Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng?6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng?6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng?6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95?%) between 0.07 and 0.52, a PCR efficiency of 105?% and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng?6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng?6 DNA were observed.     

Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng 6 DNA were observed.     

Next-Generation Sequencing as a Tool for Detailed Molecular Characterisation of Genomic Insertions and Flanking Regions in Genetically Modified Plants: a Pilot Study Using a Rice Event Unauthorised in the EU

Precise molecular characterisation of genetic modifications integrated into the genomes of genetically modified organisms (GMOs) and of their flanking genomic regions forms a key component for the development of event-specific detection methods. In the EU, this information is of particular importance for risk management in cases where genetic modifications of unauthorised GM food, feed or seeds are detected. PCR-based chromosome walking approaches are commonly used for DNA sequence determination of the genetic modifications and of the flanking genomic regions in yet undescribed GM plants. If the plant contains complex and re-arranged modifications, sequencing and molecular characterisation are often difficult and laborious. Next-generation sequencing (NGS) of DNA is a powerful alternative tool to rapidly generate primary sequence data on the genome of so far uncharacterised sample material if pure GMO material is available. Recently, robust NGS platforms and affordable sequencing services are accessible for food and feed control laboratories. We here present a NGS-based study for whole-genome sequencing of the GM rice event LLRice62 as a proof-of-principle experiment to develop bioinformatics easy-to-use data analysis tools for rapid molecular characterisation. A total of 171,657,155 read mate pairs of approximately 75 bp each were obtained. Sequence reads belonging to the genetic modifications and their flanking genomic regions in LLRice62 were identified by bioinformatic comparison to the corresponding Oryza sativa ssp. japonica reference genome sequence using the Illumina InDel caller software and subsequent iterative mapping of retrieved NGS reads. An entire genetic modification of 1,493 bp in the genome of the LLRice62 sample material was determined and correctly mapped on chromosome 6. The determined nucleotide sequence coincides to the genetic modification described by the developer of this rice event. This study demonstrates for the first time the applicability of NGS for molecular characterisation of uncharacterised GMOs.     

Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.)

The cry1Ab gene is a foreign gene which encodes Bt insecticidal Cry1Ab protein and was transferred into genomic DNA of plants to acquire insect resistance. Here a loop-mediated isothermal amplification (LAMP) assay with high specificity and rapidity under isothermal conditions was developed for detecting cry1Ab gene in transgenic rice. Partial sequence of cry1Ab gene was used as the target template to design LAMP primers. The reaction conditions were optimized as follows: 60 min of reaction time, 1:3 of outer primer and inner primer concentration ratio, 25 μL of reaction volume and 0.6 μM of betaine. The results of detection could be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with that of regular PCR and real-time PCR. The results showed that the LAMP assay exhibited high specificity and the sensitivity of 3 × 10² copies number of the positive control plasmid, and of 0.5 % genetically modified (GM) contents. In comparison with real-time PCR method, LAMP showed the same results with simple instruments. The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for detecting cry1Ab gene from transgenic rice plants and rice ingredients in processed foods aimed at screening the growing transgenic crops in the fields and detecting genetically modified (GM) ingredients in imported and domestic foods.     

DNA extraction and fingerprinting of commercial rice cereal products

DNA was extracted from commercial rice cereal products using modified conventional methods (CTAB, SDS and a commercial kit) in large fragments (>3 kb) and with relatively high yields (1.4–10.7 μg DNA per g of sample) and was used as template for the amplification of a single copy rice gene (i.e. MIPS) fragment (ca. 850 bp) and microsatellite DNAs (ca. 120–400 bp). The cereal products were further discriminated by using six microsatellite markers. The usefulness of DNA analysis was discussed for quality control and authenticity testing of raw rice materials in rice-based food production, and to monitor genetically modified (GM) rice ingredients in commercial food products.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Growth and toxin production of proteolytic Clostridium botulinum in aseptically steamed rice products at pH 4.6 to 6.8, packed under modified atmosphere, using a deoxidant pack

