发酵肉制品菌群的非培养鉴定技术研究进展

随着分子生物学技术的快速发展,以聚合酶链反应(polymerase chain reaction,PCR)为基础的非培养鉴定技术在发酵肉制品菌群鉴定中得到越来越广泛的应用。其方法主要有种特异性PCR、聚合酶链反应-变性梯度凝胶电泳等。与传统的分离培养鉴定方法相比,非培养方法具有直接、快速、准确、实时等优点,但同时也存在一些缺点,如样品复杂程度、DNA(RNA)提取条件、PCR反应过程的差异等均可影响鉴定结果。非培养鉴定技术与传统微生物鉴定技术相结合,能够对发酵肉制品菌群多态性和动态变化做出准确的鉴定。     

乌贼DNA条形码的筛选与验证

目的筛选适于鉴定乌贼的DNA条形码,建立鉴定舟山常见乌贼种类的DNA条形码技术体系。方法用聚合酶链反应(polymerase chain reaction, PCR)对现有5组DNA条形码引物组合进行了筛选,设计一对新的DNA条形码引物以供筛选备用。结果现有引物与部分乌贼的DNA模板存在错配而缺乏通用性,在部分乌贼DNA的扩增受阻。本实验设计的12S rRNA基因引物可以特异性扩增乌贼的DNA,提高了乌贼鉴定的准确度。结论本研究设计的12S rRNA基因引物可以作为现有乌贼DNA条形码鉴定的补充。     

酿酒葡萄DNA的提取方法

采用琼脂糖凝胶电泳、核酸蛋白检测和聚合酶链反应扩增(polymerase chain reaction,PCR)检测DNA的浓度和纯度,对改良十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)法与2种商业试剂盒提取4种不同品种酿酒葡萄DNA的方法进行了对比分析,结果表明改良的CTAB法提取DNA完整性较好,基因组DNA样品的OD260/OD280值均在1. 8左右,4个酿酒葡萄均能扩增出PCR条带,且条带清晰,无拖尾现象。说明改良的CTAB法提取的酿酒葡萄基因组DNA浓度和纯度均较高,可以进行PCR扩增、电泳检测、遗传多样性分析以及基因图谱构建等下游分子生物学研究试验。     

水产品中特定腐败菌分析技术的研究进展

随着我国居民生活水平的提高,人们已经不再满足于单一的饮食品种,所以,近年来我国的水产品消费呈现出逐年增长的趋势。由于水产品在捕捞后的贮藏过程中极易发生腐败变质,其中引起水产品腐败的主要原因是特定腐败菌的代谢和生长,因此,对水产品中特定腐败菌的分析技术进行研究具有重要意义。该文主要针对国内外水产品特定腐败菌分析技术的研究现状进行综述,总结分析聚合酶链反应基因组指纹(polymerase chain reaction-repetitiveextra-genic palindromic,Rep-PCR)分析技术、聚合酶链反应变性梯度凝胶电泳(polymerase chain reaction-denaturinggradient gel electrophoresis,PCR-DGGE)分析技术、聚合酶链反应温度梯度凝胶电泳(polymerase chain reaction-temperature gradient gel electrophoresis,PCR-TGGE)分析技术、聚合酶链反应限制性片段长度多态性(polymerase chainreaction-restriction fragment length polymorphism,PCR-RFLP)分析技术和高通量测序技术各自的特点,以期为这方面的研究提供一定的参考。     

玉米包衣对种子鉴定过程中PCR扩增的影响

目的探讨玉米包衣内含有的杀虫剂、杀菌剂是否影响种子检测过程中的聚合酶链式反应(polymerase chain reaction,PCR)扩增效果及检测结果。方法利用紫外吸收法、凝胶电泳法以及PCR.扩增法对提取的包衣玉米DNA浓度和纯度进行测定,考察PCR扩增效果并对检测结果进行判定。结果不同清洗时间所提取的包衣玉米DNA与未包衣种子提取的DNA质量相比几乎没有差异,浓度和纯度均很好,DNA溶液OD_(260)/OD_(280)比值均在1.7~2.0之间,其DNA质量符合国家种子检测标准要求。玉米包衣对PCR扩增效果及检测结果没有影响。结论包衣玉米样品的种子进行检测时可不作清洗而直接进行DNA提取和PCR扩增,仍可得到准确可靠的结果,从而节约人力和物力并缩短检测时间。     

