Cloth-based hybridization array system for expanded identification of the animal species origin of derived materials in feeds

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.     

Cloth-based hybridization array system for the detection of Clostridium botulinum type A, B, E, and F neurotoxin genes

A simple cloth-based hybridization array system was developed for the characterization of Clostridium botulinum isolates based on the botulinum neurotoxin serotype. Bacterial isolates were subjected to a multiplex PCR incorporating digoxigenin-dUTP and primers targeting the four botulinum neurotoxin gene serotypes (A, B, E, and F) predominantly involved in human illness, followed by hybridization of the amplicons with an array of toxin gene-specific oligonucleotide probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound digoxigenin label. This system provided sensitive and specific detection of the different botulinum neurotoxin gene markers in a variety of C. botulinum strains, exhibiting the expected patterns of reactivity with a panel of target and nontarget organisms.     

Development of primers for detection of meat and bone meal in ruminant feed and identification of the animal of origin

The safe use of cattle feed free from meat and bone meal is an important prerequisite to prevent further spread of bovine spongiform encephalopathy. We designed primers to detect very small amounts of meat and bone meal in ruminant feed. Mitochondrial subunit 8 of the ATP synthase gene was used as a target sequence. PCR-based assays revealed amplification of DNA from mammals, ruminants, and individual species using these primers. The method allowed detection of the presence of meat and bone meal in ruminant feed from 0.1 to 0.01%. Sensitivity and effectiveness of the method for detecting prohibited animal proteins in ruminant feed was evaluated.     

反刍动物饲料中农药残留研究进展

农药在农业和畜牧业生产中作为预防和治疗农作物病虫草害和料仓中卫生害虫等有害生物侵害的重要投入品,在保障农作物质优、高产及保护饲料贮存环境卫生等方面发挥着突出作用。由于其广泛应用,不可避免的残留在饲料当中。然而,反刍动物的主要饲料原料来源于农作物,致使农药进入体内的可能性增加。使用含有农药残留的饲料,可能降低动物的生长性能,严重者可危害动物健康甚至导致动物死亡,且残留在动物产品中的农药可能危害人类健康。饲料中农药残留问题应引起社会各界的关注和高度重视。本文针对反刍动物饲料原料种植过程中的使用情况,综述了饲料中农药残留情况、代谢情况及对人畜健康安全的影响,对反刍动物饲料中农药残留情况的摸底调查和风险评估具有重要的现实意义。     

Species-specific PCR for the identification of ruminant species in feedstuffs

A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA mitochondrial gene has been developed for the specific identification of bovine, ovine and caprine DNAs in feedstuffs. The primers designed generated specific fragments of 84, 121 and 122pb length for bovine, ovine and caprine species, respectively. The specificity of the primers designed was tested against 30 animal species including mammals, birds and fish, as well as eight plant species. Analysis of experimental feedstuffs demonstrated that 0.1% of raw and heated bovine, ovine or caprine tissues can be easily detected using the species-specific primers developed. The performance of this method is not affected by prolonged heat treatment, and consequently it could be very useful to verify the origin of the raw materials in products submitted to denaturing technologies, for which other methods cannot be applied.     

PCR-RFLP identification of prohibited animal derived DNA in animal feed

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.     

Development of a PCR assay for the detection of animal tissues in ruminant feeds

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.     

PCR assay for the identification of animal species in cooked sausages

A species-specific PCR assay was developed for the detection of low levels of pork, horse and donkey meat in cooked sausages. Oligonucleotid primers were designed for amplification of species-specific mitochondrial DNA sequences of each species and detected the presence of 0.01 ng of template DNA in water. When applying the assay to DNA extracts from sausages samples that were prepared from binary meat mixtures, it was possible to detect each species when spiked in any other species at the 0.1% level. In conclusion, it can be suggested that this assay can be used to determine mislabelled and/or fraudulent species substitution in comminuted meat products.     

Evaluation of two commercial lateral-flow test kits for detection of animal proteins in animal feed

Performance characteristics were evaluated for two lateral-flow test kits, Reveal for Ruminant in Feed (Neogen Corporation) and FeedChek (Strategic Diagnostics Inc.), designed to detect ruminant or terrestrial animal proteins in feeds. The stringent acceptance criteria used were developed by the Center for Veterinary Medicine Office of Research to identify test kits with comparable selectivity and sensitivity to microscopy and PCR assay, the analytical methods used by the U.S. Food and Drug Administration (FDA). Guidelines were developed for evaluating the selectivity, sensitivity, ruggedness, and specificity of these kits. These guidelines further stated that ruggedness and specificity testing would be performed only after a test passed both the selectivity and sensitivity assessments. Acceptance criteria for determining success were developed using a statistical approach requiring 90% probability of achieving the correct response, within a 95% confidence interval. A minimum detection level of 0.1% bovine meat and bone meal, consistent with the sensitivity of the methods used by the FDA, was required. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of the same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% bovine meat and bone meal. The Reveal test passed the selectivity assessment but failed the sensitivity assessment, detecting only samples fortified at the 2% level and then only 17 to 33% of those samples, when read according to the label directions. The FeedChek test passed the sensitivity assessment but failed the selectivity assessment, with rates for false-positive results ranging from 34 to 38%, depending on the user. The sensitivity of the Reveal test was affected by the concentration of trace minerals present in the feed; concentrations toward the high end of the normal range prevented the detection of true positive feed samples containing bovine meat and bone meal. Better sensitivity assessments were obtained when lamb meal was used either alone or in combination with bovine meat and bone meal. The FeedChek test was not affected by the concentration of trace minerals or by the type of animal meal used. These results indicate that neither of the two tests is adequate for routine regulatory use.     

