Fluorescence-based method, exploiting lipofuscin, for real-time detection of central nervous system tissues on bovine carcasses

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.     

Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control

Spreading transmissible spongiform encephalopathies (TSE) have been widely attributed to transmission by ingestion of mammalian central nervous system (CNS) tissue. Reliable exclusion of this epidemiological important route of transmission relies on an effective surveillance of food contamination. Here, myelin proteolipid protein (PLP) is identified as a specific and largely heat-resistant marker for detection of food contaminations by CNS tissue. PLP is a component of oligodendritic glial sheaths of neuronal processes that is specifically expressed in the CNS. A highly selective polyclonal antibody was developed directed against an epitope present in the full-length PLP protein, but absent from the developmentally regulated splice variant DM-20. In combination with a hydrophobic extraction of PLP from tissue samples, the antibody reliably detected PLP from spinal cord, cerebellum, and cortex of different mammalian species. Consistent with earlier reports on PLP expression, no cross-reactivity was observed with peripheral nerve or extraneural tissue, except for a very faint signal obtained with heart. When applied to an artificial CNS contamination present in sausages, the antibody reliably detected a low concentration (1%) of the contaminant. Application of heat, as used during conventional sausage manufacturing, led to a predominant alteration of arginine residues in the PLP protein and a partial loss of immunoreactivity. In contrast, a stretch of hydrophilic amino acids(112-122) proved to be heat-resistant, preserving the immunogenicity of this PLP epitope during heating. Taken together, the excellent CNS specificity of PLP immunodetection and the presence of a heat-resistant epitope have permitted the development of a highly sensitive immunoassay for CNS contamination in routine food control.     

Detection of beef and poultry by serological field screening tests (ORBIT and PROFIT): collaborative study

Ten laboratories each analyzed 30 raw meat and raw meat product samples in a collaborative study of the ORBIT (overnight rapid bovine identification test) and PROFIT (poultry rapid overnight field identification test) serological field screening tests for the detection of beef and poultry. Versatility of the tests was shown in the analysis of whole tissue, ground, or emulsified raw meat products. Both tests were demonstrated to be reliable and were capable of detecting adulterants present at the 10% level. The method has been adopted official first action.     

牛肉中疯牛病特殊风险物质荧光RT-PCR检测方法的建立

本研究建立了检测牛肉中疯牛病特殊风险物质（主要是中枢神经组织）标记物GFAP mRNA的荧光RT-PCR检测技术。试验结果表明：该技术具有良好的种属特异性和组织特异性，只能从牛源和羊源的中枢神经组织中检测到GFAP，猪源和禽源检测结果为阴性，而且只有脑和脊髓等中枢神经组织产生阳性反应，其他内脏组织以及不同部位牛肉检测结果均为阴性；敏感性检测结果表明，该方法最低检测限达到0.001%以下；稳定性试验结果表明，100°C 加热处理30min对检测结果无明显影响，中枢神经组织在30°C以上室温可以稳定存放4天， 在4°C可以稳定冷藏2周，检测结果仍然为阳性。该结果显示，所建立的荧光RT-PCR技术用于牛肉中疯牛病特殊风险物质的检测具有特异性强、敏感性高、稳定性好及快速方便等优点，适合于在日常检测工作中推广使用。     

Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed

The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Novel method for detecting bovine immunoglobulin G in dried porcine plasma as an indicator of bovine plasma contamination

Current U.S. Food and Drug Administration regulations prohibit feeding of protein derived from mammalian tissue, excluding blood and blood products and any product that consists entirely of porcine or equine protein. A novel lateral flow immunoassay device has been developed that can quickly and qualitatively determine the presence of bovine immunoglobulin G (IgG), a major component in blood products, at very low levels (0.01% v/v). The device can be used to test for bovine IgG commingling in spray-dried porcine plasma used in the feed industry. Producers and consumers alike could use this device to verify product content at threshold levels.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Bacteriological and genetic assessment of game meat from Japanese wild boars

Bacterial tests were used to assess bacterial contamination of game meat from Japanese wild boars. The bacterial contamination of wild boar meat was less than that of domestic pork, as determined by aerobic plate counts (APC) and coliform counts. None of the meat examined in this study was contaminated by Salmonella or E. coli O-157. To detect adulteration by domestic pig meat or European wild boar meat, 46 samples of game meat sold as Japanese wild boar were examined genetically. A total of 17 samples showed genetic haplotypes of European and Asian domestic pigs in the D-loop of mitochondrial DNA (mtDNA), and 16 samples showed nuclear glucosephosphate isomerase-processed pseudogene (GPIP) genotypes of European domestic pigs. The European GPIP genotypes of these samples were confirmed by PCR-RFLP analysis. These results indicate that some game meat sold as Japanese wild boar is adulterated by cross-breeding between pigs and wild boars or by contamination with meat from domestic pigs or European wild boars.     

