Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

基于DNA条形码技术对常见石首鱼科鱼胶的物种鉴定

摘　要：目的　运用DNA条形码技术对常见石首鱼鱼胶进行物种鉴定。方法　通过对26份鱼胶样品基因组DNA提取，PCR扩增COI基因、测序，用BOLD物种鉴定系统，与数据库中已有鱼类序列进行比对分析，鉴定出各鱼胶的物种；根据Kimura双参数模型计算样品序列遗传距离，并将所得序列构建NJ和MP系统发育树，进行聚类分析。结果　26份鱼胶样品通过鉴定引物“Fish-F”、“Fish-R”均可实现扩增，条带清晰单一，扩增和测序成功率均为100%；BOLD鉴定结果显示，26份鱼胶样品中23份能够确定物种来源（相似性达98%以上），包括石首鱼科12属15种鱼类，且多数为外来物种，另外3份鱼胶可推测其近缘物种。此外，系统发育树聚类分析结果与物种鉴定结果一致。结论　目前石首鱼类鱼胶来源物种较多，且多为外来基原鱼种。DNA条形码技术与BOLD鉴定系统相结合，可对大部分鱼胶进行准确的物种鉴定。     

进境多肉植物细菌性褐腐病的病原分离与鉴定

目的通过对病变叶片进行细菌的分离鉴定,明确韩国进境多肉植物上叶片褐色腐烂病斑的病原。方法采用组织分离法,从韩国进境多肉植物叶片中分离纯化到1株细菌菌株,对该菌株进行了鉴定和生物学特性研究。结果形态及培养特征、生理生化特性的研究结果表明该菌株在营养琼脂(nutrient agar,NA)培养基上单菌落圆形淡黄色,边缘整齐。菌体直杆状,周生鞭毛运动,无芽孢,为革兰氏阴性菌。经形态鉴定、培养特征、生理生化、致病性测定及16S rDNA序列分析,确认引起该多肉植物发生褐腐病的病原是菠萝泛菌(Pantoea ananatis)。结论经过对发生细菌性褐腐病的叶片进行病原分离与鉴定,有效阻止了菠萝泛菌的远距离传播。     

Molecular identification of five commercial flatfish species by PCR–RFLP analysis of a 12S rRNA gene fragment

Refrigerated or frozen fillets of commercial flatfish species are sometimes mislabelled, and identification of those products is needed to avoid fraudulent substitution. Molecular identification of five commercial flatfish species (order Pleuronectiformes), ie Lepidorhombus whiffiagonis (megrim), Platichthys flesus (flounder), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot) and Solea vulgaris (= S solea) (sole), has been carried out on the basis of the amplification of an approximately 433 bp segment from the mitochondrial 12S rRNA gene using the polymerase chain reaction (PCR) and universal primers. Direct DNA sequencing from two PCR products for each flatfish species was carried out, and sequences were used to select six restriction enzymes. PCR products of 15 individuals of each species were cut with each enzyme, resulting in species‐specific restriction fragment length polymorphism (RFLP). The five flatfish species could be identified by application of the restriction enzyme AluI as well as by using different combinations of a pair of enzymes, ie DdeI and either AciI or MwoI. No intraspecific genetic polymorphism was found for any of the six enzymes. Results confirmed the usefulness of this technique to distinguish and genetically characterise refrigerated or frozen pieces of these five flatfish species. Copyright © 2003 Society of Chemical Industry     

超微粉碎对红米理化性质和加工特性的影响

采用超微粉碎技术加工红米,对超微红米粉的理化性质和加工特性进行了研究,以期改善红米的加工适用性。结果发现,随着红米粉粒径的减小,粉体的堆积密度、溶解度逐渐增大,糊化温度降低;冻融稳定性、酶解性质、高温持水能力、透明度、沉降性能和流动性得到显著改善。这表明超微粉碎技术可以改善红米粉的粉体性质和加工特性。在制作冷冻和冷藏食品时,红米超微粉可作为主料,但在制作速溶和方便食品时,则适合用做辅料。     

