实时荧光PCR法检测肉制品中鸡、鸭源性成分

根据鸡、鸭线粒体DNA(mt DNA)序列设计了鸡、鸭特异性引物及Taqman探针,建立了肉制品中鸡、鸭源性成分的实时荧光聚合酶链式(real-time PCR)检测方法。结果发现:利用此方法,两种动物源性的最低检出限值(质量分数)分别为0.01%和0.001%。实时荧光PCR法能够作为肉制品中检测鸡、鸭源性成分的有效方法。     

基于G-四联体比色生物传感器检测肉制品中羊源性成分

近年来,肉制品掺假现象已成为社会关注的热点,鉴定畜产品中动物源性成分是打击掺假的一种重要技术手段。基于此,利用不对称PCR技术(asymmetric PCR,As-PCR),并结合G-四联体的功能特点,建立了一种G-四联体比色生物传感器以鉴定肉制品中羊源性成分。以羊的基因组DNA为模板,并在限制性正向引物的5′端加上一段G-四联体的反向互补序列,通过As-PCR扩增出大量含有G-四联体序列的单链DNA(single strand,ssDNA),生成的ssDNA在K~+的作用下可与氯高铁血红素(hemin)结合形成具有DNA核酶(DNAzyme)活性的G-四联体-hemin复合物。该复合物可催化H_2O_2与2,2′-联氮-双(3-乙基-苯并噻唑琳-6-磺酸)二胺盐[2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt,ABTS~(2-)]的反应,从而生成绿色的反应产物,结果可用肉眼观察,无需电泳、测序等常规检测步骤。通过对As-PCR反应条件和体系的优化,该生物传感器可实现对肉制品中羊源性成分准确、快速的鉴定,可稳定检测出的混合肉样中羊源性成分的最低质量分数为5%。研究所建立的G-四联体比色生物传感器具有简便、灵敏、可视化、低成本等优点,可用于肉制品中动物源性成分的检测,为肉制品的安全检测提供了强有力的技术支撑。     

普通牛及其近缘种特异性序列筛选及环介导等温扩增检测方法

近年来,肉制品掺假事件频频发生,亟需建立快速、可靠的肉制品动物源性成分检测方法,保障肉类食品安全。与常规PCR等检测方法相比,环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法具有高特异性、高灵敏度、反应快、易操作等优势。基于此,利用生物信息算法筛选普通牛及其近缘种（牦牛、水牛、美洲野牛）的全基因组序列,以获得种间相似性序列,进而建立并优化可用于检测普通牛及其近缘种成分的特异性LAMP方法。最终优化后的LAMP反应体系为：10 μmol·L-1外引物F3和B3各0.25 μL,10 μmol·L-1内引物FIP和BIP各2 μL,2 mmol·L-1 dNTP 2.5 μL,25 mmol·L-1 MgSO4 4 μL,5 mol·L-1 甜菜碱 3.5 μL,8.0 U·μL-1 Bst DNA 聚合酶 1 μL,10×ThermoPol Buffer 2.5 μL,DNA模板2 μL,加双蒸水至25 μL;LAMP反应条件为：65 ℃恒温扩增1 h,80 ℃ 10 min。在扩增产物中加入SYBR GreenⅠ荧光染料后,检测结果可用肉眼直接观察。优化后的LAMP方法在1 h内能特异性地检测出普通牛、牦牛、水牛及美洲野牛成分,检测灵敏度达到0.020 ng·μL-1,且可特异性地检测出市售肉制品中含有牛肉成分的样品。研究所建立的LAMP方法具有高特异性、高灵敏度、反应快速、操作简便和无需精密仪器等特点,可应用于肉制品中普通牛及其近缘种成分的实际检测,为我国肉类食品安全提供了有力保障。     

肉类掺假检测技术研究进展

肉类掺假现象严重威胁公共卫生安全。快速、准确、可靠的动物源性成分检测技术是有效监管与检测肉类掺假的关键。总结了常见的掺假形式,并对物理技术、光谱技术、免疫学技术、DNA分析等检测方法进行了全面的总结归纳和比较分析,尤其是对当前热门的DNA分析方法,详细阐述了常规PCR、实时荧光定量PCR、数字PCR和等温扩增PCR这4种主要分子检测技术的优缺点和应用情况,旨在为未来动物源性检测技术的发展指明方向。     