Development of poultry rapid overnight field identification test (PROFIT)

A poultry rapid overnight field identification test (PROFIT) has been developed as a screening test which is practical, economical, and easy to perform and interpret for use in field environments to determine the presence of poultry tissue (chicken and turkey) in raw whole tissue or ground/formulated meat products. The basis of the test is an agar-gel immunodiffusion technique used with a printed template pattern and stabilized reagent paper discs. The test shows adequate sensitivity and specificity for its intended purpose. Key components are stable for at least 1 year if they are stored at refrigerator conditions. The design of the test is such that it can be made commercially available as a complete, stable, test kit suitable for use by any type of inspection program concerned with verification of poultry species in meat and/or poultry products that are subject to regulatory or quality controls.     

Comparison of virus recovery techniques for assessing the virucidal activity of disinfectant spray products.

A comparison was made of virus recovery from inoculated glass surfaces by 3 different viral recovery techniques used in spray test methods. On a total of 7, 5, and 5 observations, for the cover glass crushing, cotton swab rubbing, and microscopic slide shaking techniques, respectively, the log reduction of the median tissue culture infective dose (log TCID-50/ml) averaged 1.48, 2.57, and 2.22, the mean percentage recovery was 77.98, 61.55, and 66.94%, and the coefficient of variation was 4.37, 10.01, and 7.42%. The cover glass technique was found to be the least variable and the most effective and precise of the 3 methods.     

Evaluation of microbial indicators for the determination of bacterial groundwater contamination sources

Several different Microbial source tracking methods (MSTs) can be used to distinguish human from animal fecal contamination in water; In this study, experiments were carried out to test the effectiveness and reliability of three bacteria based approaches (the fecal coliforms to fecal streptococci ratio, antibiotic-resistant Clostridium perfringens, and human bifidobacteria) in a simulated groundwater micro-environment. The methods were evaluated in three phases: initially, the specificity of each indicator was validated on groundwater samples affected by known pollution source; then the variation of performance with time of each method was determined, and finally, the die-off coefficients for pure species of Clostridium perfringens and Bifidobacterium adolescentis were measured. The results indicate that only the determination of human bifidobacteria concentration can be considered reliable in distinguishing human from animal pollution in groundwater at the conditions applied. Nevertheless, human bifidobacteria were detectable only for two weeks after the contamination event. This study also shows that antibiotic resistant Clostridium perfringens detected using the Shahidi-Ferguson medium is not source specific, whereas it confirms that this species can be useful for timing general fecal contamination events.     

Supercritical fluid extraction/enzyme assay: a novel technique to screen for pesticide residues in meat products.

The novel combination of supercritical fluid extraction (SFE) with an enzyme assay system has been used to screen meat products to detect the presence of pesticides. Analytes are collected in water by expanding supercritical carbon dioxide to atmospheric pressure through a restrictor and into an aqueous phase. The solution is then tested for the presence of pesticide residues by enzyme assay. Two experimental approaches have been used. Alachlor-fortified lard and bovine liver were monitored by static SFE coupled with an enzyme immunoassay. SFE of carbofuran-fortified frankfurters was coupled with an enzyme assay based on cholinesterase inhibition. A major benefit of the SFE/enzyme assay technique over conventional screening techniques is that the analyst is not exposed to organic solvents.     

Effect of glutathione on oxymyoglobin oxidation

The oxidation of oxymyoglobin (OxyMb) to metmyoglobin (MetMb) is responsible for fresh meat discoloration. Glutathione (GSH) is an important tripeptide reductant that can protect lipid and protein from oxidation. The objective of this research was to investigate the effect of GSH on MetMb formation in vitro and in bovine skeletal muscle cytosol. Equine MetMb formation was greater in the presence of GSH than controls at pH 5.6 or 7.2 and 25 or 37 degrees C (p < 0.05); GSH addition to purified bovine OxyMb solution also resulted in more MetMb formation at pH 7.2 and 25 or 37 degrees C (p < 0.05). This effect on MetMb formation was partly or completely inhibited by EDTA or catalase in the GSH-equine OxyMb system (p < 0.05). The addition of GSH to bovine muscle cytosol inhibited MetMb formation at pH 5.6 or 7.2 and 4 or 25 degrees C (p < 0.05); the effect was concentration-dependent. The inhibitory effect was observed in a high molecular weight (HMW) but not low molecular weight fraction of cytosol at pH 7.2 and 25 degrees C (p < 0.05); there was no effect when HMW was heated at 90 degrees C for 15 min. These results suggest the antioxidant effect of GSH on bovine OxyMb is dependent on heat-sensitive HMW cytosolic component(s).     

