Detection of porcine DNA in Korean processed foods by real-time PCR

Food Science and Biotechnology - In this study, a commercial DNA extraction kit and a real-time Polymerase Chain Reaction (PCR) kit were applied to detect porcine DNA in Korean processed foods. The...     

Detection of salmonella by flow-through immunocapture real-time PCR in selected foods within 8 hours

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTI-XLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P < 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P < 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 hfaster than did the modified BAM Salmonella culture method.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

Detection of olive oil using the Evagreen real-time PCR method

A sensitive real-time PCR method using the novel fluorescence stain Evagreen was established for detection of olive oil. First, a comparative study was made of two different methods for the recovery of high quality DNA from oil samples. Thereby, the Promega wizard DPSF kit proved to be the most suitable for recovery of DNA from oil samples. Second, an olive-specific pair of primers was designed based on the sequence of an olive plasma intrinsic protein cDNA sequence. Experiments with 13 samples including olive, soybean, sesame, sunflower seed, pumpkinseed, walnut, rapeseed, rice, peanut, maize, pig, chicken and fish confirmed that this pair of primers is highly specific for olive. Additional experiments indicated that the absolute detection limit of our test is 0.07 ng olive DNA and that 0.5% olive DNA can be detected against background of 2 ng/μl sesame DNA.     

A robotic DNA purification protocol and real-time PCR for the detection of Campylobacter jejuni in foods

Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42 degrees C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 x g for 10 min at 4 degrees C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.     

电子鼻在芝麻油掺芝麻油香精识别中的应用

采用电子鼻对芝麻油中掺入芝麻油香精进行识别.通过对所获得的数据进行主成分分析(Principal Component Analysis,PCA)、判别因子分析(Discriminant Factor Analysis,DFA)、偏最小二乘回归分析(Partial Least-squares Analysis,PLS)和统计质量控制分析(Statistical Quality Control,SQC).结果表明:不同样品在电子鼻传感器上有不同的特征性响应图谱,电子鼻能够有效识别不同掺入比例的芝麻油样品;DFA方法的区分效果比PCA方法更好;SQC模型对于掺入芝麻油香精超过50％的芝麻油能明显区分;采用PLS对数据进行处理,电子鼻响应信号和芝麻油香精掺入比例之间有很好的相关性(相关系数为0.992 1),PLS方法能有效识别掺入比例为0％～ 100％的试验样品.试验证明电子鼻可用于芝麻油掺假的识别.     

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。     

Application of real-time PCR for tree nut allergen detection in processed foods

Abstract

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to accidental contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. Real-time PCR allowed a specific and accurate amplification of allergen sequences. Some processing methods could induce the fragmentation and/or degradation of genomic DNA and some studies have been performed to analyze the effect of processing on the detection of different targets, as thermal treatment, with and without applying pressure. In this review, we give an updated overview of the applications of real-time PCR for the detection of allergens of tree nut in processed food products. The different variables that contribute to the performance of PCR methodology for allergen detection are also review and discussed.     

实时荧光定量技术在食品微生物检测和研究中的应用

实时荧光定量PCR技术是通过检测PCR产物中荧光讯号强度来达到定量的目的,不仅实现了对核酸信息量的分析比较,而且与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点。文章概述了实时荧光定量PCR技术的原理、优缺点及其在食品微生物检测中的应用与研究进展,并探讨了它的技术发展和应用前景。     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

Salmonella in sesame seed products

In the context of an international outbreak of multiresistant Salmonella Typhimurium DT 104 that was correlated to the consumption of halvah ("helva," an Asian candy made from sesame seed), we examined several sesame seed products for the occurrence of Salmonella. Of 117 ready-to-eat food items containing sesame, we isolated salmonellae from 11 (9.4%) samples. In addition to finding Salmonella Typhimurium DT 104 in the halvah involved in the outbreak, we also isolated different Salmonella Typhimurium strains out of halvah from other manufacturers and countries of origin, as well as Salmonella Offa, Salmonella Tennessee, and Salmonella Poona from sesame paste (tahini) and sesame seed, which is sold for raw consumption in cereals.     

单增李斯特菌毒力因子多重荧光PCR法 建立与应用

目的建立同时检测单增李斯特菌(Listeria monocytogenes)及其3种毒力因子的多重荧光PCR快速检测方法,并应用于日常食品的检测。方法根据单增李斯特菌溶血素基因hly A、内化素基因inl A和表面蛋白act A基因的保守序列,分别设计合成特异性引物和探针,优化多重荧光PCR反应体系。对该方法的特异性、敏感性和重复性进行评估。结果该法特异性强、敏感性高,对单增李斯特菌纯培养物的最低检出限410cfu/m L;重复性好,变异系数均小于2%。对84份食品检测结果与传统国标法相符,共检出单增李斯特菌4份,检出率为4.76%。多重荧光PCR检测方法耗时1 h,比传统方法节约2~5 d。4株单增李斯特菌分离株中2株同时含有inl A、act A、hly A 3种毒力基因,另2株为毒力基因act A缺失株,提示目前流行株并非同一来源。结论本研究建立的多重实时荧光PCR方法能同时对单增李斯特菌及其3种毒力因子进行快速检测,且灵敏度高、特异性好,为食源性疾病的病原学检测提供了快速可靠的方法。     

TaqMan探针双重荧光PCR技术检测食源性沙门氏菌和志贺氏菌

目的 建立TaqMan探针双重荧光聚合酶链式反应(polymerase chain reaction,PCR)技术检测食源性沙门氏菌和志贺氏菌的方法。方法 通过比对沙门氏菌和志贺氏菌的基因组序列,选择同源性高、保守性好的区域设计特异性引物和探针,经过引探筛选、浓度调试等一系列优化,建立了食源性沙门氏菌和志贺氏菌双重荧光PCR核酸检测方法,并对其特异性、质粒最低检出限、菌液敏感性、重复性、稳定性以及实际样品进行了验证。结果 该方法与大部分食源性致病菌无交叉反应,特异性强;沙门氏菌和志贺氏菌质粒的最低检出限可达5 copies/μL,沙门氏菌菌液敏感性为1.0×102 cfu/mL,志贺氏菌菌液敏感性10 cfu/mL;质粒和菌液核酸梯度的批内和批间变异系数均在0.177%~1.958%之间,小于5.0%,具有较强的稳定性;标准曲线相关系数(R2)均大于0.999。结论 本研究成功建立了一种TaqMan探针双重荧光PCR技术同时检测食源性沙门氏菌和志贺氏菌的方法,该方法扩增时间短,只需要30min即可,而且特异性强、稳定性好,可用于疑似沙门氏菌和志贺氏菌污染样品的检测,为食品安全的快速检测提供技术支撑。     

数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究

目的建立微滴式数字PCR技术(droplet digital PCR,ddPCR)定量检测食品中单核细胞增生李斯特氏菌的分析方法。方法选择基因hly A为靶序列,设计PCR扩增引物和Taq Man探针,优化反应条件和反应体系,通过细菌分离和dd PCR方法对靶标基因的检测特异性、灵敏度和重复性进行实验,并对定量结果进行分析。结果 ddPCR反应中的最佳探针浓度为5 pmol/μL,特异性好,检出限为(3.6±0.1)copies/20μL,重复性实验良好,标准偏差为0.067%。ddPCR的拷贝数(copy number)与细菌密度(colony forming units,CFU/mL)形成了较好的线性关系。结论本研究建立dd PCR的拷贝数和菌液密度或菌落数的线性关系,可以为单核细胞增生李斯特氏菌的快速定量检测提供参考。     

实时荧光PCR法检测食品中亚利桑那沙门氏菌

沙门氏菌（Salmonella）是食品检测过程中最常见的致病菌之一,亚利桑那沙门氏菌（Salmonella arizonae）又是沙门氏菌中比 较难鉴定的亚种。该实验通过实时荧光聚合酶链式反应（PCR）方法快速准确的检测亚利桑那沙门氏菌和其他沙门氏菌。根据GenBank 公布的亚利桑那沙门氏菌和其他沙门氏菌gud D基因序列, 分别设计引物和Taqman探针。 使用10株不同血清型的沙门氏菌标准菌 株、88株沙门氏菌分离株和29株食品中常见食源性致病菌进行实时荧光PCR特异性实验。结果显示,该实验所设计的引物探针特异 性非常好。 实时荧光PCR灵敏性试验结果表明,检测灵敏度可达到1～10 CFU/mL的添加浓度。 经模拟污染样品验证,所建立的实时 荧光PCR方法与传统方法的检测结果相一致,具有检测周期短、操作简便的优势。     

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/w_tf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/w_tf GM soybean powders was found to have a correlation coefficient ( r ) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/w_tf GMO). The average GMO amount ranged from 0.35% to 0.63% w/w_tf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay.     

实时荧光PCR法检测鼠伤寒沙门氏菌

目的 建立实时荧光PCR法检测鼠伤寒沙门氏菌的方法。方法 基于鼠伤寒沙门氏菌II型限制酶基因, 设计引物及Taqman探针, 利用实时荧光PCR进行特异性、灵敏性及模拟样品的检测实验。结果 特异性探针可从25种血清型沙门氏菌(共49株)及11株阴性对照菌株中检测出全部的11株鼠伤寒沙门氏菌。以鼠伤寒沙门氏菌梯度稀释菌液DNA为模板进行实时荧光PCR实验, 菌株模板浓度与Ct值呈良好线性关系, 线性系数(R2)为0.998, 扩增效率90%, 最低检测浓度300 cfu/mL。对已接种鼠伤寒沙门氏菌的4种模拟样品同时进行实时荧光PCR检测和传统方法鉴定, 两者结果一致。结论 此方法特异、灵敏、准确, 适于食品中鼠伤寒沙门氏菌的检测。