微滴式数字聚合酶链式反应在食品安全检测领域的应用

微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction, ddPCR)是一种新型核酸扩增技术,可对DNA或RNA分子采用绝对定量的方式进行分析。其结果具有更高的精准度、准确性和灵敏度,大大提升了数字PCR技术的可扩展性与实用性,促进了现代分子生物学在精准定量检测方面的发展和应用。本文重点论述了ddPCR法的技术原理、优势以及在食源性致病微生物定量检测、转基因成分分析、食品源性成分检测等食品安全检测领域的应用研究进展情况。     

马来西亚研究人员利用聚合酶链反应检测靶细胞色素b基因来检测肉丸配方中掺假狗肉

<正>马来西亚研究人员利用聚合酶链反应(polymerase chain reaction,PCR)法检测肉丸中狗肉掺假(拟发表于2014年8月Meat Science)。该法选择性地从纯、原始、处理后和混合背景中扩增出100 bp区域的犬线粒体细胞色素b基因。利用肉丸配方常见的11种动物和3种植物进行该法的特异性测试。在广泛的高压灭菌条件下能够击穿靶DNA证实该法具有稳定     

PCR技术在酿酒中的应用

PCR技术即利用聚合酶链反应，进行DNA片段的体外扩增，可用于发酵过程的特定菌的检验和鉴定，该法快速、准确。PCR技术有特异引物PCR技术、RAPD-PCR技术和Nested-PCR几种。PCR技术在酿酒中可用于啤酒中的腐败菌的鉴定、对葡萄酒中苹果酸一乳酸发酵(MLF)的检测和控制以及对葡萄酒中生物胺的检测。(孙悟)     

基于核酸分子学方法的肉类成分鉴别技术研究进展

近年来,肉类掺假问题频繁发生。基于核酸的分子生物学肉类成分鉴别技术已成为研究热点,其具有灵敏度高、特异性强、检测时间短以及成本低的优点。本文综述了基于核酸分子学的肉类成分种属鉴别技术在肉类掺假检验中的应用,着重于量化各种方法的检测限,并重点对实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)和数字PCR技术在动物成分鉴别定量分析的研究现状与前景做介绍。探讨不同来源的靶基因(核DNA和线粒体DNA)在动物成分鉴别中,定性和定量检测灵敏度与特异性的区别。     

DNA条形码技术在肉品防欺诈鉴别中的应用

以DNA条形码技术鉴别进出口监督抽检的鱼肉等水产制品的种类来源,用以判别其与申报或产品标签是否相符.分别提取鱼肉等样品的基因组DNA,以目前国际上比较公认的动物线粒体细胞色素氧化酶COⅠ基因通用引物进行PCR扩增.PCR产物经测序分析后,将得到的扩增片段序列与Genbank数据库进行序列比对,同时提交Barcoding Life DNA条形码数据库(BOLD)进行鉴定分析.本批次监督抽检的16份鱼肉、鱼丸等水产制品中除1份样品未能成功获得鱼肉COⅠPCR扩增外,其余15份样品均顺利得到种类来源鉴定,鉴定结果约有31.25％的样品与产品标签标示不符.作为一种简单、快速、有效的分子鉴定技术,DNA条形码可以直接应用于鱼肉等动物源性食品的种类鉴定.     