Demand for aseptically steamed rice products has been increasing rapidly in Japan over the past 10 years. In our previous study, we showed that proteolytic Clostridium botulinum produce toxins in steamed rice products packaged under a modified atmosphere of < or =0.3% oxygen. In the present study, we examined the effect of pH to control botulism risk in steamed rice products packaged under modified atmosphere (5% CO2 and 95% N2 as the balance) with the inclusion of a deoxidant pack to produce an oxygen concentration of < or =0.3%. A mixture of 10 strains of proteolytic C. botulinum (5 type A strains and 5 type B strains) was inoculated into steamed rice products at pH values between 4.6 and 6.8 prior to packaging. All samples were stored at 30 degrees C for 24 weeks. Samples at higher pH showed earlier starts of neurotoxin production. Neurotoxin was detected after 2 weeks of incubation in samples at pH 5.4 or above, whereas it took 4 weeks for the toxin to be detected in samples at pH 5.2 to 5.3 and 12 weeks in samples at pH 5.0 to 5.1. In samples at pH 4.9 or below, no toxin was detected during the experimental period. Apparent sample spoilage did not occur before C. botulinum produced neurotoxin in most of the samples. Based on these results, we conclude that aseptically steamed rice products must be packaged at pH 4.9 or below under modified atmosphere containing < or =0.3% oxygen, with the inclusion of a deoxidant pack.     

转基因大米及其安全性评价研究进展

近年来,转基因稻米的迅速发展引起了广泛关注,尤其是与食品密切相关的营养与安全问题。分析转基因大米的基因来源及对大米品质的影响,总结转基因大米可能存在的危害及其安全性评价的方法,可使人们科学的对待转基因大米,并为研究与开发新型转基因大米提供参考。     

In-depth analysis of the endogenous reference genes used in the quantitative PCR detection systems for rice

To standardize the rice-specific PCR detection methods, five previously reported rice (Oryza sativa) taxon-specific genes were compared and evaluated. The investigated genes included the rice root-specific gene (gos9), the ppi phosphofructokinase gene (ppi-PPF), the phospholipase D gene (PLD), the starch branching enzyme gene (RBE4) and the sucrose phosphate synthase gene (SPS). Sequencing analyses revealed that among the tested rice cultivars, single-nucleotide polymorphisms (SNPs) existed in the gos9, PLD, ppi-PPF and SPS amplicons, though no statistically significant effect on their Ct values was found. The ppi-PPF and PLD systems were found to produce amplicons in non-rice species, such as sugarcane and broomcorn. The quantitative real-time PCR results revealed that this cross-reaction led to an underestimate of the GM (genetically modified) rice content. With the exception of the aforementioned shortcomings, these five endogenous reference genes all have acceptable amplification efficiencies, which ranged from 98 to 108?%, and high sensitivity within the limit of detection (LOD) values, which ranged from 5 to 10 copies of the haploid genome. In estimating the GM content in blinded rice samples, these five systems produce relatively accurate quantitative results with deviations less than 15?%, but the RBE4 system produced the most accurate quantitative results. Therefore, we have determined that the RBE4 gene is the most suitable rice reference gene, and the gos9 and SPS genes can also be used as rice reference genes because they have good commutability with the RBE4 gene. Care should be taken when interpreting results based on the PLD and ppi-PPF genes owing to their cross-reaction with other species.     

Multiplex real-time PCR for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference molecule

According to the EU and Swiss legislation for food, only approved traits of transgene plants are allowed to be imported and sold to the consumer. In order to control imports of rice and rice products from retailers, dealers and importing companies, efficient and reliable methods for the detection and quantification are a prerequisite. Therefore, a novel pentaplex real-time polymerase chain reaction system was developed and validated for the quantitative determination of three genetically modified rice lines at once. This system simultaneously determines DNA contents of the phospholipase-gen, a rice species specific gene, the 35S:BAR-construct, as the promotor of different transgene rice lines and the specific systems for LL62, LL601 and Bt-63-rice (Shanyou63). The test exhibits a good specificity and sensitivity for the transgenes in the range of 0.01–1%. It proved its efficiency and reliability in daily routine. Due to the lack of appropriate reference material for the Bt-63-rice, a reference oligonucleotide was artificially constructed. This oligonucleotide proved its applicability in diagnostic analysis.     

The use of modified atmospheres to control Sitophilus zeamais and Sitophilus oryzae on stored rice in Portugal

This study reports the efficacy of using CO₂ against Sitophilus zeamais and Sitophilus oryzae as an alternative treatment to fumigation for rice stored in a rice mill in Portugal. The trials were conducted in a silo containing 40 tonnes of polished rice and in four hermetic big bags of 1 tonne capacity; two with paddy and two with polished rice. The composition of the atmosphere ranged from 90 to 95% CO₂ and 0.7–2.1% O₂. Three trials were carried out at different temperatures and treatment times; stored rice in the silo at 29.6 ± 0.1 °C for 26 days (first trial), at 34.1 ± 0.2 °C for 10 days (second trial), and in big bags at 22 °C for 26 days (third trial).To evaluate the efficacy of each treatment, metal cages with 16 g of infested rice where placed at bottom, middle, top and surface of the polished rice in the silo. Four replications of each type of infested rice containing one-week-old S. zeamais adults, or eggs of S. zeamais or S. oryzae, were incubated in the laboratory, at the same temperature as in the silo, to serve as a control.In all modified atmosphere treatments adults of S. zeamais, and eggs of both S. oryzae and S. zeamais, showed mortality close to 100% and no F1 emergence was recorded in any treatment sample. This was the first time that a Portuguese rice mill used modified atmospheres.     

