Accuracy of a real-time polymerase-chain-reaction assay for a quantitative estimation of genetically modified sources in food products

The accuracy of a real-time polymerase-chain-reaction assay for genetically modified sources in food products was determined using two official test systems (kits) of primers and samples. These kits were recommended by the Federal Center of State Sanitary and Epidemiological Surveillance (Russian Ministry of Health) and the European Commission. We used the following three models of thermocyclers: iCycler iQ (Bio-Rad, United States), Rotor-Gene 3000 (Corbett Research, Australia), and DT-322 (DNA-Technology, Russia). Studies of samples that contained 1% genetically modified sources showed that the error of a quantitative assay for genetically modified sources in food products corresponds to 20–30% and does not depend on the kit type and the thermocycler model used.     

Random amplified polymorphic DNA analysis of genetically modified organisms

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.     

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.     

Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.     

Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.     

Comparative evaluation of different DNA extraction procedures from food samples

Five methodologies for extracting DNA from food samples are described. The food products analyzed are from either soybean or maize. They were selected on the basis of the mechanical, thermal, and chemical treatments that they had been subjected to during industrial processing. DNA preparations were evaluated for purity, yield, and average fragment size. Two endogenous genes, soybean lectin gene and alcohol dehydrogenase gene (adh1), were used to assess the degree of DNA degradation at different stages of the transformation chain. The goal of this study was to determine the role that extraction methods play in DNA amplification in order to select the best protocol for a food sample. This comparative evaluation can be specifically useful for detection of genetically modified ingredients in a variety of food matrices.     

Effects of petroleum on mink applied as a model for reproductive success in sea otters.

Ranch-reared mink (Mustela vison) were used as a model in an experimental trial to investigate the potential effects of exposure to two petroleum products on sea otters (Enhydra lutris). Mink were exposed either dermally on one occasion 60 days prior to breeding or via low level contamination of their diets daily from 60 days prior to breeding (January 1994) until weaning of kits (June 1994). For dermal exposure, we placed mink in either a slick of Alaskan North Slope crude oil (n = 24) or bunker C fuel oil (n = 24) on sea water or sea water alone (n = 10) for 1 min. For dietary exposure, we fed mink rations containing 500 ppm of either Alaskan North Slope crude oil (n = 24) or bunker C fuel oil (n = 24; control, n = 15). The number of liveborn kits did not differ significantly among mink exposed dermally (5.0 kits/female for crude oil and 6.5 kits/female for bunker C fuel oil) and unexposed controls (5.3 kits/female). However, only 2.3 and 0.7 kits were produced per female for those exposed through the diet to crude oil and bunker C fuel oil, respectively. Females with reduced reproductive success had no clinical signs of toxicosis or behavioral abnormalities. In addition, kits of females exposed through the diet had poor survival to weaning. Once mature, kits born to females exposed to bunker C fuel oil in the diet had significantly reduced reproductive success (3.4 kits/female) although their only exposure to the petroleum products was in utero or during nursing. Therefore, it is possible that sea otter populations consuming contaminated food sources or colonizing previously oiled habitats will have reduced reproductive success.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Detection of genetically modified DNA in fresh and processed foods sold in Kuwait

Developments in genetic engineering technology have led to an increase in number of food products that contain genetically engineered crops in the global market. However, due to lack of scientific studies, the presence of genetically modified organisms (GMOs) in the Kuwaiti food market is currently ambiguous. Foods both for human and animal consumption are being imported from countries that are known to produce GM food. Therefore, an attempt has been made to screen foods sold in the Kuwaiti market to detect GMOs in the food. For this purpose, samples collected from various markets in Kuwait have been screened by SYBR green-based real time polymerase chain reaction (RT-PCR) method. Further confirmation and GMO quantification was performed by TaqMan-based RT-PCR. Results indicated that a significant number of food commodities sold in Kuwait were tested positive for the presence of GMO. Interestingly, certain processed foods were tested positive for more than one transgenic events showing complex nature of GMOs in food samples. Results of this study clearly indicate the need for well-defined legislations and regulations on the marketing of approved GM food and its labeling to protect consumer's rights.     

Methods of identification and assessment of safety of genetically modified microorganisms in manufacture food production

Methods of identification of genetically modified microorganisms (GMM), used in manufacture food on control probes are presented. Results of microbiological and molecular and genetic analyses of food products and their components important in microbiological and genetic expert examination of GMM in foods are considered. Examination of biosafety of GMM are indicated.     