鱼胶基原鱼种鉴定技术研究进展

鱼胶作为我国传统滋补药材,近年来其消费市场日益繁荣,鱼胶基原鱼种来源范围不断扩大,尤其是进口基原鱼种占比不断提升,部分市售鱼胶真伪难辨,鱼胶基原鱼种鉴定需求不断增加。本文概述了鱼胶基原鱼种形态学鉴定法、光学分析法和分子生物学鉴定法的发展历程、应用情况,对比了分子生物学方法中不同基因片段、不同引物等的适用性,分析了上述方法的优缺点。传统形态学鉴定具有快速、不需要鉴定人员具备较高实验操作能力的优势,但依赖专家个人经验和观察手段,且对鱼胶残片等形态特征缺失的样品鉴定能力有限;光学分析法无法精确到种属;而以DNA条形码技术为主的分子生物学方法是对某一特定区域的DNA序列排列顺序进行对比分析,鉴定结果准确,但较为耗时。由于鱼胶是动物组织干制品,在物种鉴定方面具有形态信息较少、样品处理时间较长等特点;本文有针对性地综合国内外相关研究,填补了特殊动物组织物种鉴定研究方法综述的空白,为以后进口贸易或市场监管中如何优化鉴定技术提供理论依据。     

Differentiation of fish species by PCR-based DNA analysis of nuclear genes

Food control laboratories are confronted with new challenges in respect to fishery product authentication; examples are identification of hybrids (e.g. in case of catfish, tilapia, sturgeon, snapper) and assignment of fish to certified stocks. Against this background, differentiation of fish species and populations by polymerase chain reaction (PCR)–based DNA analysis of nuclear genes has become of considerable importance as a tool for the completion of mitochondrial gene analysis. Four applications of nuclear gene analysis are presented: (1) fish species identification using the intronless rhodopsin RH1 gene; (2) detection of “fish” as an allergenic foodstuff by means of universal primers amplifying a segment of a parvalbumin gene; (3) differentiation of cod (Gadus morhua) from various fishing grounds by exon-primed intron-crossing PCR of a parvalbumin gene intron; (4) detection of hybridization between North Atlantic redfish species (genus: Sebastes) by restriction fragment length polymorphism analysis of amplicons obtained from the second intron of the 7S ribosomal protein gene.     

Quantitative competitive (QC) PCR for quantification of porcine DNA

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.     

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy^® Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.     

IDENTIFICATION OF GOAT MEAT USING HIGHLY SPECIES‐SPECIFIC POLYMERASE CHAIN REACTION

ABSTRACT

A highly species‐specific polymerase chain reaction (PCR) assay was developed for the authentic identification of goat. A product of 436 bp was amplified using newly designed primers against mitochondrial D‐loop region. The possibility of cross‐amplification was ruled out by considering as many as 25 other animal species. Suitability of the developed goat species‐specific PCR assay was confirmed for in raw, cooked (60, 80 and 100C for 30 min) and micro‐oven‐processed meat samples (n = 20 each). A sensitivity of 0.1% was established for detection of adulteration and limit of detection of goat DNA was 0.1 pg. This investigation presents a novel PCR assay with its newly designed primers that could be used for the authentic identification of goat species.

PRACTICAL APPLICATIONS

This work details about a novel diagnostic polymerase chain reaction, which could be used for authentic identification of goat species. This approach could be used for the confirmation of goat tissues in raw, cooked, as well as adulterated samples. The developed technique has also applications in the forensic analysis of wild animal‐related disputes, where this work could solve the problem of goat‐related issues.     

Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay.     

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100 bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.     

Polymerase chain reaction (PCR) identification of Aspergillus niger and Aspergillus tubingensis based on the calmodulin gene.

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly found in plant products and processed food, such as grapes, cereals, coffee, and derived products. These two species are very difficult to differentiate by classical morphological criteria and some isolates are known to produce ochratoxin A. The exact identification of these two species is very important to avoid the overestimation of toxicological contamination and related risks. A polymerase chain reaction (PCR)-based identification and detection assay was developed as a tool to identify A. niger and A. tubingensis, using molecular differences obtained by sequencing the calmodulin gene. Two pairs of species-specific primers were designed and empirically evaluated for PCR identification of A. niger and A. tubingensis. Species-specific PCR products generated by each primer set were 505 bp (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in a 25-microl PCR reaction volume, using pure total DNA of the two species. The method described in this study represents a rapid and reliable procedure to assess the presence in food products of two ochratoxigenic species of section Nigri.