转基因大豆MON89788实时荧光PCR检测方法的建立

为实现转基因大豆MON89788的标识管理,针对转基因大豆MON89788的品系特异性序列设计引物和TaqMan探针,建立转基因大豆MON89788实时荧光聚合酶链式反应（polymerase chain reaction,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。结果显示：建立的转基因大豆MON89788实时荧光PCR检测方法能扩增出127 bp的产物,特异性强,灵敏度达到0.1%,约为40 个单倍体基因组拷贝,检测重复性好,可成功应用于实际样品检测。因此,建立的转基因大豆MON89788实时荧光PCR检测方法可以应用于转基因大豆MON89788大豆及其制品的检测。     

LAMP实时浊度法快速检测转基因玉米MON810

LAMP实时浊度法是采用环介导等温扩增（Loop Mediated Isothermal Amplification,LAMP）技术通过实时浊度仪实时检测反应过程中所产生的白色沉淀,从而实现对扩增全过程的监控,弥补了显色法只能观看反应终点的缺陷,使引物筛选和反应体系的优化有数据可依。本研究以转基因玉米MON810为研究对象,针对外源基因苏云金芽孢杆菌杀虫毒蛋白CryIA(b)与内源基因边界序列设计6条特异性引物,通过实时浊度法在63 ℃的恒温条件下完成检测,对检测的灵敏度、特异性、稳定性进行了评价。建立了转基因玉米MON810的LAMP实时浊度检测方法,该方法最低检出限为0.5%,与LAMP显色法和实时荧光PCR法进行结果比对,符合率为100%,经评价具有特异性高、稳定性强、准确简便等优点,非常适合转基因玉米MON810的快速检测,有较好的应用价值。     

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

PCR-based methods are widely used in the European Union and in other countries for the detection, identification, and quantification of genetically modified organisms (GMOs). The preparation of good-quality DNA from plant samples for GMO detection can be a challenging task, particularly if the DNA will be used for quantitative analysis. Two DNA extraction methods, namely manual (NucleoSpin Food kit from Machery-Nagel) and high-throughput partially automated (NucleoMag Plant kit from Machery-Nagel) methods, which utilize different DNA separation principles, were used for the isolation of DNA from maize flour samples. Despite the higher DNA recovery obtained using the high-throughput isolation method, a lower PCR efficiency was achieved, most likely due to the presence of PCR inhibitors in the extracts. We found both DNA extraction methods suitable for GMO analysis.     

A Qualitative Approach for the Assessment of the Genetic Stability of the MON 810 Trait in Commercial Seed Maize Varieties

Maize MON 810 is one of the European Union’s (EU) authorized genetically modified organisms (GMO) for placing on the food and feed market. The total number of MON 810 varieties registered in the European Common Catalogue of varieties of agricultural plant species has almost tripled since 2005. One of the requirements described in EU legislation, namely the genetic stability of GM seed varieties, was thus assessed by analyzing the intactness of the entire MON 810 integration and its genotypic stability in commercial varieties available on the market for at least the last 2 years. A combined strategy using qualitative analytical methods made possible to determine the presence/absence of the individual genetic elements and of the whole GM construct. The restriction fragment length polymorphism patterns obtained from amplified whole constructs by long polymerase chain reaction (PCR) were compared side by side. CryIA(b) protein expression levels were determined by enzyme-linked immunosorbent assay. Twenty-four out of the 26 analyzed varieties met the expected stability features. One variety gave negative results in all assays, and one variety contained the necessary genetic elements for expressing CryIA(b) protein although giving negative results for the long PCR product. To our knowledge, this study is the first post-marketing stability analysis performed on GM commercial seed varieties.     