Comparison of dry matter losses and aflatoxin B1 contamination of paddy and brown rice stored naturally or after inoculation with Aspergillus flavus at different environmental conditions

The objective of this study was to compare the effect of different storage moisture conditions (0.70, 0.85, 0.90 and 0.95 water activity, a_w) and temperatures (20, 25, 30 °C) on (a) respiration rates (R) and dry matter loss (DML) of paddy and brown rice and (b) quantify aflatoxin B₁ (AFB₁) production by isolates of Aspergillus flavus from the rice samples and (c) inoculation of both rice types with A. flavus under these storage conditions on R, DML and AFB₁ contamination. There was an increase in temporal CO₂ production with wetter and warmer conditions in naturally contaminated rice. Higher R and consequently, % DML, were generally found in the brown rice (21%) while in paddy rice this was only up to 3.5% DML. From both rice types, 15 (83.3%) of 18 A. flavus isolates produced detectable levels of AFB₁ in a range 2.5–1979.6 μg/kg. There was an increase in DML in both rice types inoculated with A. flavus as temperature and a_w were increased. Interestingly very little AFB₁ was detected in paddy rice, but significant contamination occurred in the brown rice. The %DML in the control and A. flavus inoculated rice increased with temperature and a_w at both 25 and 30 °C from 1-2% to 15–20% DML at 30 °C and 0.95 a_w. All the inoculated rice samples had AFB₁ levels above the EU legislative limits for contamination in other temperate cereals and products derived from cereals (=2 μg/kg). Even samples with % DML as low as 0.2% had AFB₁ contamination levels twice the limits for other cereals. These results suggest that the mycotoxin contamination risk in staple commodities like rice, is influenced by whether the rice is processed or not, and that measurement of R rates can be used to predict the relative risk of AFB₁ contamination in such staple commodities.     

采用GS1系统建立非转基因大米制品溯源体系

2011年欧盟出台2011/884/EU决议,针对中国出口的大米及米制品中转基因成分扩大范围进行检测,加强了对中国出口米制品的转基因管理。自该决议出台以来,中国米制品生产企业产品出口欧盟屡屡受阻。本研究以上海市C食品有限公司生产的非转基因年糕及白粿干为例,从水稻种植、收割、大米碾磨、年糕和白粿干加工、包装、仓储6 个关键环节进行信息的收集,并按照GS1系统的格式分别对转基因年糕及白粿干的6 个关键环节收集到的信息进行设计和编码,并按照产品的生产加工流程将6 组设计的编码组合在一起,形成一套完整的GS1-128码。通过对GS1-128码进行扫描和解读,可以对该公司生产的非转基因年糕和白粿干产品进行溯源,为出口米制品的出口贸易提供溯源保障。     

Molecular analysis of yeasts from Indonesian cassava and glutinous rice tapé

Tapé is a popular delicacy in Indonesia that is prepared by fermenting starch-rich material. Yeast plays an important role in the making of tapé especially in the aroma and taste of the product. In this study, yeasts were isolated from tapé and subjected to diversity analysis using the restriction fragment length polymorphism (RFLP) technique on 5.8S rRNA encoding gene and the internal transcribed spacer (ITS). A total of 21 yeast isolates were obtained from 3 different types of Indonesian tapé (cassava tapé, white-, and black-glutinous rice tapé). The identified yeast species from cassava tapé were Pichia jadinii, Candida glabrata, and Clavispora lusitaniae. P. fabianii, Rhodotorula mucilaginosa, and Ogataea polymorpha were obtained from white glutinous rice tapé, while Saccharomycopsis fibuligera and Wickerhamomyces anomalus were obtained from black glutinous rice tapé.     

Detection of genetically modified soy in processed foods sold commercially in Malaysia by PCR-based method

Regulations for the use and labeling of genetically modified organism (GMO) products and derived ingredients are being implemented worldwide, that demands reliable and accurate methods to detect GMO in raw materials and food products. In this study, polymerase chain reaction (PCR) method was established for monitoring products derived from GMO that are sold in the markets in Malaysia, which specifically amplify the 35S promoter, nos (nopaline synthase-terminator), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) and RRS (CTP/CP4EPSPS). Using this method, we investigated the incidence of genetically modified soy (GM-soy) and specifically the presence of roundup ready soy (RRS). All the soybean samples were evidenced by presence of the lectin gene. Out of 85 samples examined, the 18 positive GM samples were raw bean (9), tofu (8) and tempe (1) (a traditional Malay food). The results demonstrate for the first time the presence of GM-soy in Malaysian food products, reinforcing the need for the development of accurate quantitative methods for routine analyses.     

