6,486 research outputs found
Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.
BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform
Detection of genetically modified organisms (GMOs) using molecular techniques in food and feed samples from Malaysia and Vietnam
Food labeling in accordance with Novel Food Regulation has been enforced in the European Community since 1997 with a series of updated legislations namely, EC/258/97, EC/1139/98, EC/49/2000, EC/50/2000 and EC/1829/2003. Guidelines and labeling regulations for the use of GMOs materials in food and feed products has also been introduced in Malaysia and Vietnam. Therefore, the demand for the establishment and development of a robust and rapid operation procedure for GMO detection has increased recently in both countries. The procedure of GMO detection emphasizes not only on detection tests but also on confirmation assays. This study employed PCR technology for detection and direct DNA sequencing for confirmation procedures respectively. The results demonstrated for the first time the presence of GM plants with glyphosate-resistant trait led by the control of P35S promoter and NOS terminator in either Malaysian or Vietnamese feed with high frequency (20 positive samples out of 24 analyzed samples).
The P35S promoter, EPSPS gene and NOS terminator sequences obtained showed some mutations on single-stranded and double-stranded targeted sequences caused by single nucleotide insertion or single nucleotide changes. These results reinforce the need for development of detection procedures to comply with food/feed labeling system
Molecular Identification of Genetically Modified Crops for Biosafety and Legitimacy of Transgenes
Crops undergo artificially DNA modifications for improvements are considered as genetically modified (GM) crops. These modifications could be in indigenous DNA or by introduction of foreign DNA as transgenes. There are 29 different crops and fruit trees in 42 countries, which have been successfully modified for various traits like herbicide tolerance, insect/pest resistance, disease resistance and quality improvement. GM crops are grown worldwide and its area is significantly increasing every year. Many countries have very strict rules and regulations for GM crops and are also a trade barrier in some situations. Hence, identification and testing of crops for GM contents is important for identity and legitimacy of transgene to simplify the international trade. Normally, molecular identification is performed at three different levels, i.e., DNA, RNA and protein, and each level has its own importance in testing about the nature and type of GM crops. In this chapter, current scenario of GM crops and different molecular testing tools are described in brief
Biosensores y plataformas sensoras de ADN con transducción electroquímica para la detección y cuantificación de soja genéticamente modificada en alimentos y piensos
Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Analítica, leída el 06/04/2017. Tesis formato europeo (compendio de artículos)In the nearly two decades since they were first commercialized, genetically engineered crops have gained ground on their conventional counterparts, reaching nearly 180 million hectares worldwide in 2015. The technology has bestowed most of its benefits on enhancing crop productivity with two main traits currently dominating the market: insect-resistant and herbicide-tolerant crops. Genetically modified organisms (GMOs) are conventionally obtained through the introduction of foreign DNA fragments into the host genome via genetically engineering techniques. The modified organism, i.e. plant, will then be able to express new protein(s) confering it with the novel, desired trait(s), e.g. herbicide tolerance. Plants such as maize and soybean have been modified to withstand weed-killing chemicals or resist insect pests to increase yields and improve profits to farmers. Despite their rapid and vast adoption by farmers worldwide, GMOs have generated heated debates, especially in European countries, driven mostly by consumers concerned about safety of transgenic foods and about the potential impact of their release into the environment. The European Union (EU) has established the mandatory labeling of GMOs in food and feed above a certain threshold (0.9%, based on the ingredient). In the list of ingredients the term "genetically modified" must appear (next to the ingredient in question). Below such level, labeling is not mandatory provided that the presence of GM material is proven to be accidental or technically unavoidable. The need to monitor GMOs and to verify compliance with EU legislation has driven the development of analytical methods able to detect and quantify GMOs in crops, and in food and feed products...Los cultivos genéticamente modificados han ganado terreno desde que han sido comercializados hace casi dos décadas, habiendo alcanzado en 2015 casi 180 millones de hectáreas en el mundo, lo que sobrepasa el área destinada a cultivos convencionales. La tecnología ha permitido mejorar la productividad de los cultivos, existiendo dos tipos de modificación genética que actualmente dominan el mercado: la resistencia a insectos y la tolerancia a herbicidas. Los organismos genéticamente modificados (OGMs) se obtienen, normalmente, mediante la inserción de un fragmento de ADN de una especie diferente a la especie receptora, a través de la introducción de fragmentos de ADN al genoma receptor, a través de técnicas de ingeniería genética. El organismo modificado (planta) será capaz de expresar una o más proteínas nuevas que le conferirá la característica deseada (p. ej., tolerancia a herbicidas). Plantas como el maíz o la soja han sido modificadas para tolerar agentes químicos que eliminan malas hierbas o para expresar proteínas insecticidas y resistir enfermedades, siendo el resultado un aumento en el rendimiento de los cultivos y en los beneficios económicos adquiridos por los agricultores...Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEunpu
Prevalence and diversity of Arcobacter spp. in poultry meat in New Zealand : a thesis presented in the partial fulfillment of the requirements for the degree of Master of Science in Veterinary Microbiology and Public Health at Massey University, Palmerston North, New Zealand