Development of an Event-Specific Detection Method for Genetically Modified Maize IE034 by Quantitative Real-Time PCR

The insect-resistant transgenic maize event IE034 has been proved to be one of the most commercially developed transgenic maize events in China. This study was aimed to develop a stable and reliable quantitative detection method to monitor this new transgenic maize event. Here, we developed a novel event-specific real-time PCR method for this genetically modified maize event IE034. The resulting 134 base pair (bp) amplicon was designed according to the 5′ junction of inserted sequence and flanking maize genome sequence. Standard curve of the IE034 5′ event-specific sequence showed good linear regression and high PCR efficiency when using the IE034 pure line samples as calibrator. The limit of detection (LOD) for the IE034 detection method was estimated at approximately 8 initial template copies, and the limit of quantification (LOQ) was estimated at about 40 copies. The accuracy of this quantitative real-time PCR method was verified by screening four mixed DNA samples with known levels of the IE034 event (5, 1, 0.5, and 0.23 %, respectively). The quantified biases deviated from 8.7 to ?12.2 %, and the relative standard deviation (RSD) ranged from 2.7 to 12.7 %. These data indicated that this new-developed IE034 event-specific real-time PCR method is suitable and reliable for the quantification of IE034 maize and its derivates.     

A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat

It has been developed a method for quantitative detection of Salmonella enterica in poultry meat based on real-time PCR (qtPCR) with species-specific primers and SYBR® GreenER? chemistry. Two methods for bacterial DNA extraction were compared: one based on a commercial kit (AccuPrep®) and the other on silica–magnetite nanoparticles. Primers were designed on sequence of invA gene encoding for an inner membrane protein associated with invasiveness of Salmonella. Serial dilutions of DNA from pure cultures of Salmonella and from broiler breast samples spiked with serial dilutions of Salmonella were analyzed in different replicates and with different PCR equipments. Robustness of the method was evaluated and compared in terms of repeatability, reproducibility, and consistency with conventional plate count methods and for applicability to the different equipments. The matrix effect upon each reaction specificity was assessed with addition of DNA from a noncompetitive internal amplification control. The limit of detection (LOD) was determined between 10 and 40 colony-forming units (CFUs)/ml; whereas, the limit of quantification (LOQ) was 10² CFUs/ml. Quantification with qtPCR was in the same order of magnitude as enumeration with plate counting but with an overestimation.     

The Use of Infrared Thermography as a Novel Approach for Real-Time Validation of PCR Thermocyclers

Validation of PCR thermocycler performance is crucial to obtain reliable results. In this study, infrared (IR) thermography was evaluated as a novel validation tool. After stabilisation, no significant difference in the temperatures recorded using thermography and a reference block-based system was found. By employing IR thermography, information about the length of the time until temperature stabilisation in the sample could be obtained. This study shows the potential of using IR thermography for validation of thermocyclers.     

环介导等温扩增法检测转基因玉米MON89034

根据MON89034外源插入片段与植物基因组序列设计特异性引物,筛选最佳引物并对反应体系和反应条件进行优化,最终建立转基因玉米MON89034转化体特异性LAMP检测方法。对该方法进行了特异性、灵敏度、稳定性和重复性测试。结果表明:该方法能够特异性检测出MON89034玉米;检测其灵敏度达到1 pg;以转基因玉米MON89034 DNA标准品质量分数为1.00%,0.10%,0.05%的样品为模板,其稳定性好、重复性高,假阴性率为0。本试验设计的LAMP方法适用于特异性检测转基因玉米MON89034。     

Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

Coliforms are a major group of bacteria associated with spoilage of refrigerated pork. It is necessary to develop an efficient and fast method to detect total coliforms in chilled pork because the traditional methods are laborious and time-consuming. In this study, a quantitative real-time polymerase chain reaction (qRT-PCR) targeting the lacZ gene was developed for rapid enumeration of coliforms from chilled pork. Of the 106 strains tested, 35 coliform strains and one Shigella sonnei strain were correctly identified compared with 70 control strains which failed to amplify. Detection sensitivity was 112 CFU/g. The assay was able to detect and accurately quantify the total coliforms in chilled meat within 2 h without preenrichment. The results indicate that the real-time PCR is an efficient and time-saving method that could be adopted by the meat industry to detect coliforms to replace the traditional most probable number and the rapid coliform count plate methods.     

Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44?×?10³ copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47?±?0.73)?×?10³ colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named “Rapid Amplification of genomic DNA Ends” (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.     