Assessment of two enzyme-linked immunosorbent assay tests marketed for detection of ruminant proteins in finished feed

The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

Development of immunoassay for detection of meat and bone meal in animal feed

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Dairy products for the animal feed markets

The uses of non-fat milk products in the feeding of calves and pigs are summarized, and it is shown that the feeding of calves in particular is an important outlet for milk solids. However, the effects of quotas will reduce the amount of milk solids available, and adjustments will have to be made in consequence. It is suggested that the feeding of liquid skimmed milk to pigs may have to be phased out and supplies for calf feeding maintained by phased withdrawal of stocks from intervention.     

食品中沙门氏菌分离、鉴别方法的比较研究

目的筛选用于分离、鉴别食物样本沙门氏菌的适宜方法。方法比较了3种沙门氏菌显色培养基与亚硫酸铋(BS)琼脂、HE琼脂和木糖赖氨酸脱氧胆盐(XLD)琼脂传统培养基的检测敏感性、特异性、准确性和食品样本的适用性。结果 3种沙门氏菌显色培养基对杂菌的抑制效果不同,但检测结果直观,检测灵敏度高;BS琼脂方法分离沙门氏菌特异性强;HE琼脂及XLD琼脂分离沙门氏菌,当样品污染菌数量多时,鉴别结果易受影响。结论建议使用传统培养基分离、鉴别沙门氏菌时,补充使用一种抑制杂菌效果好的沙门氏菌显色培养基,以提高沙门氏菌的检测效率。     

Optimization of multiplex PCR for the identification of animal species using mitochondrial genes in sausages

Detection of species fraud in meat products is very important in order to protect consumers from undesirable adulteration, as well as for the economic, religious and health aspects. The most important reason for verification of the labeling statements is to detect fraudulent substitution of expensive meat components with other cheaper animals or mislabeling. The aim of this study was to develop a multiplex PCR that could be used in the simultaneous identification of multiple meat species. In this study, ten sausages with a minimum beef content of 55 %, from ten different manufacturing companies, and five samples of cow, chicken, goat, camel and donkey raw meats, for the purpose of positive control, were collected from food markets in Tehran, Iran. Total DNA was extracted from each sausage and the raw meats. Primers were selected in different regions of mitochondrial DNA (12S rRNA, cytochrome b and NADH dehydrogenase subunits 2) for identification of meat species. 12S rRNA and NADH dehydrogenase subunits 2 primers generated specific fragments of 183 and 145 bp length, for chicken and donkey, respectively. Three different specific primers were used for amplification of cytochrome b gene in goat, camel and cattle species and amplified species-specific DNA fragments of 157, 200 and 274 bp, respectively. The results proved that half of the specimens were contaminated with chicken meat, and this was greater than the proportion of beef stated on the label, while the other half only had chicken residuals, and no beef content. No contamination was found with goat, donkey or camel meats. These findings showed that molecular methods, such as multiplex PCR, is a potentially reliable, sensitive and accurate assay for the detection of adulterated meat species in mixed meat products.     

PCR-based fingerprinting techniques for rapid detection of animal species in meat products

A reproducible, rapid, and simple method for simultaneous identification of multiple meat species in a single step DNA-based test has been developed based on the generation of species-specific fingerprintings by two different arbitrary DNA amplification approaches (RAPD- and AP-PCR). Samples representative of various species and meat products submitted to different processing conditions were selected to verify the applicability of the techniques. RAPD-PCR fingerprintings allowed the discrimination amongst pork, beef, lamb, chicken and turkey in all cases. Samples corresponding to each species were clustered together at similarity levels ?75%. The DNA profiles consisted of a discrete but reproducible number of bands, which made possible the interpretation of the results by simple visual inspection. AP-PCR also allowed identification of the five tested species in every sample although more complex patterns were generated, including some low intensity bands. In both cases, a ramp time between annealing and extension temperatures was introduced to achieve good reproducibility. Overall, the simplicity of RAPD-PCR patterns could make this technique suitable for meat authentication in routine analysis.