Comparison of hepatitis B,core, HBc,and hepatitis B antibody,anti HBs,in a presumed low risk donor population

Donors screened by medical social history interview negative for high risk behavior or communicable disease history, but subsequently exhibiting reactive serological markers, emphasize importance of duel safe guarding factors for determining donor suitability. This report examines a relationship between two immunoabsorption assay tests, hepatitis B core (HBc) antibody, a required food and drug administration (FDA) test, and hepatitis B antibody (anti HBs), non-required test. Reactive serology results, 129 cases, 3,581 donors (2008–2012) for HBc as the only initially positive serological marker were subjected to anti HBs testing in this history pre-screened donor population. Enzyme linked immunoabsorption assay kits hepatitis B, core and antibody, were used in this study. All samples were initially tested for human immunodeficiency virus, hepatitis B, and hepatitis C, utilizing nucleic acid testing and antigen antibody immunoabsorption assay. Testing was performed by a FDA-registered CLEA-certified reference laboratory. Samples were deceased donor blood samples and a limited number of pre-mortem samples, separated, stored and analyzed according to manufacturer recommendation and FDA regulations. 129 reactive HBc only samples, were subsequently tested for anti HBs. Of these 129, 94 were found to be reactive for anti HBs. This represented 72 % of samples tested for antibody, a higher percentage than anticipated for a medical history negative, low risk population.     

玉米蛋白粉DNA提取方法比较研究

饲料样本本身的复杂性给进出口产品转基因（genetically modified,GM）成分的检测工作带来了巨大的压力和挑战。玉米蛋白粉主要包含蛋白质、淀粉和脂类等成分,从中提取高品质的DNA比较困难,而高品质的DNA是转基因检测研究的关键,能够大大降低进出口检验中的“假阴性”结果。采用市售主流品牌常用的7种试剂盒（Promega公司、Biotecon公司、天根生化科技有限公司、Invitrogen公司、Qiagen公司、TaKaRa公司）、CTAB法以及改良SDS?CTAB法提取玉米蛋白粉中的DNA。通过对玉米内源基因进行实时荧光PCR检测,发现改良SDS?CTAB法提取的玉米蛋白粉DNA质量明显优于其他方法,改良SDS?CTAB法内源基因zSSIIb的C_t值为28.14,较CTAB法提高了27%。研究建立的改良SDS?CTAB法在国内外尚属首次应用于饲料转基因产品中,并对7批次实验室送检样品进行转基因成分检测,成功检出2批次阳性样品。     

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS 200 . The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55°C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 μl of 1 mmol 1^-1 AttoPhos^TM (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1–10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR. assay described here can be useful to screen a large number of food samples for contamination by salmonellas.     

Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.     

Rapid and Economic DNA Extraction from a Single Salmon Egg for Real-Time PCR Amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

Monitoring the presence of genetically modified food on the market of the Republic of Croatia

From the beginning of the human race people have been applying different methods to change the genetic material of either plants or animals in order to increase their yield as well as to improve the quality and quantity of food. Genetically modified organism (GMO) means an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination. Analysing the presence of GMO in food is done by detecting the presence of either specific DNA sequences inserted in the genome of transgenic organism, or detecting proteins as a result of the expression of the inserted DNA. In this work food testing for the presence of genetically modified organisms was conducted during the period from 2004 to 2007 in the GMO laboratory of the Croatian National Institute of Public Health. According to the regulations, among the samples in which the presence of GMO was detected, all those which had more than 0.9% of GMO content were either rejected from the border or removed from the market, because such GM food has to be appropriately labelled. Among the food samples which were analysed in 2004: 127 (2.37%) of a total of 1226 samples contained more than 0.9% of GMOs; in 2005 there was only one in 512 (0.20%) samples in total; in 2006 there were 4 out of 404 samples (0.99%), and in 2007: 7 of a total of 655 samples (1.07%) had GMO content above the allowed threshold of 0.9%.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

PCR detection of genetically modified soya and maize in foodstuffs

The detection of genetically modified foodstuffs is becoming both a food sales and legal necessity. This study reports a rapid DNA extraction/PCR-based method for the detection of genetically modified soya (GMS) and maize (GMM) in mixed samples of transgenic and unmodified soybeans and maize kernels, and a variety of processed samples including soya flour, soya protein isolates, extruded defatted soya, acid- and alcohol-precipitated soya concentrates, soya lecithin, maize grits, seasoned corn puffs and salted corn chips. The presence of GMS DNA was determined with two pairs of primers directed towards different GMS target sequences and GMM by one primer pair. In addition, a multiplex PCR reaction which utilises an internal positive control was developed for both genetically modified organisms (GMOs). Results indicated that the methods are sensitive and specific enough to detect GMS down to a level of 0.01% dry weight in single-product PCRs and 0.1% in multiplex PCRs and GMM down to 0.001% dry weight in single-product PCRs and 0.01% in multiplex PCR. The methods are considered to represent a viable route for the commercial detection of GMS and GMM in foodstuffs.