DNA微阵列在食品营养与安全中的应用与展望

DNA微阵列是一种高通量基因检测和分析技术,主要概括介绍DNA微阵列在营养基因组学、食源性病原体检测、转基因食品和饮食补充剂安全性评价中的应用。     

Microbiological quality of raw milk used for small-scale artisan cheese production in Vermont: Effect of farm characteristics and practices

This study 1) evaluated the overall milk quality and prevalence of 4 target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk used for small-scale artisan cheesemaking and 2) examined specific farm characteristics and practices and their effect on bacterial and somatic cell counts (SCC). Raw milk samples were collected weekly from 21 artisan cheese operations (6 organic) in the state of Vermont that manufactured raw-milk cheese from cow (12), goat (5), or sheep (4) milk during the summer of 2008. Individual samples were examined for standard plate counts (SPC), coliform counts (CC), and SCC. Samples were also screened for target pathogens both quantitatively and qualitatively by direct plating and PCR. Overall, 86% of samples had SPC <10,000 cfu/mL, with 42% <1,000 cfu/mL. Additionally, 68% of samples tested were within pasteurized milk standards for coliform bacteria under the United States’ Grade A Pasteurized Milk Ordinance at <10 cfu/mL. Log₁₀ SPC and CC did not differ significantly among species. Similarly, method of sample delivery (shipped or picked up), farm type (organic or conventional), and duration of milking (year-round or seasonal) did not have significant effects on farm aggregated mean log₁₀ SPC, CC, or SCC. Strong positive correlations were observed between herd size and mean log₁₀ SPC and between log₁₀ SPC and CC as well as SCC when data from all animal species were combined. Although SCC for cow milk were significantly lower than those for goat and sheep milk, 98, 71, and 92% of cow, sheep, and goat milk samples, respectively, were within the compliance limits of the United States’ Grade A Pasteurized Milk Ordinance for SCC. Fourteen of the 21 farms (67%) were positive for Staph. aureus, detected in 38% of samples at an average level of 20 cfu/mL. Neither L. monocytogenes, E. coli O157:H7, or Salmonella spp. were detected or recovered from any of the 101 samples tested. Our results indicate that the majority of raw milk produced for small-scale artisan cheesemaking was of high microbiological quality with no detectable target pathogens despite the repeat sampling of farms. These data will help to inform risk assessments that evaluate the microbiological safety of artisan and farmstead cheeses, particularly those manufactured from raw milk.     

基因探针技术及其在食品卫生检测中的应用

建立在DNA杂交基础上的基因探针技术是现代分子生物学中的一种常规技术。用基因探针技术检测食品中的有害微生物具有特异性强、灵敏度高和操作简便、省时等优点。近年来 ,有关非放射性基因探针、DNA生物传感器探针及分子信标探针等技术研究取得的重要进展 ,必将加速基因探针技术在食品微生物检测中的应用。本文对多种基因探针的技术原理与研究应用概况及最新进展进行了综述讨论     

Pathogen-specific production losses in bovine mastitis

Reduction in long-term milk yields represents a notable share of the economic losses caused by bovine mastitis. Efficient, economic, and safe measures to prevent these losses require knowledge of the causal agent of the disease. The aim of this study was to investigate pathogen-specific impacts of mastitis on milk production of dairy cows. The materials consisted of milk and health recording data and microbiological diagnoses of mastitic quarter milk samples of 20,234 Finnish dairy cows during 2010, 2011, and 2012. The 6 most common udder pathogens were included in the study: Staphylococcus aureus, non-aureus staphylococci (NAS), Escherichia coli, Corynebacterium bovis, Streptococcus uberis, and Streptococcus dysgalactiae. We used a 2-level multilevel model to estimate curves for lactations with and without mastitis. The data on lactation periods to be compared were collected from the same cow. To enable comparison among lactations representing diverse parities, the estimated lactation curves were adjusted to describe the cow's third lactation. Mastitis caused by each pathogen resulted in milk production loss. The extent of the reduction depended on the pathogen, the timing of mastitis during lactation, and the type of mastitis (clinical vs. subclinical). The 2 most commonly detected pathogens were NAS and Staph. aureus. Escherichia coli clinical mastitis diagnosed before peak lactation caused the largest loss, 10.6% of the 305-d milk yield (3.5 kg/d). The corresponding loss for Staph. aureus mastitis was 7.1% (2.3 kg/d). In Staph. aureus mastitis diagnosed between 54 and 120 d in milk, the loss was 4.3% (1.4 kg/d). The loss was almost equal in both clinical and subclinical mastitis caused by Staph. aureus. Mastitis caused by Strep. uberis and Strep. dysgalactiae resulted in losses ranging from 3.7% (1.2 kg/d) to 6.6% (2.1 kg/d) depending on type and timing of mastitis. Clinical mastitis caused by the minor pathogens C. bovis and NAS also had a negative effect on milk production: 7.4% (2.4 kg/d) in C. bovis and 5.7% (1.8 kg/d) in NAS when both were diagnosed before peak lactation. In conclusion, minor pathogens should not be underestimated as a cause of milk yield reduction. On single dairy farms, control of E. coli mastitis would bring about a significant increase in milk production. Reducing Staph. aureus mastitis is the greatest challenge for the Finnish dairy sector.     