转基因玉米的七重PCR鉴定方法

以玉米内源基因IVR、外源抗除草剂基因(BAR、PAT)、抗虫基因Cry1Ab、筛选基因NPTII、CaMV35S启动子和NOS终止子为检测的目的片段,分别设计了7对引物,通过研究较佳引物终浓度配比和退火温度,建立了玉米转基因成分七重PCR检测体系。结果表明,建立的七重PCR体系用于同时检测内源基因和转基因成分是可行的,检测方法效率高、稳定性好。     

Molecular Identification of Four Genetically Modified Maize (Bt11, Bt176, Mon810 and T25) by Duplex Quantitative Real-Time PCR

In response to the increasing number of genetically modified (GM) events released on the market, control laboratories explore various strategies to simplify and reduce the number of tests needed to characterise the content in genetically modified organism (GMO) of a given sample. Lastly, multiplexing is considered as one of the possible ways to decrease the time and cost of analysis. Here, we report the development of four duplex polymerase chain reaction (PCR) tests for the identification and the quantification of four maize transformation events from which commercial lines have been authorised in Europe namely, Bt11 and Bt176 (Syngenta, DE, USA), Mon810 MaisGard? (Monsanto, MO, USA) and T25 Liberty Link? (Bayer CropScience, Monheim, Germany). The duplex PCR tests combine a maize-specific PCR test hybridising in the Adh1 locus with an event-specific detection system designed on a junction fragment for each of these four GM maize. Real-time PCR tests, suitable to comply with the European regulation, were designed by using Taqman® chemistry.     

实时荧光PCR法在鱼翅鉴别中的应用

为有效鉴别市售鱼翅的真伪,针对鱼翅中鲨鱼源性成分的线粒体基因组的部分序列,利用实时荧光聚合酶链式反应(Polymerase Chain Reaction,PCR)技术,通过特异性引物和探针的设计建立起一种有效的真伪鉴别方法。该方法中对样品脱氧核糖核酸(Deoxyribonucleic acid,DNA)的提取方法、DNA检测的浓度灵敏度、质量灵敏度以及方法的特异性进行研究。结果表明鱼翅样品用试剂盒进行提取效果良好,其检测的DNA浓度灵敏度可达1×10~(-5) ng/μL,质量灵敏度可达0.01%。利用该方法对市场上的购买的47份鱼翅类样品和18份非鱼翅样品进行了检测,43样品检测出鲨鱼成分,包括4份仿鱼翅在内的22份未检出鲨鱼成分。     

Statistical Evaluation of Real-Time PCR Protocols Applied to Quantify Genetically Modified Maize

Real-time PCR (RTi-PCR) is the technique of choice for event-specific quantification of genetically modified organisms (GMOs) by determining the amount of event with respect to a species-specific reference gene. Reference genes can be amplified from the genome extracted from Certified Reference Materials (CRMs) or from ad hoc designed plasmids. In the present study, we statistically evaluate the performance of RTi-PCR protocols for GM maize MON810 event by using both genomic DNA from conventional CRMs and a plasmid containing sequences representative of four maize species-specific reference genes. The significance of simple and interaction effects of several variables included in the experimental design on DNA quantification methods and RTi-PCR were evaluated and discussed. Statistically significant differences on Ct values may have an impact on the GMOs quantification and consequently on the compliance of GM quantification-established legal thresholds. Our results confirm the reliability of the plasmid as alternative calibrant for the calculation of GMOs copy number.     

Validation of a 1-Day Analytical Diagnostic Real-Time PCR for the Detection of Salmonella in Different Food Meat Categories

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.     

转基因大豆MON89788双重数字PCR通用定量检测方法的建立

建立一种特异、稳定、灵敏、通用的转基因大豆MON89788品系双重数字聚合酶链式反应（digital polymerase chain reaction,dPCR）定量检测方法,在一个体系内同时进行内外源基因的定量检测,适用于微滴式数字PCR（droplet digital PCR,ddPCR）和芯片式数字PCR（chip digital PCR,cdPCR）平台,并通过了食品分析能力评价体系国际能力验证项目的盲样检测评价。ddPCR平台对大豆MON89788品系和内源Lectin的绝对定量限分别为8.0copies/μL和8.2copies/μL;cdPCR平台对大豆MON89788品系和内源Lectin的绝对定量限分别为7.443copies/μL和7.646copies/μL;ddPCR和cdPCR对转基因大豆MON89788品系成分相对含量的相对定量限均为0.1%。