基于骨蛋白拉曼光谱特异性的肉骨粉种属鉴别方法

为了快速检测肉骨粉的种属来源,该研究开发了一种简便、可靠、科学、高效的肉骨粉种属鉴别方法。以87个肉骨粉(猪,鸡,牛和羊肉骨粉)为研究对象,利用拉曼光谱技术,结合化学计量学方法,建立了基于骨蛋白拉曼光谱特性的肉骨粉种属鉴别分析方法与模型。研究结果表明:根据偏最小二乘判别分析(PartialLeastSquaresDiscriminant Analysis,PLS-DA)模型,发现鸡和哺乳动物(猪,牛和羊)肉骨粉主要在1 659、2 930、2 852、1 246和1 455 cm-1附近的特征谱带具有差异性;猪和反刍动物(牛和羊)肉骨粉主要是在2 852、1 659和1 246 cm-1附近的特征谱带具有差异性;牛和羊肉骨粉主要是在878、853、2 930、2 852、1 246、1 455和1 659 cm-1附近的特征谱带具有差异性,并且PLS-DA模型鉴别肉骨粉的灵敏度和特异度均大于93.8%。研究结果可以丰富肉骨粉种属鉴别方法体系以及为开发便携式拉曼光谱仪提供参考。     

Establishment and application of a fluorescent polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying porcine,caprine, and bovine meats

A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the species-specific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy.     

Monitoring of denaturation processes in aged beef loin by Fourier transform infrared microspectroscopy

We present the results of a Fourier transform infrared (FT-IR) microspectroscopic study using conventional FT-IR microscopy and FT-IR imaging to detect the denaturation process during four different heating temperatures (raw, 45, 60, and 70 degrees C) spatially resolved in bovine cryosections from longissimus dorsi muscle. FT-IR imaging, employing a focal plane array detector, which allowed the simultaneous collection of spectra at 4096 pixels, enabled the investigation of the heat-induced changes in the two major meat constituents, i.e., myofibrillar and connective tissue proteins, spatially resolved. The infrared spectra of both compounds revealed that the major spectral changes involved an increase in beta-sheet and a decrease in alpha-helical structures, which appeared to be much more pronounced for the myofibers than for the connective tissue. These conformational changes could be correlated to the denaturation of the major meat proteins, such as myosin, actin, and collagen.     

Genotoxicity of glycidamide in comparison to 3-N-nitroso-oxazolidin-2-one

Acrylamide (AA) is generated by thermal processing of foods, depending on processing conditions and precursor availability. AA is not genotoxic by itself but becomes activated to its genotoxic metabolite glycidamide (GA) via epoxidation, mediated primarily by cytochrome P450 2E1. In the Comet assay in V79 cells and in human lymphocytes, GA induced DNA damage down to 300 microM concentration (4 h). After post-treatment with the DNA repair enzyme formamidopyrimidine-DNA-glycosylase (FPG), DNA damage became already detectable at 10 microM (4 h). By comparison, the N-nitroso compound 3- N-nitroso-oxazolidin-2-one (NOZ-2) is a much stronger genotoxic agent, significantly inducing DNA damage already at 15 min (3 microM). Post-treatment with FPG in this case did not enhance response. GA induced DNA damage in V79 cells rather slowly, with first response detectable at 4 h. The hPRT forward mutation test encompasses 5 days of expression time during which also repair can take place. GA-induced hPRT mutations only became detectable at concentrations of 800 microM and above. This is 80-fold higher than the lowest significant response to GA in the Comet assay (10 microM with FPG). In contrast, NOZ-2 was as effective in the hPRT test as in the Comet assay (3 microM). These results demonstrate substantial differences in the genotoxic potency of GA and NOZ-2. Whereas NOZ-2 is a pontent genotoxic mutagen, GA in comparison shows only low genotoxic and mutagenic potential, presumably as a result, at least in part, of preferential N7-G alkylation.     

反刍动物饲料中乳源性成分与牛羊肉骨粉的区分

利用奶粉的溶解性,通过水和0.5%次氯酸钠溶液洗涤混有乳源性成分和牛、羊肉骨粉的饲料样品,去除饲料中的乳粉成分后,再使用16S rDNA PCR方法进行动物源性检测。结果表明,实验所建立的方法能够完全区分饲料中的乳粉与肉骨粉。当乳粉含量分别为25%、50%和75%时,混合饲料中牛、羊肉骨粉的检出限分别为2%、0.5%和0.1%。此方法操作简单,容易掌握,可用于鉴别反刍动物饲料中非乳源性成分的牛、羊源性成分。     

Development of serological ovine field test (SOFT) by modified agar-gel immunodiffusion

A serological ovine field test (SOFT) has been developed for detection of lamb or sheep tissue in a wide variety of raw meat products. The test is an adaptation of previously developed field screening immunodiffusion tests for beef, poultry, and pork detection. The SOFT test was demonstrated to be specific, sensitive, and accurate in the analysis of 104 samples.