Identification of virgin olive oil from different cultivars by analysis of DNA microsatellites

DNA analysis enables genome fingerprinting with consequent identification of different individuals. In the agro-food industry, this can have interesting applications for the identification of species and cultivars of both raw materials and processed food. In this investigation, the efficiency of DNA microsatellite analysis in identifying virgin olive oils from different cultivars was evaluated. Ten virgin oils were obtained in the laboratory from olives of 10 different cultivars and the DNA extracted from the cell residues, recovered by oil centrifugation, was used as a template with seven different primer pairs of microsatellite sequences. The electrophoretic patterns showed an adequate level of amplification and were identical to those obtained from leaves and drupes of the same cultivar. By analyzing all the patterns obtained, the smallest number of microsatellites able to distinguish the examined oils was established and an identification key for the different oils was developed.     

Development of a DNA microarray for authentication of ginseng drugs based on 18S rRNA gene sequence

Ginseng drugs, derived from underground parts of Panax species (Araliaceae), are the most important group of herbal medicines in the Orient. Previously, the nucleotide sequences of the nuclear 18S rRNA gene of 13 Panax taxa were determined, as were the specific polymorphic nucleotides for identification of each species. On the basis of the nucleotide difference, a DNA microarray (PNX array) was developed for the identification of various Panax plants and drugs. Thirty-five kinds of specific oligonucleotide were designed and synthesized as probes spotting on a decorated glass slide, which included 33 probes corresponding to the species-specific nucleotide substitutions and 2 probes as positive and negative controls. The species-specific probes were of 23-26 bp in length, in which the substitution nucleotide was located at the central part. Triplicate probes were spotted to warrant accuracy by correcting variation of fluorescent intensity. Partial 18S rRNA gene sequences amplified from Panax plants and drugs as well as their derived health foods were fluorescently labeled as targets to hybridize to the PNX array. After hybridization under optimal condition, specific fluorescent patterns were detected for each Panax species, and the analyzed results could be indicated as barcode patterns for quick distinction. The developed PNX array provided an objective and reliable method for the authentication of Panax plants and drugs as well as their derived health foods.     

Empirical Bayes analysis of variance component models for microarray data

A gene-by-gene mixed model analysis is a useful statistical method for assessing significance for microarray gene differential expression. While a large amount of data on thousands of genes are collected in a microarray experiment, the sample size for each gene is usually small, which could limit the statistical power of this analysis. In this report, we introduce an empirical Bayes (EB) approach for general variance component models applied to microarray data. Within a linear mixed model framework, the restricted maximum likelihood (REML) estimates of variance components of each gene are adjusted by integrating information on variance components estimated from all genes. The approach starts with a series of single-gene analyses. The estimated variance components from each gene are transformed to the “ANOVA components”. This transformation makes it possible to independently estimate the marginal distribution of each “ANOVA component.” The modes of the posterior distributions are estimated and inversely transformed to compute the posterior estimates of the variance components. The EB statistic is constructed by replacing the REML variance estimates with the EB variance estimates in the usual t statistic. The EB approach is illustrated with a real data example which compares the effects of five different genotypes of male flies on post-mating gene expression in female flies. In a simulation study, the ROC curves are applied to compare the EB statistic and two other statistics. The EB statistic was found to be the most powerful of the three. Though the null distribution of the EB statistic is unknown, a t distribution may be used to provide conservative control of the false positive rate.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

Identification of a novel glycoside, leptosin, as a chemical marker of manuka honey

As a preliminary study, we have found that honey from manuka (Leptospermum scoparium) in New Zealand inhibits myeloperoxidase (MPO) activity. In this study, using a chromatographic technique, we isolated two active compounds for MPO-inhibition from manuka honey. One is methyl syringate (MSYR), and the other was identified as a novel glycoside of MSYR, methyl syringate 4-O-β-D-gentiobiose, which has been named "leptosin" after the genus Leptospermum . The amount of the glycoside ranged from 0.2 to 1.2 μmol/g honey. Leptosin was only found in honeys from the Oceania region, and abundantly in manuka honey including jelly bush honey from Leptospermum polygalifolium in Australia. Therefore, leptosin may be a good chemical marker for manuka honey. Interestingly, the concentration of leptosin in manuka honey was positively correlated with the unique manuka factor (UMF) value, which is expressed as phenol equivalents of its bactericidal activity.     

