Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase A gene

The presence of cyanobacterial bloom in water supply reservoirs can cause potential health hazards. In this study, we aimed at the quantification of microcystin-producing cyanobacteria based on the microcystin synthetase A (mcyA) gene using real-time PCR. To perform a highly sensitive real-time PCR assay, the novel primer MSR-2R was designed and a coprecipitation DNA extraction method was used in this study. Cyanobacterial cells could be collected efficiently by coprecipitation with other bacteria suspended in solution even in the case of low concentrations of cyanobacteria. The detection limit of the method was found to be 8.8 cells per reaction. When cyanobacterial growth was monitored in pure culture, the cell concentration determined by real-time PCR positively correlated with the cell concentration determined from direct microscopic count. Furthermore, we could detect and quantify the mcyA gene in lake water samples using real-time PCR. It was concluded that the quantification of the mcyA gene based on real-time PCR is a powerful tool for the rapid quantification of microcystin-producing cyanobacteria in environmental samples.     

检测食品中大肠杆菌O157的多重荧光PCR方法的建立

针对大肠杆菌O157的志贺毒素编码基因stx1、stx2和O157血清型的标志基因wzy的保守序列,设计特异性的引物和探针,反复优化反应体系和条件,建立了一种新的多重荧光PCR检验方法.结果显示,该方法灵敏度高,stx1、stx2的扩增效率分别为96.4%和94.6%,实际样品检测时stx1、six2的最低检出限分别为4.2×102cfu/mL和4.2×103cfu/mL.该方法特异性强,扩增结果与各参考菌株基因型一致,能良好的区分出O157菌株和非O157犁大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

食品中3种致病菌多重PCR检测方法的建立及初步应用

建立用多重聚合酶链式反应(Multiplex polymerase chain reaction,mPCR)同时检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的方法。以沙门氏菌的gyrB基因、单核细胞增生性李斯特菌的gyrB基因、金黄色葡萄球菌的coa基因作为目的基因,分别设计3对引物,通过优化反应体系,建立3种致病菌的多重PCR检测体系。采用单重PCR检测时,灵敏度可达0.423ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)和0.36ng/mL(单核细胞增生性李斯特菌);而采用三重PCR检测时,灵敏度较单重PCR有所下降,分别为2.43ng/mL(沙门氏菌)、2.5ng/mL(金黄色葡萄球菌)、3.6ng/mL(单核细胞增生性李斯特菌)。初步建立能同时、快速、灵敏地检测食品中沙门氏菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的三重PCR方法。     

Development and validation of a real-time PCR detection method for celery in food

As from 25 November 2005 onwards, a list of ingredients with known allergenic potential has to be labeled according to Directive 2003/89/EC, including celery and products thereof. In order to provide appropriate detection methods a novel real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of DNA from celery (Apium graveolens) was developed and validated. Specificity was confirmed by testing DNA derived from more than 50 food relevant organisms. Sensitivity was demonstrated on the basis of a calibration curve plotting the corresponding Ct-values against DNA amounts ranging from 1 to 1000 copies. Due to the lack of certified reference material the applicability of the method was assessed by analysis of sausages spiked with defined amounts of grounded celery seed. The limit of detection (LOD) examined exemplarily for emulsion-type sausages was 5–10 mg/kg. Analysis of celery-containing commercial products demonstrated the performance potential and limitations of the new real-time PCR system.     

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

The present work is focused on the development of a TaqMan multiplex real-time PCR method for the detection of Salmonella, Shigella and L. monocytogenes in seafood, meat and ready-to-eat products. The aim of this study is to detect the three pathogens in one single test including an enrichment medium for the simultaneous growth of the bacteria of interest and an Internal Amplification Control (IAC) to monitor PCR inhibitors. For this purpose, three genes were selected, invA for Salmonella, ipaH for Shigella and hlyA for L. monocytogenes. Also, no. 17 broth without dextrose and further modified by adding Tween 80 was used for the enrichment step. Specificity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR efficiency obtained for the simultaneous amplification of all three pathogens was 102.5% for Salmonella, 108.9% for Shigella and 106.4% for L. monocytogenes. The limit of detection (LOD) was evaluated in seafood, meat and ready-to-eat products, being established within 3 and 22 cfu in 25 g of sample for the three bacteria analyzed. Seventy-eight samples were analyzed with multiplex RT-PCR including spiked and natural samples collected from different laboratories. Even though several RT-PCR methods have been developed for the detection of Salmonella, Shigella and L. monocytogenes, as far as we know this is the first method developed for the simultaneous detection of these three pathogens, coupling RT-PCR with an enrichment in the same broth and being tested in a wide range of different processed food samples with a low LOD. The application of this method can significantly reduce costs and time of analysis in laboratories, what would be reflected in a faster response in those risk situations when they are detected.     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

