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[The biological time].

Fabio Colonna's De glossopetris dissertatio is part of the biological time. Announces the dawn of a paleontology that understood nature as a closed and orderly system ruled by a perfect, natural codex. The Neapolitan intellectual climate, characteristic of the transit from XVIIth to XVIIIth century, converge in a process of institutionalization of science.

Biology↗

Ajoene, a garlic-derived natural compound, enhances chemotherapy-induced apoptosis in human myeloid leukaemia CD34-positive resistant cells.

UNLABELLED: The reputation of garlic as an effective remedy for tumours extends back to the Egyptian Codex Ebers of 1550 BC. Several garlic compounds, including allicin and its corresponding sulfide, inhibit the proliferation of several human malignant cells. Ajoene is a garlic-derived compound produced most efficiently from pure allicin and has the advantage of a greater chemical stability than allicin. Recently, ajoene was shown to inhibit proliferation and induce apoptosis of human leukaemia CD34-negative cells including HL-60, U937, HEL and OCIM-I. More significantly, ajoene was shown to induce 30% apoptosis in myeloblasts from a chronic myeloid leukaemia patient in blastic crisis. Acute myeloid leukaemia (AML) is a heterogeneous malignant disease in which disease progression at the level of CD34-positive cells has a major impact on resistance to chemotherapy and relapse. MATERIALS AND METHODS: The aim of the present study was to investigate the effect of ajoene on changes in the expression of apoptosis-related proteins: bcl-2 and caspase-3, induced by two principal drugs used in treatment of AML, cytarabine and fludarabine, in KGI human myeloid leukaemia CD34-positive-resistant cells. Both quantitative ELISA measurement of bcl-2 and colourimetric measurement of active caspase-3 were used. RESULTS: Quantitative ELISA measurement of bcl-2 (units per million cells) showed treatment of KG1-resistant leukaemia cells with 40 microM ajoene alone to significantly reduce the bcl-2-expression from 239.5 +/- 1.5 in control cultures to only 22.0 +/- 4.0 in ajoene-treated cultures. Fludarabine had significantly more inhibitory effect on bcl-2-expression than cytarabine in KGI-resistant myeloid leukaemia cells. Ajoene significantly enhanced the inhibitory effect of the two chemotherapeutic drugs, cytarabine and fludarabine, on bcl-2-expression in KGI cells. Bcl-2-expression could not be detected in fludarabine + ajoene-treated cultures. The Western blot of bcl-2-expression in KGI control and treated cells confirmed the quantitative ELISA measurements. Quantitative measurement of activated caspase-3 (pg per million cells) showed the two drugs, cytarabine and fludarabine, significantly increased the activated caspase-3 level in KGI myeloid leukaemia cells. CONCLUSION: The addition of ajoene enhanced the activation of caspase-3 in both cytarabine- and fludarabine-treated KGI cells. In conclusion, the present results suggest a potential role for the combination of ajoene with fludarabine-based chemotherapy in the treatment of refractory and/or relapsed AML patients. Further studies are warranted to evaluate a similar enhancing effect for ajoene in blast cells from AML patients in primary cultures.

Antigens, CD34↗

[Meat and potential risks].

By the term "meat" the consumer understands muscle meat, that is skeletal muscle. The german regulations, the "Leitsätze für Fleisch und Fleischerzeugnisse" as well as the Codex Alimentarius define meat in a much broader sense to include all edible parts of slaughtered or shot warm-blooded animals (skeletal muscles, fat, heart, gut, liver etc). Therefore, a differentiated procedure is required when the risk of BSE transmission by meat has to be estimated. This must be based on knowledge of occurrence, amount, and persistence of BSE agents in the organs of animals. The risk evaluation has to include further factors: differences between animal species, age of the animal at the time of slaughter, the possibility of contamination during the slaughtering and cutting process, and--as far as meat products are concerned--the processing technology. To date there are no indications for the existence of transmissible spongiform encephalopathy in pigs, poultry and fish, even though BSE has been transmitted to pigs experimentally by intracranial administration. Muscle meat of these species can be considered safe. Muscle meat of experimentally infected cattle in the preclinical stage and even of animals with clinically manifest BSE has not shown infectivity in homologous and autologous bioassays performed so far. This finding justifies the assumption that the risk of BSE exposure by the consumption of beef can be classified as extremely low. Nevertheless, the absence of a proof of infectivity can at present not be equated with absence of BSE agents. This is because of the limits of sensitivity of the bioassays and because muscle meat does contain non-muscle tissue such as connective tissue collagen, nerve and lymph tissue and blood vessels.