Flexible microarray construction and fast DNA hybridization conducted on a microfluidic chip for greenhouse plant fungal pathogen detection

This study employed a microfluidic method in which probe creation does not require pin-spotting and fast hybridization is conducted on the same microarray chip for the detection of three greenhouse pathogens ( Botrytis cinerea, Didymella bryoniae, and Botrytis squamosa). In this method, 16 oligonucleotide probe line arrays were created on a glass substrate by a microfluidic printing method. Then, low amounts of the DNA samples (1 fmol of oligonucelotides or 1.4 ng of PCR products) were introduced into the microchannels that were orthogonal to these probe lines. The hybridizations of 16 samples (21-mer complementary oligonuleotides and approximately 260 bp PCR products) were fulfilled at the channel-probe line intersections and in a short time (minutes). The optimization of probe immobilization and sample hybridization are described in detail. The method successfully detected and discriminated between two 260 bp PCR products with a one-base-pair difference from closely related greenhouse plant fungal pathogens (B. cinerea and B. squamosa).     

利用羽毛对鸽子进行分子性别鉴定

鸽子是一种单态鸟,并且是典型的"一夫一妻制",给配对和人工繁殖带来了很大的困难.以雏鸽(Columbalivia Gmelin)的羽毛为材料,采用PCR方法扩增染色体螺旋蛋白基因.结果显示,雏鸽羽毛可以提取出高质量的DNA,雄鸽仅有CHD-Z(450 bp)1条带,而雌鸽有CHD-W(380 bp)和CHD-Z(450 bp)2条带.这种性别鉴定技术准确,并且对动物无伤害性,在生产管理上有广阔的应用前景.     

PCR-RFLP analysis of mitochondrial DNA: a reliable method for species identification

A method for identification of game species has been developed on the basis of the amplification of a specific part of the mitochondrial genome (tRNA(Glu)/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several game species, the obtained 464-bp-long PCR products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphism (RFLP). Even closely related deer species could be distinguished by application of one or two RE. Natural polymorphisms of the target sequence within one species were examined for red deer (Cervus elaphus), and base pair substitutions were identified affecting the RFLP pattern.     

Profiling of hepatic gene expression of mice fed with edible japanese mushrooms by DNA microarray analysis: comparison among Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus

To compare and estimate the effects of dietary intake of three kinds of mushrooms (Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus), mice were fed a diet containing 10-14% of each mushroom for 4 weeks. Triacylglycerol in the liver and plasma decreased and plasma cholesterol increased in the P. ostreatus-fed group compared with those in the control group. Cholesterol in the liver was lower in the G. frondosa-fed group than in the control group, but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the liver revealed differences of gene expression patterns among mushrooms. Ctp1a and Fabp families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes, such as Mx1, Rsad2, and Oas1, were upregulated.     

Identification of monoclonal antibodies against 2,4-D herbicide by ELISA and DNA sequencing

Seven hybridoma clones, E2/G2, E2/B5, E4/C2, G5/E10, F6/C10, B5/C3, and B7, produced within one fusion experiment in 1991 and the clone E4/C2 originated from 1995 were characterized by sequencing of genes coding for variable domains of the antibodies against 2,4-D herbicide. Amino acid sequences of selected antibodies, deduced from DNA analysis, were confirmed by mass spectrometry. Surprisingly, nucleotide sequence analysis of the clones E2/G2 and E2/B5, producing the most sensitive antibodies, proved to have sequence homology of their variable domains, although the IC(50) values determined for these antibodies 9 years prior to the DNA analysis were 2.0 and 8.2 ng/mL, respectively. The same findings arose from the comparison of the immunochemical to DNA data established for G5/E10, F6/C10, and B5/C3 clones producing antibodies with IC(50) values in the range of 26.3-43.1 ng /mL. The clone E4/C2, originating from the later fusion experiment, did not share nucleotide homology with any of the examined clones. Data obtained by ELISA, immunosensor, and DNA analysis within a 9 year period are discussed with respect to hybridoma stability, methodic artifacts, measurement reliability, and other possible factors influencing the result interpretation.     

