荞麦源性成分实时荧光PCR检测方法的建立

为检测荞麦类食品及原料中的荞麦源性成分,根据荞麦品种DW-1核酸序列信息(KY945277.1)设计了荞麦品种特异性引物;以苦荞和甜荞品种基因组为模板,建立了荞麦源性成分的实时荧光PCR(qPCR)的检测方法,并对购自市场的多个粮食产品进行检测,检测结果与商品标识相符方法特异性强、灵敏度高,为食品中荞麦源性成分的检测提供了新思路。     

实时荧光PCR法检测食品和饮料中胡萝卜成分的研究

胡萝卜是一种质脆味美的家常蔬菜,同时也是一种常见的食品过敏原。针对胡萝卜抗冻蛋白基因的保守序列,设计特异性引物和探针,经验证和条件优化,建立胡萝卜成分的实时荧光PCR检测方法。经实验比较,针对胡萝卜抗冻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1 ng DNA,而且和常见的食物种类无交叉反应,适于胡萝卜成分的检测。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。     

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。     

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。      

荧光定量PCR方法检测榛果过敏原成分

目的建立榛果过敏原成分的荧光定量PCR检测方法,并将此方法与酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)方法进行比对实验。方法根据榛果成分oleosin特异性基因设计并筛选合适的引物和探针,优化反应体系和反应条件,建立榛果过敏原成分的荧光定量PCR检测方法,对荧光定量PCR方法与ELISA方法检测结果进行分析。结果建立的榛果过敏原成分荧光定量PCR方法特异性良好,可用于榛果过敏原成分的定量检测,但检测灵敏度低于ELISA检测方法。结论所建立的榛果过敏原成分的荧光定量PCR检测方法特异性好,灵敏度达到10 mg/kg,具有较好的实用性,ELISA检测方法灵敏度高于荧光定量PCR法,但当榛果过敏蛋白被破坏后有可能出现假阴性结果。     

实时荧光PCR法检测食品中红枣成分

目的:分析不同品种枣样本的LOC112491224基因序列,设计枣源性成分的特异性引物和探针,为检测市售食品中枣源性成分提供一种分子生物学检测方法.方法:将提取好的DNA 10倍稀释至0.0001 ng/μL,进行荧光PCR反应,确定其检测灵敏度.对苹果、梨、香蕉、草莓、葡萄、杏、樱桃和橘子等8种常见的非红枣植物样品进...     

食品过敏原羽扇豆成分的实时荧光PCR检测方法

羽扇豆是欧盟法规标签要求强制性标识的食品过敏原。采用现有参考文献TaqMan实时荧光PCR方法,研究建立食品过敏原羽扇豆成分的检测方法。研究表明建立的研究方法具有特异性,灵敏度高,检测限为1 mg/kg。此方法适用于食品中过敏原羽扇豆成分检测。     

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。     

实时荧光定量PCR在食品检测领域中应用

实时荧光定量PCR是在定性PCR技术基础上发展起来的核酸定量技术;该技术不仅实现对DNA模板定量,且具有灵敏度高、特异性强、无污染性、及实时性和准确性等特点。该文主要介绍实时荧光定量PCR原理和在食品检测中应用,及其在食品领域发展前景。     

小麦和榛子过敏原成分检测的实时荧光PCR方法

过敏原风险问题已成为重要的食品安全问题,如何快速准确地检测出食物中的过敏原成为当前亟需解决的关键。目前常用的过敏原检测方法有免疫学检测、质谱及SPR等技术,但这些方法均有一定程度的局限性。通过引入分子生物学鉴定手段,建立小麦、榛子的过敏原基因数据库,并依据数据库寻找特异性序列并设计探针引物,建立一种快速、便捷、高效的过敏原检测方法,并适当调整了方法的检出限,降低了检测误判的风险。通过建立实时荧光PCR方法,可快速筛选样品的过敏原基因,简化了食品中过敏原成分的鉴定,降低了实验成本与技能要求,降低了检测的难度,缩短了实验时间。通过适用性验证与检出限验证实验,该方法用于过敏原成分鉴定可以达到理想的效果,重复性好,准确率高,检出限为1%。     

