致敏性影响的研究进展

花生是“8大类”过敏原食物之一，因其诱发严重的临床症状及日益上升的发病率而受到广泛关注。文章概述了花生过敏原(Ara h 1-18)的结构特性、免疫特性及体内和体外评估方法，总结了不同食品加工对花生致敏性的研究进展，并展望了未来食品加工技术对消减花生致敏性的研究方向。     

热加工花生的蛋白分离及其过敏原纯化方法研究进展

花生不仅本身是一种营养丰富的食品,而且作为原料或配料广泛应用于食品加工中。然而花生及其制品是FAO/WHO认定的八大类食物过敏原之一,可导致严重的过敏反应,通常伴随终身,甚至危及生命。不同地区的人们食用花生的加工方式不同,其花生过敏的患病率也有所不同,热加工是花生的主要加工方式,因此各类热加工导致的花生致敏性变化成为研究热点。过敏原蛋白的分离作为花生热加工研究中的重要步骤,也变得十分重要。本文主要对常见的3种热加工花生(水煮、油炸和烘烤)中的花生蛋白分离及其过敏原纯化的方法研究进行综述。现有的花生热加工研究中蛋白分离技术主要是通过溶剂浸提;而过敏原纯化技术主要是借助层析法,根据各组分在物理化学性质上的差异进行纯化;此外还可以根据最终研究目的的不同采用其他的辅助方法达到分离纯化效果。通过对现有分离纯化方法进行了解和比较,可为热加工花生过敏原蛋白的分离纯化甚至进一步的分析检测提供理论参考和指导。     

Effect of roasting history and buffer composition on peanut protein extraction efficiency

Peanut is a major allergenic food. Undeclared peanut (allergens) from mis-formulation or contamination during food processing pose a potential risk for sensitized individuals and must be avoided. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. The extraction of proteins from allergenic foods and complex food products is an important step in any allergen detection method. In this study, the protein extraction efficiency of various buffers prepared in-house and some extraction buffers included in some commercial allergen enzyme-linked immunosorbent assay (ELISA) test kits for peanut determination in food products were tested. In addition, the effect of roasting history on the extractability of peanut protein was investigated by the biuret and the bicinchoninic acid (BCA) assays. Elevated roasting temperatures in food processing were found to have a major impact on protein extraction efficiency by reducing protein yields of oil and dry roasted peanuts by 50-75% and 75-80%, respectively, compared with the raw material. Extraction buffers operating in the higher pH range (pH 8-11) showed best yields.     

花生中主要过敏原的研究进展

花生是影响部分人群生活质量的过敏原食物之一。本文重点介绍了花生中主要过敏原Arah1、Arah2、Arah3的研究进展。     

A two-site monoclonal antibody immunochromatography assay for rapid detection of peanut allergen Ara h1 in Chinese imported and exported foods

Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10 ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs.