A novel real-time polymerase chain reaction method for the detection of pecan nuts in food

A real-time PCR-based method for the detection of the pecan (Carya illinoiensis) component in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pecan-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the putative gene for allergenic vicilin-like seed storage protein of pecan. The method was positive for 10 pecan varieties and negative for all other tested plant materials used in food industry, including walnut. The intrinsic detection limit of the method was 1 pg pecan DNA which corresponds to 1.2 haploid genome copies. Using a series of model pastry samples with defined pecan contents, a practical detection limit of 0.01% (w/w) pecan was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples; no discrepancies between the declared and detected pecan contents were found. The presented PCR method is useful for sensitive and selective detection of pecans in food samples and can be performed in one working day.     

A novel real-time polymerase chain reaction method for the detection of macadamia nuts in food

A real-time PCR-based method for the detection of macadamia nuts (fruits of Macadamia integrifolia or M. tetraphylla or their hybrids) in food products is described. The method consists of DNA isolation by chaotropic solid phase extraction and subsequent PCR with macadamia-specific primers and a TaqMan fluorescent probe. The primers and the probe were targeted to the gene encoding for vicilin precursor. The method was positive for M. integrifolia and M. tetraphylla and negative for 16 other plant species used in food industry, including peanuts, walnuts, hazelnuts, almonds, pistachio nuts, cashew nuts, Brazil nuts, and chestnuts. The DNA-based detection limit of the method was 1.45 pg. Using a series of model samples with defined macadamia nut contents, a practical detection limit of 0.02% (w/w) macadamia nuts was determined. Practical applicability of the PCR method was tested by the analysis of 14 confectionery samples. For all of the samples, results conforming to the labeling were obtained. The presented PCR method is useful for relatively fast, highly selective, and moderately sensitive detection of macadamia nuts in food samples.     

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.     

A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food

A real-time polymerase chain reaction (PCR)-based method for the detection of the walnut (Juglans regia) component in food is described here. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with walnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the jug r2, a major allergen gene of walnut. The method was positive for 8 varieties of walnut and negative for all other tested plant materials used in food industry, including pecan nuts. The intrinsic detection limit of the method was 0.24 ng walnut DNA. Using a series of model pastry samples with defined walnut contents, a practical detection limit of 0.01% walnut content was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples (bakery and confectionery products), out of which two cakes were found to contain walnuts although they were not adequately labelled. The presented PCR method is useful for sensitive and selective detection of walnuts in food samples and can be performed in one working day.     

Detection of pea in food by real-time polymerase chain reaction (PCR)

A qualitative 5′-nuclease real-time PCR-based method for the detection of pea (Pisum sativum) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pea-specific primers and a TaqMan fluorescent probe. The primers and the probe are oriented to the chloroplast DNA intron located between trnL and trnF exons encoding for tRNA. The analytical parameters of the method were inclusivity 100%, exclusivity 100% and the detection limit of 0.11±0.07 ng of pea DNA corresponding to 12±7 diploid pea genome copies. Using a set of model meat patés with defined pea contents, a matrix-related detection limit of 0.05% was determined and a linear calibration line was constructed. The presented analytical method was useful for qualitative detection or semiquantitative determination of pea in food products. The method was relatively fast because the analysis could be performed in one working day.     

副溶血弧菌PCR检测方法研究进展

副溶血弧菌是引起包括我国在内的世界各地沿海地区食物中毒的重要食源性致病菌,患者有典型的肠胃炎症状。及时准确地对食品中的副溶血弧菌进行检测是预防该菌引起的食物中毒的关键。分子生物学检测方法在副溶血弧菌检测中具有许多优势,现已得到广泛的应用。本文对PCR检测方法中的多重PCR、有扩增内标的PCR、实时荧光PCR(包括荧光染料法和荧光探针法)、基于DNA染料叠氮溴化乙锭和叠氮溴化丙锭的PCR、纳米粒子PCR、免疫捕获PCR、PCR-变性高效液相色谱、PCR-酶联免疫吸附等方法的国内外研究情况进行了综述,并对其检测效率、灵敏度、优点和缺点等进行了分析比较,环介导等温扩增(loop-mediated isothermal amplification,LAMP)——包括常规LAMP和原位LAMP因与PCR方法有相似之处,故一并进行了综述,为副溶血弧菌PCR检测方法的应用与开发提供参考。     

Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication

The advent of real-time polymerase chain reaction has revolutionized the field of molecular biology, but the design and optimization of these assays has been largely overlooked in the literature. This dearth of information is probably in response to the provision of assay design software and robust guidelines issued by the leading manufacturer. However, many applications require highly specific assays with no cross-amplification of non-target DNA and it has been found that the software and guidelines, whilst producing assays of great sensitivity, do not necessarily produce specific assays. Two complementary strategies were used to confer specificity on a real-time assay, first by placing the 3' end of the primers on a point of sequence heterogeneity and, second, by truncating the primers at the 5' end to lower the calculated melting temperature. Using these strategies in concert, a specific assay for a conserved region of the mitochondrial cytochrome b gene was developed that can be used for the unambiguous detection of the target species in a meat mixture. This approach can be used for any real-time polymerase chain reaction assay to increase assay selectivity and specificity.     