DNA条形码技术对鱼子酱物种溯源及鉴定

为探讨DNA条形码技术在鱼子酱物种鉴定中的适用性,利用细胞色素b（Cytochrome b,Cyt b）和细胞色素氧化酶I亚单位I（Cytochrome Oxidase I,COI）作为DNA条形码对鱼子酱样品进行DNA提取、聚合酶链式反应（Polymerase Chain Reaction,PCR）、测序、利用NCBI网站和BOLD鉴定系统进行基因比较分析,构建系统发育树,鉴定鱼子酱物种,对我国鱼子酱产品物种标签符合性情况进行检查。购买的40份样品,一致性鲟鱼物种基因序列相似性均在99%以上,涉及5个鲟鱼种,其中杂交种占比75%、西伯利亚鲟、施氏鲟、欧鳇、俄罗斯鲟占25%。说明Cyt b、COI作为DNA条形码可以对鱼子酱进行物种鉴定,检测的鱼子酱产品均为鲟鱼子酱,无造假,但是45%产品标签物种替代或物种标识不清。加强对产品物种标识重视及鉴定技术的开发,有助于我国鱼子酱对外贸易发展,保障我国鲟鱼产业可持续性健康发展。     

桃果实贮藏期间病原真菌的ITS rDNA序列分析与鉴定

目的:对桃果实采后病原菌进行分离与鉴定.方法:采用转接法分离桃果实采后病原真菌,经形态学观察和核糖体rDNA ITS(Internal transcribed spacer)区序列分析,对相关病原菌进行分类鉴定.结果:从采后贮藏过程中发生病害的桃果实分离到2株病原菌,经鉴定分别为美澳型核果链核盘菌(Monilinia fructicola)和葡萄座腔菌(Botryosphaeria dothidea).结论:通过对ITS rDNA序列的相关分析,对分离自采后贮藏果实中的病原真菌进行有效的分类鉴定.     

硫酸锌溶液浸泡对红香糙米萌发、锌积累量及其他营养成分的影响

采用不同浓度硫酸锌溶液（0²50 mg/L）对红香糙米浸泡12 h,用二次水萌发24 h后,测定了红香糙米发芽率、锌积累量、GABA（γ-氨基丁酸）、蛋白质及维生素C含量。结果表明:（25²50 mg/L）硫酸锌浸泡条件下,发芽红香糙米对锌有着较高的积累量,与未发芽红香糙米相比,锌含量提高了355%¹225%,参考RDA锌日推荐摄入量,（25¹00 mg/L）硫酸锌浸泡处理下的富锌发芽红香糙米即可满足人日锌摄入量要求;（0²50 mg/L）硫酸锌浸泡条件下,发芽红香糙米中营养成分GABA、蛋白质及维生素C含量与未发芽红香糙米相比,分别提高了134%²91%,9%²8%,131%¹76%;（0¹00 mg/L）硫酸锌范围内最适合红香糙米发芽、GABA的积累,100 mg/L硫酸锌处理下蛋白质、维生素C积累量达到最高;综合发芽率、锌膳食补充量及营养成分积累量,最适宜的硫酸锌浸泡浓度应为100 mg/L。      

发酵醋醅酵母菌的分离、生长特性及分子鉴定

从山西老陈醋发酵醋醅中分离纯化出2株酵母菌,编号分别为YW与YR-1.通过YW与YR-1在不同温度和pH值条件下的培养,采用浊度法测定培养菌液OD600值,确定最适生长温度与pH值,结果表明菌株YW最适生长温度为30℃,最适生长pH值为6.5;菌株YR-1最适生长温度为28℃,最适pH值为7.0.对菌株YW,YR-1的核糖体ITS+5.8S rDNA区的克隆与测序,获得菌株YW与YR-1的ITS+5.8S rDNA片段长度分别为607bp与602bp,序列已提交至Genbank(登录号为JX205095和JX205094).通过在Genbank数据库BLAST以及与亲缘关系较近模式菌株构建分子发育树,表明YW属于毕赤酵母属(Pichia),与Pichia guilliermondii亲缘关系最近,命名为Pichia guilliermondii YW; YR-1属于掷孢酵母属(Sporobolomyces),与Sporobolomyces camicolor亲缘关系最近,命名为Sporobolomyces carmicolor YR-1.     

Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

一株杀线虫活性海洋真菌BH-0531的培养特性及种属鉴定

从渤海湾底水中分离得到的一株具有杀线虫活性海洋真菌BH-0531,考察该菌株的培养特性并进行种属鉴定。结果表明,菌株BH-0531最适海水浓度为20%,最适培养基为20%海水琼脂培养基,最适氮源为硝酸钠,最适pH值为6～7,最适温度为26 ℃。根据菌株BH-0531的形态学观察结果和5.8S rDNA-ITS序列对比发现,菌株BH-0531与Acremonium potronii isolate 35（JX535064.1）高度同源（100%）,被鉴定为波氏枝顶孢霉（A. potronii）的海栖型变种：Acremonium potronii var. marine BH-0531。     

紫菜中类菌胞素氨基酸种类的确定及提取条件优化

类菌胞素氨基酸(MAAs)是小分子、水溶性化合物,具有抗紫外辐射功能。采用紫外光谱和质谱分析,确定了福建产条斑紫菜中所含的MAAs类化合物为Porphyra-334和Shinorine;采用单因素实验和正交实验确定了紫菜中MAAs类化合物的最佳提取工艺条件为:提取液初始pH2.0,提取时间1.5h,提取温度45℃,料液比为1∶8。在最佳提取工艺条件下Porphyra-334和Shinorine的提取率分别为5.124mg/g和3.941mg/g。     

Changes in the quality and in vitro digestibility of brown rice noodles with the addition of ultrasound-assisted enzyme-treated red lentil protein

The effects of red lentil protein with ultrasound-assisted enzymatic treatment on the quality of brown rice noodles were investigated. The thermostability and anti-retrogradation ability of brown rice flour decreased significantly (P < 0.05), while the elastic and viscous modulus increased after adding untreated red lentil protein. Meanwhile, the cooking loss rate of brown rice noodles increased from 4.41% to 5.68%. When the red lentil protein was treated with ultrasound and enzyme, the protein–starch interaction was enhanced, and the negative effects of protein on brown rice flour and noodles were weakened. Moreover, the rice noodles with ultrasound-assisted enzyme-treated red lentil protein showed a lower starch digestibility than brown rice noodles, and its protein digestibility was significantly higher than that of rice noodles with ultrasound or enzyme-treated protein. Therefore, the addition of ultrasound-assisted enzyme-treated red lentil protein could effectively decrease predicted glycaemic index value, improve nutritional value and produce high-quality brown rice noodles.     

Species identification and authentication of tissues of animal origin using mitochondrial and nuclear markers

We evaluated and compared the utility of mitochondrial markers viz. 16S rDNA and NADH dehydrogenase subunit 4 (ND4) and a nuclear marker viz. the actin gene to identify the specimens of animal origin for forensic identification, food regulatory control and to prevent illegal trading, poaching and conservation of endangered species. We also tested PCR fingerprinting methods like RAPD and actin barcoding to generate species-specific “fingerprints”. Our results suggested that mitochondrial markers are more efficient than nuclear markers for the purpose of species identification and authentication. Among PCR fingerprinting approaches, RAPD was proved to be more discriminatory, accurate and efficient than actin fingerprinting. Considering the present scenario in trading of vertebrate animal tissues like buffalo, cow, pig, goat, chicken, frogs, fishes and snakes etc., mitogenomics based technology proved to be efficient and reliable in resolving problems like meat adulteration and smuggling across countries.     

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT

Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene.

Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products.

Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.     

