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.     

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.     

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.     

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.     

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。     

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.     

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.     

Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes

For the cultivation and maintenance of seeds, genetic mutations have to be avoided as much as possible. According to the Directive 2001/18/EC, genetic stability has to be proven for the approval of GMOs [1]. The investigation of genetic stability can be performed with various methods, but Southern blots are most commonly used. In this study, a different method, a real-time PCR involving Scorpion primer, was developed for the analysis of DNA rearrangements in GMOs. The advantage of this method is that already small changes in sequence can be detected. In addition, large numbers of samples can be tested at once in a single PCR run. Possible alterations in the region of the Scorpion primer can be detected by differences in the real-time PCR plots. The sequence that was analysed contained 130 nucleotides. The sensitivity of Scorpion primers to measure genetic stability was verified in experiments with plasmids that had only minor sequence alterations in the target region. Such small differences led to a total reduction of the fluorescence signal, even in heterozygous samples where the unaltered template was present as well. Scorpion primers were then used to investigate the transgenic sequences of 567 individual seeds of MON810 for maize at the 5′ and the 3′ region of the insert. In combination with SYBR green, the screening procedure allowed a selection of samples for further analysis. If a sample showed low fluorescence with the Scorpion primer but a normal amplification with SYBR green, then the integrity of the insert was checked by sequencing. The results, however, gave no indication of a mutation or rearrangement of MON810 seeds. In additional experiments, the reliability of the method was studied by determining several parameters that are required for a validation, e.g., specificity, cross-reactivity, dynamic range, correlation of the template concentration and the corresponding Ct values, PCR efficiency, relative repeatability standard deviation (RSDr) relative reproducibility standard deviation (RSDR).     

Method Validation for Cafestol and Kahweol Quantification in Coffee Brews by HPLC-DAD

Cafestol and kahweol are two diterpenes exclusively found in coffee. Negative effects on health, like the increase in total lipid blood levels as well as positive effects like carcinogenic protective and antioxidant activity, have been reported for these two compounds. A simple and expeditious method for the quantification of total cafestol and kahweol levels in coffee brews by high-performance liquid chromatography with diode array detection was developed and validated. Esterified diterpenes were hydrolysed and resulting diterpene alcohols extracted with diethyl ether. For cafestol limit of detection was 0.01 mg L^?1 and limit of quatification was 0.04 mg L^?1. Coefficient of variation for cafestol ranged between 0.2 and 2.8 % for repeatability and between 1.4 and 15.3 % for intermediate precision. Average recoveries of cafestol-spiked coffee samples were from 96 to 110 %. Kahweol was quantified using cafestol calibration data and corrected by an appropriate conversion factor. Different kinds of coffee brews were analysed. Cafestol levels ranged from 0.04 to 0.80 mg/cup and for kahweol levels were between 0.01 and 0.40 mg/cup. Highest levels of diterpenes per cup were present in espresso coffee, while the lowest were found in filter coffee and instant coffee.     

Validation of an enzyme immunoassay for monitoring Cry1Ab toxin in soils planted with Bt-maize (MON810) in a long-term field trial on four South German sites

For monitoring the environmental impacts of a genetically modified crop well-established methods are a key requirement for the collection of appropriate data. With regard to the fate of Cry1Ab protein from Bt-maize MON810 released by root exudates, plant residues and pollen deposition into soil, a highly specific and sensitive sandwich enzyme immunoassay for detection and quantification of Cry1Ab in soil matrix was developed. The assay was validated according to the criteria of EU-Decision 2002/657/EC for a range of soils originating from four selected experimental field sites in South Germany, where Bt-maize MON810 has been cultivated. On three sites Bt-maize was grown permanently for eight years whereas one site was cropped with Bt-maize for one year only. The validation of the method showed that the assay fulfils the validation criteria as prescribed in the guidelines of the EU-Decision with minor restrictions. When the assay was applied to field soil samples collected from the selected four experimental sites six months after harvest, no Cry1Ab protein was detectable. By ELISA-determination and quantification of Cry1Ab protein in soils collected from these sites at further time points more profound knowledge about the long-term behaviour of Cry1Ab protein in soil will be obtained.     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described.     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described. Received: March 19, 2007     

In-House Validation and Comparison of Two Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

Common wheat is one of the most important staple food crops worldwide. However, unlike other important staple crops such as maize or soybean, genetically modified (GM) wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosate-tolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat samples by using both taxon-specific and screening tests. Two candidate common wheat taxon-specific real-time PCR methods were suggested, one targeting ssII-D gene coding for starch synthase and the other targeting waxy-D1 gene, coding for granule-bound starch synthase. In the present study, the two above-mentioned real-time PCR taxon-specific methods were in-house verified and compared, proposing droplet digital PCR (ddPCR) as a new tool for supporting the application of the European Network of GMO Laboratories (ENGL) established method performance criteria. Preliminary performance data of waxy-D1 and ssII-D methods in ddPCR format are shown too to give a contribution to the bridging process from the consolidated to the emerging quantitative PCR methodology.