Detection of peanut using real-time polymerase chain reaction

Preliminary results are presented on a sensitive and robust assay for the identification of peanut in commercial products using real-time PCR technology. Peanut specific primers and probe, designed using the Arah 2 gene, were optimised for real-time PCR using an ABI PRISM 7700. Commercial extraction kits employing different technological strategies were assessed for the extraction of PCR quality peanut DNA template. The specificity of the primer and probe set was determined using a wide range of food items and the limit of detection and quantification calculated using dilutions of peanut DNA. The assay was used to detect spiked or trace level of peanut in commercial samples and was finally used to detect peanut in a biscuit prepared with 2 ppm of lightly roasted peanut powder.An erratum to this article can be found at     

转基因鲑鱼AquAdvantage品系特异性实时荧光聚合酶链式反应检测方法的建立

目的实现转基因鲑鱼AquAdvantage的标识管理,建立其品系特异性实时荧光聚合酶链式反应(PCR)检测方法。方法针对转基因鲑鱼的品系特异性序列设计引物和TaqMan探针,建立转基因鲑鱼实时荧光PCR检测方法,并对该方法的特异性、灵敏度和重复性进行检测。结果建立的转基因鲑鱼实时荧光PCR方法特异性强,在600 000~60拷贝范围内呈良好的线性关系,其线性回归方程为y=-3.2194x+40.805,R~2=0.997,检测限为60拷贝,检测重复性良好。结论建立的品系特异性实时荧光PCR方法可应用于转基因鲑鱼AquAdvantage的鉴定。     

A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.     

微滴式数字聚合酶链式反应在食品安全检测领域的应用

微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction, ddPCR)是一种新型核酸扩增技术,可对DNA或RNA分子采用绝对定量的方式进行分析。其结果具有更高的精准度、准确性和灵敏度,大大提升了数字PCR技术的可扩展性与实用性,促进了现代分子生物学在精准定量检测方面的发展和应用。本文重点论述了ddPCR法的技术原理、优势以及在食源性致病微生物定量检测、转基因成分分析、食品源性成分检测等食品安全检测领域的应用研究进展情况。     

Detection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false-positive 35S-promoter screening results

 Today DNA-based techniques are very common for the detection of genetically modified organisms (GMOs) in food products. For fast and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. These techniques do not allow differentiation between GMOs and the natural occurrence of transgenic elements, such as the 35S-promoter of cauliflower mosaic virus (CaMV) or the NOS-terminator of Agrobacterium tumefaciens, and thus may result in false-positive detection of GMOs. In this study we evaluated three different existing 35S screening systems and report the development of two new CaMV-specific PCR systems. These PCR systems based on CaMV-specific genes allow the identification of positively screened 35S food samples as naturally virus-infected products or plants. Seven food samples tested positive in routine 35S screening analysis and negative in GMO specific systems were investigated using the new virus-specific PCR systems. In all seven samples CaMV was detected. Received: 26 April 1999 / Revised version: 28 June 1999     

真鲷虹彩病毒聚合酶链式反应检测方法的建立

目的:真鲷虹彩病毒一直都被列入在世界卫生组织（OIE）水生动物疫病病原的名录中,故需要对其检测方法进行深入研究,建立操作简单,普遍适用的检测技术手段。方法:本文针对RSIV片段设计了一对特异性引物,对反应体系和反应程序进行筛选和优化,建立了检测RSIV聚合酶链式反应的方法。并且用优化好的反应体系和反应程序对重组质粒p MD18-T-RSIV进行扩增。结果:研究发现该方法特异性强,与IHNV、IPNV、SVCV和VHSV无交叉反应,并且灵敏度高,可以达到0.2pg。结论:成功的建立了真鲷虹彩病毒常规PCR检测方法,可以为真鲷虹彩病毒病的诊断提供理论依据。      

Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction

A study on development of Fourier-transform infrared spectrophotometric method combined with principle component analysis as well as real-time polymerase chain reaction for determination of pork–beef mixture in meatballs has been performed. A lipid component extracted from pork and beef in meatballs is analyzed using Fourier-transform infrared spectroscopy, while DNA extracted from meatball was analyzed using real-time polymerase chain reaction. The correlation between actual and predicted concentration of lard using Fourier-transform infrared spectroscopy was performed by aid of partial least squares, while grouping of lard and beef fat components in meatball was carried out by Fourier-transform infrared spectra coupled with principle component analysis. The results showed that Fourier-transform infrared spectra at wavenumbers of 1000–1200 cm^?1 coupled with partial least square and principle component analysis are successfully used for quantification and classification of pork in beef meatballs. The relationship between actual value and predicted value of lard (lipid fraction obtained from meatballs containing pork) with Fourier-transform infrared spectrophotometric method revealed good correlation, with coefficient determination (R²) value of 0.997 and standard error of calibration of 0.04%. Principle component analysis is able to classify samples containing pork and beef meatballs. Fourier-transform infrared spectroscopy using normal spectra is fast technique for identification and quantification of lard extracted from pork in meatball. In addition, real-time polymerase chain reaction using Leptin Primer–AJ 865080 can be used for amplification of pork DNA specifically in meatballs containing pork.     

Rapid and sensitive detection of Vibrio parahaemolyticus in sea foods by electrochemiluminescence polymerase chain reaction method

Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. Because of the safety concerns, detection and characterization of V. parahaemolyticus have attracted much attention. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) method combined with universal probes hybridization technique was applied to rapid detection of V. parahaemolyticus, infected and uninfected sea foods for the first time. Whether the sea food samples were infected was discriminated by detecting the gyrase B (gyrB) gene. We detect V. parahaemolyticus both in artificially contaminated sea foods and natural samples. The experiment results show that the infected and uninfected sea food samples can be clearly identified and the detection limit for V. parahaemolyticus is 1.6 pg purified genomic DNA in the presence of 1 μg non-specific background DNA. The technique may provide a new means in V. parahaemolyticus detection due to its simplicity and high efficiency.     

实时荧光聚合酶链式反应技术与国标方法检测致病菌的应用与研究

目的比较实时荧光聚合酶链式反应技术(real-time polymerase chain reaction,RT-PCR)与国标方法在食品致病菌检测中的异同。方法应用RT-PCR法及国标GB 4789系列对采集的60件畜产品、禽产品、水产品和乳及乳制品中的沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏菌以及副溶血性弧菌同时进行检测。结果除了金黄色葡萄球菌检测结果均为阴性外,其他3种致病菌2种方法都有检出,但RT-PCR方法的阳性率均高于国标方法。对于沙门氏菌,国标方法阳性率为1.67%,RT-PCR方法阳性率3.33%;单核细胞增生李斯特氏菌国标方法阳性率为7.50%,RT-PCR方法阳性率为15.00%;副溶血性弧菌国标方法阳性率为8.33%,RT-PCR方法阳性率为11.67%。结论在本实验条件下,RT-PCR技术的阳性检测结果多于国标GB4789系列,并且结果可完全覆盖国标GB4789。各企业实验室甚至政府主导的食品风险监测项目可根据产品特点,合理应用RT-PCR技术以减少人员工作量,方便产品放行并提高工作效率。     

The detection of foodborne pathogens by the polymerase chain reaction (PCR)

Nucleic acid probes are being used increasingly in the food industry. These can be relatively insensitive but a new technique for amplification of a target sequence of nucleic acid has the potential of being rapid, sensitive and specific. The method, termed polymerase chain reaction or PCR, utilises a thermostable DNA polymerase from the bacterium Thermus aquaticus. Following thermal denaturation of double stranded DNA, small oligonucleotides, termed primers, are annealed to the single strands of DNA and these determine the starting point for replication of the DNA by the polymerase enzyme. Applications for food microbiology are being researched but problems have been encountered due to the complex nature of many foodstuffs.     

实时荧光PCR快速筛选食品中鸭源性成分

基于鸭线粒体细胞色素Cyt b基因建立了肉制品中的鸭源性成分检测的real-time PCR方法,并对模拟样本和市售样本进行了检测分析,检出限低至1%,适用于实验室的鸭源性成分的鉴定分析。      

鱼肉制品中巴沙鱼源性成分的实时荧光聚合酶链式反应快速检测法

目的 建立巴沙鱼源性成分的实时荧光聚合酶链式反应(PCR)快速检测手段.方法 根据巴沙鱼的线粒体cytb基因序列设计引物,使用实时荧光PCR进行扩增,从而达到快速检测产物的目的.结果 此方法特异性良好,巴沙鱼基因组DNA灵敏度可达到10-4 ng,在与婴幼儿米粉、儿童副食芝麻粉、鸡肉粉和大西洋鳕鱼粉混合的鱼肉制品中均可...     

实时荧光PCR技术快速检测食品中的牛源成分

目的:建立基于实时荧光PCR技术的食品中牛源性成分快速检测方法。方法:以牛线粒体细胞色素b为目的基因,设计特异性引物和探针,通过特异性、灵敏性实验,及模拟混合肉样和市售肉制品检测,对该体系进行验证。结果:该牛源荧光PCR检测体系具有很好的特异性及灵敏性,可检测1pg牛源DNA的存在,对于各模拟肉类样品中掺杂的牛源性成分,其检测限低至0.5%,且经市售加工食品验证具有较好的应用能力。结论:所建立的牛引物探针体系具有特异性好、灵敏度高,快速高效等优点,可用于对食品中牛源性成分的掺假鉴别检测。