Age Factors↗

[The concept of illness and kidney diseases in Nahuatl medicine. Synthesis of Mesoamerican pre-Columbian medicine].

Medicine in Mesoamerican cultures began in the year 1,500 BC and ended with the conquest and destruction of Mexico-Tenochtitlan in 1521 by the Spanish conquerors. Mesoamerica began with the Olmec civilization followed by the Teotihuacans, Toltecs and Mayans, and perished during the Nahoa empire. The medicine used by the Aztecs (ticiotl) is undoubtedly the sum of all Mesoamerican medicine. The medical history of the ticiotl was recovered in the years that followed the conquest from the works of Bernardino de Sahagún, Francisco Hernández and the Cruz-Badiano codex. All these works described the use of plants and herbs in the treatment of diseases, including edema, urinary retention, kidney stones, and podagra. The Aztec doctors (Titici) were well acquainted with innumerable diseases and were excellent healers of wounds and fractures. The works of modern historians confirm the theory of the ticiotl medicine. The later used a complex and philosophically elaborated medical theory based on, religion, astronomy, divination and the polarity cold/warm; different from the four humor theory of galenic medicine. They demonstrated that every culture is capable of understanding and "inventing" the meaning of disease and its cure, even when it is different from our modern medical views.

Attitude to Health↗

[Jean Guillaume Auguste Lugol (1788-1851): his life and his works: a brief encounter, 150 years after his death].

J.G.A. Lugol was born on August 18th, 1788 at Montauban. Medical student in Paris, he was admitted as an intern of the hospital 1807. Medical doctor in 1812, he was appointed acting physician at Saint-Louis hospital of Paris in 1819 and named head of a department in the same establishment, a post he held till he retired in 1851. After his death on September 16th, 1851 at Neuilly-sur-Seine, his elder child, his daughter Adèle-Augustine, married Paul Broca in 1857. French pioneer of iodine therapy, Lugol is famous for his iodine-iodurretted solution, still registered in the French Codex and present in most foreign Pharmacopoeia, and also for his four books on scrofulous diseases and their treatment (1829, 1830, 1831, 1834) These publications gather a wealth of the detailed observations of an excellent practitioner who constantly proved a great independence of spirit towards some medical concepts "à la mode", especially those sustained by Broussais. He was very close to his patients and to his medical students, who admired the quality of his lessons, the efficiency of his therapeutic innovations, his intellectual uprightness as well as his success in private practice.

France↗

NTP Carcinogenesis Studies of Food Grade Geranyl Acetate (71% Geranyl Acetate, 29% Citronellyl Acetate) (CAS No. 105-87-3) in F344/N Rats and B6C3F1 Mice (Gavage Study).

Geranyl acetate (3,7-dimethyl-2,6-octadiene-1-ol acetate) is a colorless liquid prepared by fractional distillation of selected essential oils or by acetylation of geraniol. It is a natural constituent of more than 60 essential oils, including Ceylon citronella, palmarosa, lemon grass, petit grain, neroli bigarade, geranium, coriander, carrot, and sassafras. Geranyl acetate is used primarily as a component of perfumes for creams and soaps and as a flavoring ingredient. On the U.S. Food and Drug Administration's list of substances "generally recognized as safe," the Food Chemicals Codex (1972) specifies that geranyl acetate must contain at least 90% total esters. Carcinogenesis studies of food-grade geranyl acetate (containing approximately 29% citronellyl acetate) were conducted by administering the test chemical in corn oil by gavage to groups of 50 male and 50 female F344/N rats at doses of 1,000 or 2,000 mg/kg body weight and to groups of 50 male and 50 female B6C3F1 mice at doses of 500 or 1,000 mg/kg. Doses were administered five times per week for 103 weeks. Groups of 50 rats and 50 mice of each sex received corn oil by gavage on the same dosing schedule and served as vehicle controls. The cumulative toxicity of geranyl acetate in the 2-year study was indicated by the significantly shorter survival of high dose male rats (control, 34/50; low dose, 29/50; high dose, 18/50) and of high dose male mice (control, 31/50; low dose, 32/50; high dose, 0/50) and of dosed female mice (38/50; 15/50; 0/50) when compared with controls. Throughout most of the 2-year study, mean body weights of high dose rats and mice of each sex were lower than those of the controls. The occurrence of retinopathy or cataracts in the high dose male rats and low dose female rats as compared with the controls does not appear to be related to the administration of geranyl acetate but rather the proximity of the rats to fluorescent light. The incidence of retinopathy or cataracts (combined) was: males: control, 0/50, 0%; low dose, 1/50, 2%; high dose, 11/50, 22%; females: control, 1/50, 2%; low dose, 13/50, 26%; high dose, 2/50, 4%. Kidney tubular cell adenomas, an uncommon tumor type, were found in 2/50 (4%) low dose male rats. The historical incidence of male corn oil gavage control F344/N rats with kidney tumors is 1/250 (0.4%) at this laboratory and 4/998 (0.4%) in the program. Squamous cell papillomas in the skin were increased marginally in low dose male rats (control, 0/50; low dose, 4/50, 8%; high dose, 1/50, 2%). In addition, one low dose male rat had a squamous cell carcinoma of the skin. The incidence of low dose male rats with either squamous cell papillomas or carcinomas was greater (P<0.05) in comparison with the controls. The historical incidence of squamous cell papillomas or carcinomas (combined) in gavage control male F344/N rats is 3.6% (9/250) at this laboratory and 2.5% (25/999) throughout the program. The incidence of all epidermal tumors was not significantly elevated in dosed male rats relative to controls (control, 3/50, 6%; low dose, 6/50, 12%; high dose, 1/50, 2%). All high dose (1,000 mg/kg) male and female mice were dead by week 91 as a result of accidentally being administered 2,800 mg/kg for 3 days during week 91; survival of low dose and control male mice was comparable. Survival of high dose male and dosed female mice may have been inadequate for the detection of late-appearing tumors. No evidence of any carcinogenic effect was found in either low or high dose mice of either sex. An infection of the genital tract was probably responsible for the deaths of 14/22 control and 8/32 low dose female mice before the end of the study. Cytoplasmic vacuolization was increased in the liver and in the kidney of male and female mice and was considered to be compound related (liver-- male: control, 1/50, 2%; low dose, 7/50, 14%; high dose, 47/50, 94%; female: 1/50, 2%; 27/50, 54%; 46/50, 92%; kidney or kidney tubule--male: 0/50; 0/50; 41/50, 82%; female: 0/50; 24/49, 49%; 37/50, 74%). Under the conditions of these studies, geranyl 4&percnt;; 46/50, 92&percnt;; kidney or kidney tubule--male: 0/50; 0/50; 41/50, 82&percnt;; female: 0/50; 24/49, 49&percnt;; 37/50, 74&percnt;). Under the conditions of these studies, geranyl acetate was not carcinogenic for F344/N rats or B6C3F1 mice of either sex; however, the reduced survival observed in high dose male rats, high dose male mice, and high and low dose female mice lowered the sensitivity of these studies for detecting neoplastic responses in these groups. In male rats the marginal increases of squamous cell papillomas of the skin and tubular cell adenomas of the kidney may have been related to administration of geranyl acetate. Levels of Evidence of Carcinogenicity: Male Rats: Negative Female Rats: Negative Male Mice: Negative Female Mice: Negative

Journal Article↗

[Pattern of choice in preparation of attempted suicide by poisoning--with particular reference to changes in the pattern of prescriptions].