基于实时荧光PCR定量检测肉制品猪源性成分方法的建立

为了建立肉制品中猪源性成分检测的PCR方法,以猪线粒体细胞色素b(cytochrome b,Cyt b)为目的基因,设计猪特异性PCR引物;以猪、牛、羊、鸡及鸭DNA和不同稀释浓度猪DNA进行Real-time PCR反应,证明了该引物扩增具有物种特异性,检测低限可达5×10-5 ng/μL;设计内参基因β-actin通用引物,以猪源性成分含量0%~100%的混合肉样DNA为模板扩增,建立标准曲线,2-△△Ct值与猪源性成分含量有良好线性关系(R2=0.991 4);对猪源性成分含量0%~75%的混合肉样检测,回收率为99.85%~102.60%。应用建立的PCR方法检测了市售的6种品牌火腿肠,标识为清真食品的5种均未检测到猪源性成分,未标识清真食品的猪源性成分含量为12.70%。建立的实时荧光PCR检测方法操作简便、特异性强、所得数据可靠,为肉制品猪源性成分检测提供了新的手段。     

羊肉掺假鉴别快速荧光定量PCR芯片制备及应用研究

为了建立一种基于芯片的快速鉴别羊肉掺假成分的方法,将不同动物源性成分的引物及反应所需试剂预先冻干固定到空白芯片反应池内,以制备羊肉掺假鉴别快速荧光定量PCR芯片。同时,通过模拟掺假样品（在羊肉中掺入猪肉、鸡肉、鸭肉、鼠肉成分）检测实验,对所得芯片的性能进行了评价。从与ABI7500荧光定量PCR结果对比可知,基于芯片的快速荧光定量PCR检测方法可以准确检测5种动物源性成分,具有较高的准确性及可用性,且其PCR扩增时间较短,操作简单,满足了羊肉掺假快速鉴别的要求。该芯片的研制及快速检测方法的建立将有效的简化羊肉制品掺假检测的步骤、缩短检测时间,且成本较低,仪器便于携带,使现场检测成为可能。研究结果为我国肉类食品安全监管提供了有力保障。     

运用基因芯片技术检测牛、山羊、猪和鸡源性成分

本研究通过对脊椎动物分子标记基因进行序列分析,最终选择线粒体DNA(mtDNA)16S rRNA基因为目标基因,利用一对通用引物,在该引物扩增区间设计了4条特异性基因芯片检测探针及2条质控探针用于对牛、山羊、猪、鸡等4种动物源性成分进行检测。通过对PCR扩增体系及杂交体系的优化,该检测方法能实现对上述4种动物源性成分同时进行快速、准确地检测,具有很好的特异性,灵敏度均达到1pg,最终建立了这4种动物源性基因芯片检测方法。该基因芯片检测技术将为我国进出口饲料中的动物源性成分的鉴别提供新的检测方法和技术支持。     

饲料中狐狸、水貂、貉子和狗源性的五重实时荧光PCR检测方法的建立

为了快速鉴别饲料中的狐狸、水貂、貉子和狗源性成分,根据线粒体16S r DNA种间保守序列,设计合成针对狐狸、水貂、貉子和狗的特异性引物和探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了多重实时荧光PCR方法,在同一PCR反应体系中可以同时完成4种动物源性成分检测。通过对15种其他物种的源性成分的检测,结果表明所设计的引物和探针具有很好的物种特异性,且灵敏度高,狐狸、水貂、貉子和狗的DNA检出限为0.01ng。对40份样品检测,其中5份检测出貉子、狐狸和水貂源性成分。结果表明,该方法可以有效地鉴别出饲料中狐狸、水貂、貉子和狗源性成分,同时适用于相关动物产品中。     

多重实时荧光PCR检测牛、山羊和绵羊源性成分

根据牛、山羊和绵羊线粒体细胞色素b基因序列, 设计特异性引物和以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选, 建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T 20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测, 数据显示两者检测结果符合率达100%, 特异性相当。与国标方法相比, 本试验方法不需电泳、酶切和测序, 即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分, 检测效率提高近3倍; 灵敏度更高, 比国标方法灵敏10倍; 适用性更广, 除了饲料, 还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。     

双重实时荧光PCR定量检测动物产品中牛源性成分

旨在建立一种定量检测动物产品中牛源性成分含量的双重实时荧光PCR方法。以牛卫星IV DNA为检测靶基因,以肌肉生长抑制素基因作为内对照合成引物探针,采用基于TaqMan的双重实时荧光PCR技术对已知牛肉添加比例的冻干混合肉粉标准品进行检测并建立线性模型,并应用模拟样品和送检样品进行了验证检测。结果显示,该方法可检出牛源成分含量为0.1%的冻干肉粉,不与其他种动物组织发生交叉反应,重复性试验的相对标准偏差小于5%,方法的扩增效率为93.6%。该方法适用于肉品中牛源性成分的定量检测,可用于测定市场上动物产品中的牛源性成分含量。     

蛋白质组学技术在肉类鉴别及肉质分析中的应用进展

肉制品是人体中蛋白质和多种微量元素的重要来源,但对于肉制品中肉类的鉴别及品质分析的研究受到了传统方法的限制。近年来,蛋白质组学技术的应用极大地推动了肉类鉴别技术的发展,并对肉质形成的潜在分子机制的研究有着深远的影响。主要介绍了蛋白质组学的概念及其研究策略,全面综述了蛋白质组学技术在肉类鉴别和肉质分析中的应用进展,并展望了其研究前景,以期为肉制品的质量控制及肉质影响因素的研究提供理论依据。     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