Isolation and identification of Lactobacillus and yeast species and their effect on the quality of fermented rice cakes

In this study, microbes were isolated from the rice slurry of a fermented rice cake to obtain lactic acid bacteria and yeast species. These species were identified using microbial physiology and gene sequence analyses. As the growth of the lactic acid bacterial strain R-2b and the yeast J-3a strains were found to be the best, a composite starter comprising these microbes was used for the preparation of fermented rice cakes. Based on single factor and orthogonal experiments, when the proportion of Lactobacillus plantarum, Saccharomyces cerevisiae, and Candida humilis was 1:3:6, the optimal fermentation conditions were addition of sugar and starter amounts of 20% and 6%, respectively, a fermentation temperature of 32 °C, and fermentation time of 8 h. The fermented rice cake with this optimum ratio had the most abundant volatile components and qualified physicochemical and microbial indexes. Additionally, the overall quality was better than that of commercially available products.     

High-throughput detection of genetically modified rice ingredients in foods using multiplex polymerase chain reaction coupled with high-performance liquid chromatography method

The aim of this study was to develop a method for simultaneous detection of a variety of genetically modified (GM) rice ingredients in foods using multiplex polymerase chain reaction (PCR) coupled with high-performance liquid chromatography (HPLC) assay. The following exogenous genes found in GM rice were selected as targets: CaMV35S, NOS, Cry1Ac, Bar, and Xa21. The endogenous gene PEPCex of rice was selected as an internal control. In brief, six pairs of primers for multiplex PCR were designed according to the specific region of CaMV35S, NOS, Cry1Ac, Bar, Xa21, and PEPCex, and following the optimization, a multiplex PCR assay was developed, and then the multiplex PCR products were subjected to HPLC analysis. The GM rice lines ShanYou 63, KeFeng 6, KangYou 97, and LLrice 62 were used as reference GM rice samples to evaluate the potential diagnostic capability of the method. Results demonstrated that the multiplex PCR-HPLC developed in this work was an efficient diagnostic method for simultaneous identification of the target genes with 0.15 ng/mL of high sensitivity, suggesting a better alternative for the rapid detection of many genetic modification events.     

A Bacillus thuringiensis strain producing epizootics on Plodia interpunctella: A case study

After several disease outbreaks in laboratory cultures of pyralid moths in Tabriz University, Iran, during 2004 and 2005, a new Bacillus thuringiensis aizawai strain EF495116 (BTA) was isolated from a dead Plodia interpunctella larva. A complete characterization of the strain was performed, including serological identification, protein and plasmid pattern determination, a PCR-based identification of virulence-related genes, nucleotide sequence analysis of the 16S rDNA and gyrB genes (in order to find out relationships between the species with other virulent Bacillus pathogens), and biological activity assays. These studies revealed that BTA produced a major parasporal protein band of about 135 kDa, bore seven out of the fourteen pyralid-active genes analyzed (cry1Aa, cry1Ab, cry1C, cry1D, cry1I, cry2A and cry9) and was toxic against P. interpunctella and P. xylostella larvae, with LC₅₀ values of 7.13 and 3.1 μg/mL, respectively. Although these features are common among other B. thuringiensis strains active on Lepidoptera, their role in epizootics is uncertain. However, sequence analysis of the 16S rDNA and gyrB genes revealed that BTA clustered with one of the few B. thuringiensis strains identified as a medical isolate. Interestingly, both strains, like many others reported to produce epizootics, belong to serovar aizawai. The implication of serovar or serovar-dependent genes in epizootics is discussed.     

SYBR<Superscript>®</Superscript>Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products

Identification of crops present in food and/or feed matrices represents an important step in the screening strategies targeting genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their presence in an integrated screening approach, a set of SYBR^®Green real-time polymerase chain reaction (qPCR) methods has been developed which can be used under the same assay conditions and at similar efficiency for each of the abovementioned crops. Each qPCR method is shown to meet the performance criteria (i.e. specificity, limit of detection and PCR efficiency) set by the European Network of GMO Laboratories (ENGL). When combined with the equivalent qPCR methods targeting GMO elements, these crop-specific SYBR^®Green qPCR methods can aid the development of an efficient tool for determining GMO presence in food and/or feed products.