Development of a Novel PCR Primer to Differentiate and Identify Bacillus subtilis and Closely Related Species Isolated from Thai Fermented Foods

A novel PCR assay based on 16S-23S internal transcribed spacers (ITS) length polymorphism was developed for rapid differentiation and identification of the Bacillus subtilis group, especially B. subtilis, B. licheniformis and B. amyloliquefaciens, the most frequently isolated bacilli from fermented foods. A new group-specific conserved primer pair, CITS-F and CITS-R, was designed for specific amplification of the ITS region in B. subtilis and closely related species. The fingerprints of seven reference species, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. atrophaeus, B. vallismortis and B. mojavensis using CITS-F and CITS-R primers showed the same four signature bands of 227, 400, 542 and 650 bp. They are different from those of other genera and species tested. Therefore, these four signature bands could be used to differentiate and identify the B. subtilis group. Moreover, the sequence of the 227 bp signature band could also be used to distinguish closely related species of the B. subtilis group used in this study. A novel PCR assay based on ITS length polymorphism pattern using CITS-F and CITS-R could be considered as a rapid and easy method for the primary differentiation of the B. subtilis group.     

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10² spores after 16 h of incubation.     

食品中荧光假单胞菌聚合酶链式反应检测体系的建立和评价

建立用聚合酶链式反应(polymerase chain reaction,PCR)检测食品中荧光假单胞菌的方法。分别针对16～23S rRNA基因间隔区序列、gyrB基因以及通过生物信息学方法发掘到的4个种特异性基因设计6对检测引物,通过初步特异性实验,筛选出一对种特异性最佳的引物。最终建立以gyrB基因为检测靶点的PCR扩增体系,并对体系进行系统评价。结果表明:该方法可特异检测荧光假单胞菌的存在,纯DNA检测灵敏度为14.9fg/μL(2～3拷贝/μL),纯培养物检测灵敏度为2.8×102CFU/mL。豆奶样品经15h充分增菌可提高检测灵敏度至0.28CFU/25g。     

高粱黑粉菌的分离鉴定

目的：以“吉粮105A”高粱乌米为材料,进行高粱黑粉菌的分离鉴定。方法：利用单胞分离的方法从样品中分离获得纯菌株,对分离菌株进行形态学观察及ITS(internal transcribed spacer,ITS)序列测定。结果：分离得到的菌株在天然固体培养基中形成的菌落表面呈革质,边缘呈锯齿状,棕黑色,ITS扩增片段大小为721bp;利用DNAStar软件对序列进行比较分析,鉴定出所分离菌株为丝孢堆菌。结论：成功从高粱乌米中分离出一株高粱丝孢堆黑粉菌。     

实时荧光P C R 法检测食品中芝麻过敏原成分

本实验探讨采用实时荧光定量PCR 方法检测食品中的芝麻成分,结果表明：采用芝麻的引物和探针进行扩增时;22 种样品经实时PCR 扩增,只有白芝麻和黑芝麻产生荧光信号,其余样品均不产生荧光信号。灵敏度实验结果表明能检测到10-5 稀释度的4.4 ×10-12g DNA的量,定量关系式为：y=－ 3.472018x+18.851009,R2=0.993088。这两对引物和探针具有极高的灵敏度和扩增效率,符合痕量检测要求。     

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products

Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain.     

Development of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Buckwheat Residues in Food

Abstract: Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However, buckwheat causes allergy in some individuals and must be labeled and tested accurately to protect those with allergy to buckwheat. We describe the development of a new test assay to help food producers ensure that buckwheat is not present in foods that are not intended to contain buckwheat.     

食品中乳酸杆菌的实时荧光PCR的快速检测

本文运用实时荧光PCR(Real-time PCR)技术建立了对食品中常见的乳酸杆菌进行检测的快速方法.针对乳酸杆菌16S rDNA序列,设计了通用引物和特异性探针,进行TaqManTM实时PCR检测,以非同源性参考菌株做特异性检测;把乳酸杆菌菌株稀释成不同梯度,做灵敏度检测.实验结果表明用实时荧光PCR法检测乳酸杆菌,快速、敏感、特异性高.