This investigation was initiated in 1990 to investigate the choice of preparation in cases of attempted suicide treated in the Emergency Unit in Odense University Hospital during the past decade. The results show that 1) an increase has occurred particularly among young women in the use of analgesics obtainable without prescription (e.g. 13% of young women employed paracetamol as the toxic agent), 2) the number of persons employed dextropropoxyphene did not decrease and 3) a great increase was observed in the number of cases of poisoning with ketobemodoni chloridum. In 1985, several barbiturates were withdrawn from the Danish Medical Codex and this was followed by a decrease in the number of cases of poisoning among women but not among men. At present, approximately 10% of men who poison themselves still employ barbiturates. The number of cases of poisoning with barbiturates in men is thus not reduced but phenobarbital is the preparation of choice. The authors conclude that more attention and care should be paid when prescribing dextropropoxyphene, ketobemodoni chloridum and phenobarbital and the availability of paracetamol without prescription must be debated critically.

Adult↗

[Celiac sprue (review)].

Celiac sprue may be defined as a model autoimmune disease with known trigger (gluten), a tight genetic linkage (with HLA-DQ2 and HLA-DQ8) and a specific humoral autoimmune response (autoantibodies to tissue transglutaminase, tTG). Gliadin peptides are repeatedly presented to HLA-DQ2 and HLA-DQ8 positive cells and induce an immune response in small-intestinal mucosa. tTG is a specific endomysial autoantigen released during cellular stress and by deamidation of gliadin peptides as well as by binding with them facilitates their interaction with HLA-DQ2 and HLA-DQ8 cells. CS is the consequence of an inappropriate by T-cells mediated immune reaction to gluten. The diagnosis is based on criteria of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition revised in 1990. The availability of sensitive and specific detection methods of serum antibodies to endomysium (AEA) and tTG (AtTGA) was followed by recognition of a broad spectrum of both the clinical presentation and histologic changes of intestinal mucosa in CS. The majority of patients have atypical clinical symptoms. The present prevalence of CS amounts to approximately 1:250. The following forms of CS are distinguished: classic (typical), latent, potential, subclinical, and silent. CS is frequently associated with other diseases and many of them are also of autoimmune origin. Serologic testing is indicated in subjects with atypical symptoms and autoimmune diseases. In cases with distinct suspicion biopsy should be always performed irrespective of the serologic tests. In refractory sprue the cryptic form of enteritis associated T-cell lymphoma should be excluded. Gluten-free diet is the cornerstone of CS therapy. The Alimentary Codex in individual countries admits different amounts of residual gluten in gluten-free products. In future years new basic knowledge as well as practical diagnostic and therapeutic recommendations may be expected.

Celiac Disease↗

[Books and medical knowledge in the ancient world].

Books have been used by medici in Antiquity - different kind of books for different kind of medici. Galen is the best example of a medicus with a strong interest not only in theoretical medicine, but also in a material crafting of the books, as well as in the making of the text. But he is an exception. Books in late Antiquity came in two different formats: the codex and the rotulus. The former was to be the one to survive: by the late IV century A.D., rotuli - difficult to handle and to read - had virtually disappeared. Codices were diffused in medical milieux, as well as in other milieux where culture was popularized (e.g. Christianity), probably because they were more user-friendly and manageable even for a non-cultivated public. Codices were used for practical purposes and for practical therapeutics, and allowed the reader to write on margins, thus enhancing their practical usefulness. On the contrary, books had a scanty use for didactic purposes, learning from the voice of the magister being the privileged form of transmission.

Books↗

Source of a micro-nutrient in a semi-synthetic basal diet as a causative factor in inducing urinary calculi in rats and its inhibition by PSC 833, a potent inhibitor of P-glycoprotein.

We report a serendipitous finding of urinary calculi in rats fed a semi-synthetic basal diet. This observation was made during ongoing studies to evaluate the inhibitory effect of PSC 833, a potent inhibitor of P-glycoprotein, on development of tumors in rodent tumor model systems. A large number of specific-pathogen-free (SPF) female Sprague-Dawley and SPF male Fischer 344 rats being fed the diet were euthanized when it became evident clinically that they were uremic. At necropsy, the renal pelvis, ureters, and urinary bladder contained numerous calculi. The presence of urinary calculi was determined to be related to the source of a Food Chemical Codex grade of choline bitartrate. Rats being fed the same basal diet containing the United States Pharmacopia grade of choline bitartrate failed to develop urinary calculi. Interestingly, rats treated with the P-glycoprotein inhibitor were at significantly reduced risk of developing urinary calculi. This finding highlights how something seemingly innocuous as a minor dietary constituent can have a profound impact and, thereby, affect experimental outcome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Photostimulated luminescence detection of irradiated shellfish: international interlaboratory trial.