Review: Analysis of the process and drivers for cellular meat production

Cell-based meat, also called ‘clean’, lab, synthetic or in vitro meat, has attracted much media interest recently. Consumer demand for cellular meat production derives principally from concerns over environment and animal welfare, while secondary considerations include consumer and public health aspects of animal production, and food security. The present limitations to cellular meat production include the identification of immortal cell lines, availability of cost-effective, bovine-serum-free growth medium for cell proliferation and maturation, scaffold materials for cell growth, scaling up to an industrial level, regulatory and labelling issues and at what stage mixing of myo-, adipo- and even fibrocytes can potentially occur. Consumer perceptions that cell-based meat production will result in improvements to animal welfare and the environment have been challenged, with the outcome needing to wait until the processes used in cell-based meat are close to a commercial reality. Challenges for cell-based meat products include the simulation of nutritional attributes, texture, flavour and mouthfeel of animal-derived meat products. There is some question over whether consumers will accept the technology, but likely there will be acceptance of cell-based meat products, in particular market segments. Currently, the cost of growth media, industry scale-up of specific components of the cell culture process, intellectual property sharing issues and regulatory hurdles mean that it will likely require an extended period for cellular meat to be consistently available in high-end restaurants and even longer to be available for the mass market. The progress in plant-based meat analogues is already well achieved, with products such as the Impossible^TM Burger and other products already available. These developments may make the development of cellular meat products obsolete. But the challenges remain of mimicking not only the nutritional attributes, flavour, shape and structure of real meat, but also the changes in regulation and labelling.     

PCR法快速检测肉食品污染沙门菌的实验研究

沙门菌属是引起食源性疾病的重要致病菌之一。传统的检测方法费时、费力,研究建立了检测肉食品中沙门菌的PCR检测方法。根据沙门菌侵袭基因invA设计一对引物,对肉食品中沙门菌基因组DNA进行PCR扩增,建立了沙门菌特异、敏感和快速的PCR检测方法,为食源性致病菌的检测提供参照,在食品检测领域中有良好的应用前景。     

A Next Generation Semiconductor Based Sequencing Approach for the Identification of Meat Species in DNA Mixtures

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.     

熏烤肉制品中苯并芘的危害及控制措施

本文从食品安全的角度出发,对熏烤肉制品中的有害物质苯并(a)芘的产生,对人体的危害作用,以及控制熏烤肉制品中苯并(a)芘残留的方法进行了初步的研究.这对提高熏烤肉制品品质和对苯并(a)芘进行深入的研究具有重要意义.     

Nutritional value of milk and meat products derived from cloning

The development and use of milk and meat products derived from cloning depends on their safety and on the nutritional advantages they can confer to the products as perceived by consumers. The development of such products thus implies (i) to demonstrate their safety and security, (ii) to show that their nutritional value is equivalent to the traditional products, and (iii) to identify the conditions under which cloning could allow additional nutritional and health benefit in comparison to traditional products for the consumers. Both milk and meat products are a source of high quality protein as determined from their protein content and essential amino acid profile. Milk is a source of calcium, phosphorus, zinc, magnesium and vitamin B2 and B12. Meat is a source of iron, zinc and vitamin B12. An important issue regarding the nutritional quality of meat and milk is the level and quality of fat which usually present a high content in saturated fat and some modification of the fat fraction could improve the nutritional quality of the products. The role of the dietary proteins as potential allergens has to be taken into account and an important aspect regarding this question is to evaluate whether the cloning does not produce the appearance of novel allergenic structures. The presence of bio-activities associated to specific components of milk (lactoferrin, immunoglobulins, growth factors, anti-microbial components) also represents a promising development. Preliminary results obtained in rats fed cow's milk or meat-based diets prepared from control animals or from animals derived from cloning did not show any difference between control and cloning-derived products.     

高通量测序技术在肉类掺假检测中的应用进展

肉类掺假现象普遍存在,导致严重的公共卫生风险和侵犯宗教信仰行为。快速、有效、准确和可靠的检测技术是有效监管肉类掺假的关键手段。近年来,基于高通量测序的DNA宏条形码技术发展迅速,具有高通量、高精度和速度快等特点,并且可以实现复杂样品中多个物种的同时检测,因而在肉类及其制品的掺假检测方面具有明显的优势。文中介绍了近20年来高通量测序技术的主要发展历程、DNA宏条形码的技术特点和研究方法,综述了近年来DNA宏条形码技术在肉类掺假检测中的应用,讨论分析了DNA宏条形码技术应用于肉类掺假检测领域面临的挑战,并对该技术的未来发展趋势进行了展望。