The microaerophilic bacterium Arcobacter has received increased attention in recent years as an emerging foodborne human pathogen. Although phenotypically related, arcobacters differ from campylobacters in their ability to grow aerobically and at lower temperatures. Poultry are considered a significant reservoir of this organism, with an isolation rate of up to 72% in faecal samples, and up to 100% in meat samples. To date, four species; A. butzleri, A. skirrowii, A. cryaerophilus, and A. cibarius have been isolated from poultry. The first three species have also been found to be associated with human and animal illnesses such as diarrhoea, bacteraemia, mastitis and abortions. The organisms are also found in raw meat products as well as in surface and ground water. Since most laboratories still do not use appropriate isolation techniques, the occurrence of this organism in food sources and their role in human illnesses is greatly underestimated. This is the first investigation of the prevalence of arcobacters in poultry meat in New Zealand. The aim of this study was to compare the most commonly used Arcobacter isolation methods. In addition, this study aimed to estimate the prevalence of Arcobacter spp. in retail poultry in New Zealand. Other aims include comparison of genetic diversity of Arcobacter spp. isolated from three different poultry producers, and by different methods, and estimation of overall genetic diversity of arcobacters present in New Zealand. During the period of May to October 2005, a total of 150 fresh, whole, retail poultry carcass produced by three different producers were purchased through two supermarket outlets in Palmerston North, New Zealand. Isolation of Arcobacter was done by seven different techniques. Arcobacter-like organisms were identified presumptively by phenotypic tests; temperature tolerance, aerotolerance, motility , and oxidase production. These presumptive arcobacters were confirmed by a species-specific multiplex PCR (m-PCR) either as A. butzleri, A. cryaerophilus or A. skirrowii. DNA sequencing was done for selected isolates from both species to further confirm the PCR results. The PCR positive isolates were subjected to Pulsed-Field Gel Electrophoresis (PFGE) following restriction digestion with Eagl. It was found that 55.3 % of 150 retail poultry sold in New Zealand were harbouring Arcobacter species. Two species; A. butzleri and A. cryaerophilus were detected by m-PCR which was later confirmed by sequencing. A total of 189 isolates were detected by six methods from 83 retail poultry samples. A. butzleri was the predominant species and was detected in 51.3% of the samples, whereas A. cryaerophilus was detected only in 8% of the samples. A. butzleri and A. cryaerophilus accounted for 92.6% (n=175) and 7.4% (n-14) of the isolates, respectively. A. butzleri was the only Arcobacter species present in 46.6% samples, and A. cryaerophilus only in 3.3% of the samples. Both species were detected simultaneously in 4.6% of the samples. There was a wide variation among the prevalence rate of Arcobacter spp. in retail poultry from different producers varying from 30 to 98%. There was also a wide variation among the isolation rates of different methods varying from 3.3 to 39.3%. The best isolation method was found to be Arcobacter-broth enrichment followed by passive filtration through a sterile filter of 0.45μm, onto blood-agar plates. No single isolation method detected all arcobacters. PFGE of Arcobacter isolates demonstrated the occurrence of multiple genotypes of both A. butzleri and A. cryaerophilus in the retail poultry from the same producers, and even in a single poultry. The possible explanations for the large amount of heterogeneity include multiple sources of contamination, the occurrence of multiple parent genotypes for both species in a single poultry carcass, and a high degree of genomic recombination among the progeny of historical parent genotypes. This study highlights the high prevalence of Arcobacter spp. in poultry meat in New Zealand. It also indicates prevalence of arcobacters in poultry carcass varies greatly with the choice of isolation method and none of the currently available methods are appropriate for the detection of all species of arcobacters in New Zealand. Therefore, two or more methods should be used in parallel. The level of contamination of poultry carcass may vary with the processing practices of a slaughterhouse. To eliminate or reduce arcobacters in retail poultry, maintenance of slaughter hygiene is of utmost importance. This may be achieved by regular microbiological monitoring of carcasses according to the HACCP principles. Further studies comparing the fingerprinting pattern of Arcobacter spp. isolates obtained from retails poultry with human isolates are necessary to test the hypothesis that poultry meal is an important source for Arcobacter infection in human
Comparison of DNA extraction efficiencies using various methods for the detection of genetically modified organisms (GMOs)
The ability to detect the presence of transgenes in crop-derived foods depends on the quantity and quality of DNA obtained from a product to be analyzed. The efficiency of DNA extraction protocols differs due to the nature of each food product. In this paper, we described two main DNA extraction protocols and their modifications that have been applied and evaluated for DNA extraction from raw and processed food as well as animal feed. The yield and quality for five categories of food and feed samples namely, raw soybean, raw maize, animal feed, smooth tofu and soymilk are discussed. The statistical interaction analyses showed that the cetyltrimethyl ammonium bromide (CTAB) method was proven to be the best method to extract DNA from raw soybean, maize and animal feed samples which not only obtained high DNA yield of 32.7, 28.4 and 33.4 ng DNA/mg sample respectively, but also produced high quality DNA with the absorbance A260/A280 ratio of 1.9, 1.9 and 2.0, respectively. These DNA were suitable for PCR amplification which produced a 164 bp DNA fragment of the lectin gene from soybean, and a 277 bp DNA fragment of the zein gene from maize. In the processed food category, the Wizard isolation method was found to be the best for the extraction of DNA from smooth tofu and soymilk with the yield of 13.2 and 3.4 ng DNA/mg sample, and the quality of the DNA at the absorbance A260/A280 ratio ranged from 1.9 to 1.7. These DNA were successfully amplified using primers specific to the lectin gene of soybean
- …