An interlaboratory trial was conducted to validate photostimulated luminescence (PSL) detection of irradiated shellfish. Five species of shellfish (Nephrops norvegicus, mussels, black tiger prawns, brown shrimps, and king scallops) were presented blind as nonirradiated and irradiated to 0.5 and 2.5 kGy. Precharacterization analysis of each product and treatment was performed on both whole (including shell) and intestinal samples. The results for whole samples (including shell) confirmed that the method was able to distinguish between nonirradiated and irradiated samples, regardless of dose. Intestinal data have identified that the method is dependent on the quantity and sensitivity of grits present within the intestinal tract, which can be assessed using calibration by normalization to 1 kGy. Five laboratories returned both initial screening and calibrated data and sample classification. All laboratories correctly identified all irradiated products using the screening criteria. There were no false positives. The results confirm the validity of the PSL method for shellfish, which has been adopted as a European standard method and by the Codex Alimentarius Commission. Calibration is required where only intestinal material is available. For whole samples with shell, screening alone is sufficient.

Calibration↗

Photostimulated luminescence detection of irradiated herbs, spices, and seasonings: international interlaboratory trial.

An interlaboratory trial was conducted to validate photostimulated luminescence (PSL) methods for herbs, spices, and seasonings. Forty products (11 herbs, 17 spices, and 12 seasonings) were purchased from a local commercial source, and randomly selected samples were irradiated with 10 kGy. Four blended products were prepared at Scottish Universities Research and Reactor Centre, mixing varying proportions of irradiated material with the untreated product. Precharacterization against a predefined threshold identified low sensitivity products (black and white peppers) and products with high natural signals (thyme, sage, parsley, and mixed herbs), both of which might be susceptible to misclassification. Precharacterization also revealed whether calibration was likely to resolve overlap between classification categories. Eight sets of screening data and 5 sets of calibrated data were returned by participants. Of the 840 samples sent, 1593 screening measurements and 788 calibrated measurements were received from 662 samples. In screening mode, participants reached definitive conclusions in 87% of cases, 99% of which were correct. Of the remaining 13%, calibration to identify low-sensitivity resolved 60% of cases. Overall, 94% of samples were correctly identified by either screening alone, or screening plus calibration; 6% remained unclassified and therefore required further investigation by thermoluminescence. The results confirm the validity of the PSL method for herbs, spices, seasonings, and blends, and emphasize the need for calibration to identify low-sensitivity samples. This method has now been adopted by the European Committee for Standardization (CEN) and the Codex Alimentarius Commission.

Calibration↗

[Estimation of the intake of food additives--methods applied in the European Union].

Guidelines for estimation of food additives intake were prepared by Codex Committee on Food Additives and Contaminants (CCFAC) in 1989. During the period 1994, 1995 three specific directives have been adopted, each of them including an obligation for Member States to introduce systems to monitor the usage and consumption of food additives and to report their findings to the Commission. Recommendations of Commission's experts which should be taken into account for preparing the monitoring system by Member States are based on decision tree, which systematizes proceeding procedures. Above mentioned decision tree might be a tool for elimination of the food additive which intake does not pose a health hazard. ADI (Acceptable Daily Intake) plays the essential role in the risk assessment of food additives intake. The food additives, which were not eliminated during the decision tree considerations require detailed revision of the conditions of their use. Poland, as a Candidate Country for EU, after the accession will also be obligated to introduce the system to monitor the usage and consumption of food additives and to report relevant report to the Commission.

Decision Trees↗

[Design of a Hazard Analysis and Critical Control Points (HACCP) plan to assure the safety of a bologna product produced by a meat processing plant].