The impact of the postharvest environment on the viability and virulence of decay fungi

Postharvest decay of fruits, vegetables, and grains by fungal pathogens causes significant economic losses. Infected produce presents a potential health risk since some decay fungi produce mycotoxins that are hazardous to human health. Infections are the result of the interplay between host resistance and pathogen virulence. Both of these processes, however, are significantly impacted by environmental factors, such as temperature, UV, oxidative stress, and water activity. In the present review, the impact of various physical postharvest treatments (e.g., heat and UV) on the viability and virulence of postharvest pathogens is reviewed and discussed. Oxidative injury, protein impairment, and cell wall degradation have all been proposed as the mechanisms by which these abiotic stresses reduce fungal viability and pathogenicity. The response of decay fungi to pH and the ability of pathogens to modulate the pH of the host environment also affect pathogenicity. The effects of the manipulation of the postharvest environment by ethylene, natural edible coatings, and controlled atmosphere storage on fungal viability are also discussed. Lastly, avenues of future research are proposed.     

Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in “cuajada” (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10°C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10°C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.     

肉中4种致病菌的PCR快速检测方法的建立

目的:建立一种能同时检测肉中金黄色葡萄球菌、志贺氏菌、沙门氏菌和单核增生李斯特菌的多重PCR检测方法。方法:根据金黄色葡萄球菌的耐热核酸酶基因(nuc)、沙门氏菌的侵袭蛋白基因(invA)、志贺氏菌的侵袭性质粒抗原基因(ipaH)和单核细胞增生性李斯特菌的内化素基因(inlA)设计引物,通过优化好的反应体系进行多重聚合酶链反应(PCR)扩增目的基因。结果:特异性实验结果表明4种菌均能在相应位置扩增出特异性条带。对污染4种菌的猪肉进行检测,确定出金黄色葡萄球菌和沙门氏菌的检出限是102CFU/mL,志贺氏菌和单核增生李斯特菌的检出限是101CFU/mL。结论:本实验建立的多重PCR方法比传统细菌检测方法更特异、快速、灵敏,适用于肉中金黄色葡萄球菌、志贺氏菌、沙门菌和单核增生李斯特菌的快速检测。     

Response of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus to the thermal stress occurring in model manufactures of Grana Padano cheese

The purpose of this research was to investigate the effect of temperature in the technology of production of Grana cheese against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. According to the technology of production, the cheese curds are cooked at 55°C and then cooled at room temperature (25°C). A curd-cooling model was developed to estimate the temperature variation across the curd during cooling, and the thermal stress was applied to the pathogens according to the model in model-scale productions of Grana cheese artificially contaminated with approximately 10⁴ cfu/mL of the selected pathogens. According to the numerical results, the initial temperature inside the cheese is kept at almost the initial value (above 50°C) for at least 4 h during cooling, whereas the crust of the curd cools rapidly to 30°C in the first hour. The best case was that of the core of the cheese where the high temperature was able to efficiently eliminate the contaminating pathogens. Moreover, the worst case was where the external ring of the curd in which a more rapid cooling allowed bacterial survival. Therefore, the thermal stress in the technology of production of Grana cheese can be only partially effective in the control of the selected pathogens. However, the whole technology of production includes other hurdles that can affect the survival of the pathogens and that need to be taken into account as a whole to evaluate the safety of Grana Padano cheese.     

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.     

食源性致病细菌检测技术研究进展

对几种常见的食源性致病细菌检测技术进行了综述,对以聚合酶链式反应(PCR)方法为基础的检测技术进行了分析,将PCR检测食源性致病菌所需要用到的靶基因及其引物等进行了归纳,并对食源性致病菌检测技术的发展提出了建议。