三种食源性致病菌多重PCR检测方法的建立

目的:建立同时快速检测沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7的多重PCR方法。方法:根据沙门氏菌的invA基因、志贺氏菌的ipaH基因及肠出血性大肠杆菌O157∶H7的uidA基因设计3对引物,通过单因素实验、L9(34)正交实验优化反应体系,并对多重PCR扩增的敏感性进行分析。结果:3对引物能特异性扩增出495、620、252bp的目的片段;在最优多重PCR反应体系下,多重PCR检测3种致病菌的灵敏度达104CFU/mL;将该法应用于人工污染实验,可在5h内得到准确、稳定的检测结果。结论:该方法操作简单、检测特异性和灵敏度较高,能够实现对沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7 3种食源性致病菌的快速监控和诊断。      

One-step multiplex PCR method for the determination of pecan and Brazil nut allergens in food products

BACKGROUND: A one‐step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components. RESULTS: The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin‐like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL^?1 pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg^?1). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration. CONCLUSION: The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food. Copyright © 2011 Society of Chemical Industry     

加工食品中动物源DNA的提取和多重PCR检测方法的建立

为了从深加工食品中提取高质量和数量的动物源性DNA片段,以优化后CTAB方法提取,用于PCR检测。根据鸭线粒体基因序列,设计合成检测鸭源性成分引物,进行PCR体系和反应条件的优化筛选,建立三重PCR方法;同时扩增出牛源性116bp、猪源性212bp和鸭源性322bp目的基因片段。建立的三重PCR方法特异地鉴定牛肉及其制品中掺假猪和鸭源性成分,对打击食品掺假伪造、审核食品标签、保障消费者知情权、维护消费者健康等方面有重要作用。     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

Real-time PCR multiplex method for the quantification of Roundup Ready soybean in raw material and processed food

This paper describes a quantitative real-time multiplex PCR method optimised for the ABI PRISM^® 7700 Sequence Detection System (SDS) and TaqMan^® chemistry for Roundup Ready^® Soybean (RRS) in raw material and processed food. This method has the advantage of performing the amplification of the target taxon lectin gene and the genetically modified (GM) target sequence in the same test tube. The quantification is based on a calibration curve obtained with the DNA extracted from Certified Reference Material standards (CRM IRMM-410: R) in the range 0.1–5% RRS. The method was validated in-house by using CRMs and the applicability was verified on three mixtures of soybean flour at 1, 5, 10% of RRS respectively and on prepared baked products (biscuits and plum cakes). The statistical parameters of the method were found to be satisfactory both for soybean flour and baked products. In the process the absolute LOD and LOQ was 7.1 and 35.5 copy number respectively; the relative LOD and LOQ was 0.03 and 0.01% of RRS respectively.     

基于双启动引物特异性检测单核细胞增生李斯特氏菌的PCR方法

以单核细胞增生李斯特氏菌iap基因为靶基因,利用一新型PCR引物设计方法--双启动引物（Dual-priming oligonucleotide,DPO）,建立了特异性检测单核细胞增生李斯特氏菌的DPO-PCR方法,测试了DPO-PCR方法退火温度不敏感性、特异性及灵敏度,并在实践检测中进行了初步应用。结果显示:该方法检测单核细胞增生李斯特氏菌的灵敏度为1.51×102CFU/mL;退火温度不敏感性测试中,与常规PCR引物相比,DPO引物在48⁶8℃退火温度范围内均能够高效率地扩增靶基因;特异性测试中,DPO-PCR方法能特异地检测出目标菌,与其他菌株无非特异性扩增反应,比常规PCR方法显示出更强的特异性。实践应用证明,利用DPO-PCR方法对130份样本进行检测,共计检出9份单核细胞增生李斯特氏菌阳性样本,经国标法（GB/T 4789.30-2008）复检,两者检测结果一致,显示出良好的实用性,为单核细胞增生李斯特氏菌的快速准确检测提供了新方法。      

食品中单核细胞增生性李斯特氏菌PCR快速检测方法

为建立食品中单核细胞增生性李斯特氏菌 (单增李斯特氏菌 )的快速、敏感、特异的PCR检测方法 ,选取hlyA基因作为靶序列设计一对引物 ,用该引物对 6 3株从国内食品中分离的李斯特氏菌 (进行传统方法验证 )和 2 0株非李斯特氏菌进行PCR扩增 ,并用此方法对人工污染食品进行检测 ,扩增片段表现出极好的单增李斯特氏菌种特异性 ,人工污染的生肉、冷冻虾仁、卷心菜的检出限为 39cfu g ,为食品中单增李斯特氏菌的快速、敏感并且特异的检测方法。     

6类致泻大肠埃希氏菌多重PCR检测体系的建

目的 构建食源性致泻大肠埃希氏菌高效、快速、特异性强的检验方法。方法 根据6类致泻大肠埃希菌11种毒力基因引物, 采用煮沸法制备细菌基因组DNA作为模板构建致泻大肠埃希氏菌单菌多重PCR检测体系; 采用试剂盒法制备细菌基因组DNA作为模板构建6类菌十重PCR检测体系, 对2种检测体系进行优化并对其进行特异性、重复性验证。结果 构建了6类致泻大肠埃希氏菌多重PCR检测体系, 包括单菌多重PCR、6类菌十重PCR检测体系。仅6类致泻大肠埃希氏菌阳性菌株有特异性条带产生; 不同类型致泻大肠埃希氏菌菌株均检出其特异性阳性条带, 该多重PCR检测方法重复性好, 稳定性强。结论 该方法可高效快速地确定待测样是否为大肠埃希氏菌及其致病型, 为食品中致泻大肠埃希氏菌的监控、预警及爆发引起食物中毒后溯源等提供参考。     

Novel touchdown-PCR method for the detection of putrescine producing Gram-negative bacteria in food products

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99–91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.     

产黄曲霉毒素真菌多重PCR检测体系的建立

依据标准SN/T 2582—2010《产黄曲霉毒素真菌PCR检测方法》推荐的产黄曲霉毒素调节基因afl R、柄曲霉素转甲氧基酶基因omt-1、杂色曲霉素A脱氢酶基因ver-1及真菌共有的5.8S r DNA的ITS序列分别设计4对特异性引物,对4对引物进行多重PCR反应体系构建与优化,建立快速检测产黄曲霉毒素真菌的多重PCR方法,并应用10株试验菌株对所建立的检测方法进行验证。结果表明,建立的多重PCR方法具有灵敏度高、特异性强、方便快捷的优点,为产黄曲霉毒素真菌快速检测试剂盒的研发及应用提供了重要技术保障。     

Development of multiplex PCR assays to identify Escherichia coli pathogenic genes in food

In order to rapidly screen for the virulence factor that produces pathogenic Escherichia coli in food, we have developed multiplex polymerase chain reaction (PCR) assays. The multiplex PCR assays detect 4 pathogenic genes of enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). This allows for the generation of specific fragments 150, 179, 218, 401, 465, 584, and 881 bp for VT1, ST, LT, 16S rRNA, inV, VT2, and eaeA genes, respectively. The detection limit of 3 log CFU/mL for eaeA, LT, VT1, VT2, 4 log CFU/mL for inV, 6 log CFU/mL for ST by single PCR, while 5 log CFU/mL for VT1, VT2, 6 log CFU/mL for eaeA, LT, 7 log CFU/mL for ST, inV by multiplex PCR. This optimized detection method of pathogenic E. coli can be used as supportive data to revise the microbiological analytical manuals for the Korean Food Code.     

Development and validation of a duplex real-time PCR method for the simultaneous detection of celery and white mustard in food

The developed duplex real-time PCR method allows the simultaneous detection of traces of potentially allergenic white mustard (Sinapis alba) and celery roots (Apium graveolens var. rapaceum), celery stalks (A. g. var. dulce) and leaf celery (A. g. var. secalinum). The duplex assay does not show any cross-reactivity with 64 different biological species, including various members of the Brassicaceae and Apiaceae family. In raw model sausages spiked with white mustard and celery roots, the LOD was found to be 0.001% white mustard and 0.005% celery. In model sausages brewed at 75–78 °C for 15 min the LOD was found to be 0.005% white mustard and 0.005% celery. The duplex real-time PCR assay was applied to check if commercial food products are labelled in compliance with the legal regulations.     

Development of real-time PCR method for rapid and accurate detection of Centipedes (Scolopendra mutilans) in food

Food Science and Biotechnology - Centipedes contain pharmacologically active compounds used as important medicinal material. However, the poisons produced by centipedes can cause human diseases;...