运用DNA条形码技术分析市售鱼类及制品的物种真实性

目的:运用准确快速的鱼类品种鉴定方法,对上海市售鱼类制品标识符合性进行调查。方法:利用DNA条形码技术,以动物线粒体细胞色素C氧化酶(Cytochrome C Oxidase Subunit I,COI)基因序列为鉴定靶标,对采集的63种市售鱼类样本进行序列分析比对后,分析样品的标注名称与真实物种名的一致性。结果:经序列测定和比对,13份样品的鱼类品种名称与标注名称不一致,占20.63%,此外有19份样品标注名为一类鱼的总称或俗称,占30.16%。结论:目前,我国对鱼类及其制品物种真实性鉴定研究亟待发展,鱼类标注物种名称混乱,分类不清,概念模糊,急需规范化。      

Species Identification of Beans, Peas and Other Legumes by RAPD-PCR after DNA Isolation using Membrane Columns

Forty legume seed samples representing 11 species were selected to investigate the identification of food legume species by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Template DNA was extracted from seed meal and purified using a commercial membrane column kit. Amplification was performed with commercial RAPD analysis beads and six commercially available decamer nucleotide primers. Electrophoresis of the amplicons on polyacrylamide gels and subsequent silver staining resulted in RAPD profiles from all samples of one species with a given primer that differed from those of the other species studied. Unambiguous identification of six food legume species, common beans (Phaseolus vulgaris), soybeans (Glycine max), peas (Pisum sativum), chickpeas (Cicer arietinum), lentils (Lens culinaris), and alfalfa (Medicago sativa), was obtained applying three of the primers. The technique may also be suitable to identify the remaining five species investigated, scarlet runner beans (Phaseolus coccineus), lima beans (P. lunatus), green gram (Vigna radiata), broadbeans (Vicia faba), and blue lupin seeds (Lupinus angustifolius). Using a standard protocol, amplification with Taq DNA polymerase instead of RAPD beads generated RAPD profiles only from soybeans, peas, two of four chickpea samples, green gram, and lupin seeds.     

柽柳核纤孔菌分子鉴定及基于ITS序列的系统发育分析

在对形态特征进行初步鉴定的基础上,对河南省郑州市分离的柽柳核纤孔菌6-27菌株的rDNA ITS区段进行克隆测序,并对ITS序列进行核酸序列数据库GenBank同源性检索比对.将从GenBank检索获得的22个相关分类元真菌的ITS序列连同柽柳核纤孔菌6-27菌株ITS序列一起用于系统发育分析,结果表明:形态鉴定与ITS序列分析结果一致,测定菌株为柽柳核纤孔菌.供试的23份材料被聚为6个类群,其中7个核纤孔菌属菌株和1个纤孔菌属菌株被聚在类群A;另一个来自南半球阿根廷的Inocutis jamaicensis 4508与来自印度的Fomitiporella caryophylli CBS 448.76被聚类在类群B,与类群A为大姊妹类群,与类群C为小姊妹类群;类群C包括2个Fulvifomes真菌;纤孔菌属,木层孔菌属,Mensularia,Fuscoporia等不同属菌株被聚类在类群D,E和F.相关分类元系统发育分析支持核纤孔菌为独立的属分类单元,Fulvifomes属分类地位比较稳定,纤孔菌属与木层孔菌属真菌具有较大的异质性,其分类地位有待于进一步规范和统一.     

Detection and identification of marine fish mislabeling in Guangzhou's supermarkets and sushi restaurants using DNA barcoding

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.     

烟草废水中红色酵母菌的分离鉴定及其胞内色素和油脂的初步研究

该研究采用马铃薯葡萄糖琼脂（PDA）培养基从烟草废水中分离红色酵母菌,通过形态观察、生理生化试验及分子生物学技术对其进行鉴定,并初步探讨其发酵马铃薯葡萄糖水（PDW）产油脂和色素的潜力。结果表明,从烟草废水中分离到一株红色酵母菌YH0902,并鉴定其为一株近玫色锁掷孢酵母（Sporidiobolus pararoseus）。菌株YH0902在马铃薯葡萄糖水（PDW）中培养时,细胞内可积累类胡萝卜素和油脂,30 ℃、150 r/min条件下培养4 d,菌体湿质量为（48.53±0.36） g/L,干质量为（10.63±0.15） g/L,胞内色素产量为（4.33±0.19） mg/L,油脂产量为（3.30±0.17） g/L。因此,菌株YH0902在微生物源天然色素及油脂开发领域具有一定的应用前景。