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。     

微型DNA条形码在水产类物种掺假检测中的应用

水产类物种掺假导致了商业欺诈、食品安全等问题,建立一种快速高效的水产类物种掺假检测技术迫在眉睫.目前,标准DNA条形码(FDB)在水产类物种掺假检测中已经得到了广泛应用,但是食品加工中的高温、高压、反复冻融和酸碱变化等因素均会使DNA发生降解,导致标准DNA条形码扩增失败.微型DNA条形码(MDB)可以利用相对较短(＜...     

核酸条形码技术：扩展蛋白质-蛋白质相互作用检测通量的新方法

蛋白质-蛋白质相互作用(protein-protein interaction, PPI)几乎参与了机体内所有重要的生物学过程,在细胞的基本生命过程中扮演了至关重要的角色,开发高通量的PPI检测新方法具有重要的生物学意义。目前,下一代测序技术(next-generation sequencing, NGS)发展快速,能在几天内测定超过10亿个模板的DNA序列。由于并行DNA测序技术所特有的敏感性、特异性、高通量和多路复用优势,其已被用作广谱分子计数器,应用于基因组测序和转录物组测序等领域。核酸条形码技术通过将寡核苷酸标签与目标蛋白质连接起来,从而标记编码蛋白质。之后,利用高通量的测序方法检测相互作用的蛋白质,实现了PPI的高通量检测。这一技术推动了PPI检测方法的飞速发展,提升了单次实验检测的通量,为构建PPI网络提供了强有力的技术支持。本文详细阐述了核酸条形码在PPI检测方法中的设计、生成和读取;通过分析核酸条形码技术在PPI研究中的应用范例,探讨了各自的优势和不足,并评估了数据的可靠性,讨论了基于核酸条形码技术的PPI检测方法未来的发展趋势。     

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。     

DNA宏条形码技术在食草动物食性研究中的应用

随着测序技术的快速发展,整合DNA条形码和高通量测序的DNA宏条形码技术已经成为当前研究热点之一,在食草动物的食性鉴定中有很大潜力.放牧动物食性研究是动物营养学和草地生态学领域的重要研究内容.而与传统食性研究方法相比,宏条形码技术可通过对植物DNA条形码的高通量测序,获得样本中的物种组成进而分析动物食性.介绍了传统食性...     

肉类及肉制品中动物源性成分鉴别方法研究进展

肉类掺假问题直接影响着人类健康、公共卫生安全以及社会稳定等方面,成为当今食品安全热点话题之一,因此,高效、精确的肉类及肉制品中动物源性成分的检测鉴定势在必行。基于此,主要介绍了对于动物源性成分检测及鉴别的不同研究方法,分析了利弊,并对后续肉类及肉制品中动物源性成分的鉴别方法的研发方向进行了展望,以期为此领域提供资料性参考。     

高通量二代测序 对感染性新生儿高胆红素血症病原菌的分析

摘要：目的 探讨高通量二代测序技术在筛选感染性新生儿高胆红素血症病原菌中的应用价值。方法 运用高通量二代测序技术筛选22例感染性新生儿高胆红素血症患者的病原菌,同时进行传统细菌培养鉴定,比较二者的区别。结果 高通量二代测序技术检测病原菌阳性率为100.00%,传统细菌培养检测阳性率为0.00%。高通量二代测序技术筛选出的感染性新生儿高胆红素血症主要病原菌为Anoxybacillus kestanbolensis、Geobacillus vulcani、Klebsiella oxytoca和Acinetobacter guillouiae。结论 高通量二代测序技术具有高通量、高特异性、高准确度和快速等特点,适合临床患者病原菌的检测。     

悬浮芯片技术应用进展

悬浮芯片技术(SAT)是一种新型、高通量的生物芯片技术,它是将流式细胞术、激光技术及应用流体学等技术结合在一起,利用悬浮在液相中的分类荧光编码微球作为检测载体,具有高通量、速度快、灵敏度高、特异性强及检测范围广等特点.近几年来,悬浮芯片技术在免疫学、基因组学、蛋白质组学及临床诊断检测等方面应用较广泛.就其原理、技术特点...     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性(single nucleotide polymorphism,SNP)作为第三代遗传标记已经广泛应用于基因作图、疾病相关性分析、群体遗传学及药物研究等领域.因此建立高度自动化和高通量的SNP检测分析技术十分重要.简要介绍了国内外几种主要SNP检测技术的原理和检测分析手段,并对SNP高通量检测技术的发展进行了展望.     

常压室温等离子体诱变育种与微生物液滴培养筛选技术应用进展

安全和高效的微生物突变及高通量筛选技术是微生物功能发掘、功能创制和生物产业技术创新的重要方向及重要支撑.有效的生物育种技术及高通量筛选技术成为该领域研究人员的关注点.其中,常压室温等离子体(atmospheric and room temperature plasma,ARTP)因具有活性粒子种类多、操作可控性强、基因...     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

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。     

Bioinformatic challenges for DNA metabarcoding of plants and animals

Almost all empirical studies in ecology have to identify the species involved in the ecological process under examination. DNA metabarcoding, which couples the principles of DNA barcoding with next generation sequencing technology, provides an opportunity to easily produce large amounts of data on biodiversity. Microbiologists have long used metabarcoding approaches, but use of this technique in the assessment of biodiversity in plant and animal communities is under-explored. Despite its relationship with DNA barcoding, several unique features of DNA metabarcoding justify the development of specific data analysis methodologies. In this review, we describe the bioinformatics tools available for DNA metabarcoding of plants and animals, and we revisit others developed for DNA barcoding or microbial metabarcoding. We also discuss the principles and associated tools for evaluating and comparing DNA barcodes in the context of DNA metabarcoding, for designing new custom-made barcodes adapted to specific ecological question, for dealing with PCR and sequencing errors, and for inferring taxonomical data from sequences.     

环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2?×?2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (>?30) of individuals per sample.     

Towards next-generation biodiversity assessment using DNA metabarcoding

Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.     

A practical guide to DNA metabarcoding for entomological ecologists

1. DNA metabarcoding is a cost-effective species identification approach with great potential to assist entomological ecologists. This review presents a practical guide to help entomological ecologists design their own DNA metabarcoding studies and ensure that sound ecological conclusions can be obtained. 2. The review considers approaches to field sampling, laboratory work, and bioinformatic analyses, with the aim of providing the background knowledge needed to make decisions at each step of a DNA metabarcoding workflow. 3. Although most conventional sampling methods can be adapted to DNA metabarcoding, this review highlights techniques that will ensure suitable DNA preservation during field sampling and laboratory storage. The review also calls for a greater understanding of the occurrence, transportation, and deposition of environmental DNA when applying DNA metabarcoding approaches for different ecosystems. 4. Accurate species detection with DNA metabarcoding needs to consider biases introduced during DNA extraction and PCR amplification, cross-contamination resulting from inappropriate amplicon library preparation, and downstream bioinformatic analyses. Quantifying species abundance with DNA metabarcoding is in its infancy, yet recent studies demonstrate promise for estimating relative species abundance from DNA sequencing reads. 5. Given that bioinformatics is one of the biggest hurdles for researchers new to DNA metabarcoding, several useful graphical user interface programs are recommended for sequence data processing, and the application of emerging sequencing technologies is discussed.