The Hazard Analysis and Critical Control Point (HACCP) is a systematic integral program used to identify and estimate the hazards (microbiological, chemical and physical) and the risks generated during the primary production, processing, storage, distribution, expense and consumption of foods. To establish a program of HACCP has advantages, being some of them: to emphasize more in the prevention than in the detection, to diminish the costs, to minimize the risk of manufacturing faulty products, to allow bigger trust to the management, to strengthen the national and international competitiveness, among others. The present work is a proposal based on the design of an HACCP program to guarantee the safety of the Bologna Special Type elaborated by a meat products industry, through the determination of hazards (microbiological, chemical or physical), the identification of critical control points (CCP), the establishment of critical limits, plan corrective actions and the establishment of documentation and verification procedures. The used methodology was based in the application of the seven basic principles settled down by the Codex Alimentarius, obtaining the design of this program. In view of the fact that recently the meat products are linked with pathogens like E. coli O157:H7 and Listeria monocytogenes, these were contemplated as microbiological hazard for the establishment of the HACCP plan whose application will guarantee the obtaining of a safe product.

Food Contamination↗

[Risk assessment of foodborne infections].

The microbiological risk assessment during production, processing and treatment of foods illustrates an important basis for the judgement of the safety of food products. Since the mid-nineties the concept of risk analysis according to Codex Alimentarius requirements ist pursued more intensely. Risk assessment is part of the risk analysis process besides risk management and risk communication. A strict separation between risk management and assessment should lead to a systematic, scientifically based and independent process without considering economic or political constraints whereas the decision on protective measures or the acceptance of risks lies in the responsibility of the managers. Risk assessment can only be successfully implemented in an interdisciplinary approach between physicians, veterinarians, microbiologists, molecularbiologists, food technologists, epidemiologists and mathematicians. Surveillance, monitoring programs and other data collections on a variety of parameters like statistics on foodborne human cases, the prevalence of zoonotic agents in animals, the distribution of micro-organisms in the environment and in foods, the behaviour of micro-organisms during food processing and the consumption habits of consumers deliver the necessary data for risk assessors. With the aim of mathematic modelling and simulation it is possible to calculate the probability of health problems in humans after the consumption of a foodstuff contaminated with a specific pathogenic micro-organism.

Animals↗

International trade in livestock and livestock products: the need for a commodity-based approach.

International animal health standards designed to facilitate safe trade in livestock and livestock products are set by the Office International des Epizooties (OIE) under the Sanitary and Phytosanitary Agreement of the World Trade Organization (WTO) and documented in the OIE's Terrestrial Animal Health Code. A core principle of the Code is the need for countries to eradicate important transboundary animal diseases (TADs) to reduce the risk of exporting disease to trading partners. International food safety standards are set by the Codex Alimentarius Commission, administered jointly by the World Health Organization and the Food and Agriculture Organization of the United Nations. The goal of global eradication of most TADs is unachievable for the foreseeable future, other than in the case of rinderpest, and this prevents many countries, especially developing nations, from engaging in international trade under WTO rules. This paper proposes an alternative, commodity-based approach to the formulation of international animal health and food safety standards, based on the fact that different commodities pose very different risks when it comes to the spread of human and animal pathogens. Therefore, the risk mitigation strategies required are equally commodity-dependent. The authors conclude that more focused commodity standards would improve access to international markets for all countries, especially those in the developing world. For this objective to be realised, credible and independent certification is required.

Animal Diseases↗

Evaluation of certain veterinary drug residues in food. Sixty-second report of the Joint FAO/WHO Expert Committee on food additives.

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues in food. The first part of the report considers conclusions on specific toxicological end-points, lipid-soluble residues of veterinary drugs with MRLs in milk, statistical methods for the estimation of MRLs, and the Committee's review and comments on documents provided by Codex Committees. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: five antibacterial agents (cefuroxime, chloramphenicol, flumequine, lincomycin, pirlimycin), four insecticides (cyhalothrin, cypermethrin and alpha-cypermethrin, doramectin, phoxim), and two production aids (melengestrol acetate, ractopamine). The Committee's comments on chloramphenicol found at low levels in animal products are also summarized. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes and proposed maximum residue limits.

Animals↗

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

Base Sequence↗