Effects of ingested turmeric oleoresin on glucose and lipid metabolisms in obese diabetic mice: a DNA microarray study

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric oleoresin is more effective than the use of either curcuminoids or the essential oil alone.     

Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis

PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.     

Volatile composition and contribution to the aroma of spanish honeydew honeys. Identification of a new chemical marker

Honeydew honeys from holm-oak, oak, and forest were isolated for aroma compounds by simultaneous distillation-extraction and analyzed by gas chromatography-mass spectrometry. In all, 66 volatile components were identified and quantified. trans-Oak lactone, a characteristic volatile component of oak wood, is proposed as a new chemical marker for the plant origin of honeydew honeys. Other compounds, such as aminoacetophenone and propylanisol, could be considered characteristic of holm-oak honeydew honeys. A total of 15 volatile compounds presented odor activity values (OAVs) greater than 1, with phenylacetaldehyde and beta-damascenone being those with the highest OAVs.     

一株具有固氮功能的烟草根际微生物的鉴定及其初步效应

应用16S rDNA序列分析构建系统发育树,结合生理指标、生化反应,对分离自烤烟根际的固氮菌菌株N05进行了分类鉴定,并通过小区试验探讨其对烤烟生产的效应。结果表明,自生固氮菌N05属于产碱菌属（Alcaligenes）,粪产碱菌（Alcaligenes faecalis）。将固氮菌N05制成菌肥,烤烟移栽时施入（30 kg/hm2）同时施用80% 的氮肥（B+80% N）,与全量施用氮肥（FN）相比,B+80% N烤烟根际固氮菌的数量平均提高3.6倍,放线菌的数量显著降低;圆顶期烤烟根际土壤中除Mg 元素的有效性略有降低外,P、K、Ca、Cu、Zn、Fe和 Mn 等元素的有效性均有不同程度的提高,提高幅度在 2.51%～ 46.08%。烤烟杀青样中 N 的平均含量也高于FN。在减施氮肥的情况下,应用固氮菌肥可提高烤烟根际固氮菌数量和矿质元素的有效性。     

2个马铃薯杂交组合F1的SSR鉴定

杂交育种是马铃薯新品种选育的重要方法, 其杂种F₁真实性鉴定是获得目标性状单株的关键环节。为选育优质、高产、抗病性及抗旱性强的马铃薯新品种, 用马铃薯品种"费乌瑞它"(Favorita)分别与"J07-6"和"陇薯3号"杂交, 获得了杂种F1代。本试验利用SSR标记技术对"Favorita"×"J07-6"、"Favorita"×"陇薯3号"2个杂交组合F1共86个单株的真实性进行了鉴定。试验从43对SSR引物中筛选出2对适宜引物STM1049和S7。利用这2对引物进行PCR扩增, 将"Favorita"×"J07-6"杂交种F1和"Favorita"×"陇薯3号"杂交种F1的SSR带型划分为双亲互补型、缺失型、父本型和母本型4种类型。依据SSR带型特征, 从"Favorita"×"J07-6"和"Favorita"×"陇薯3号"2个杂交组合F₁单株中分别鉴定出真杂种34个和27个, SSR分子标记技术用于马铃薯杂交种真实性鉴定是可靠的。该研究结果可为马铃薯杂交种优良株系选育提供依据。     

Hepatic gene expression of the insulin signaling pathway is altered by administration of persimmon peel extract: a DNA microarray study using type 2 diabetic Goto-Kakizaki rats

Persimmon (Diospyros kaki) is a very popular fruit in East Asian countries, but its peels are not consumed despite the fact that they contain many antioxidants such as carotenoids and polyphenols. We prepared a fat-soluble extract from persimmon peel (PP) and fed type 2 diabetic Goto-Kakizaki (GK) rats an AIN-93G rodent diet supplemented with persimmon peel extract (PP diet) for 12 weeks. Compared with the control AIN-93G diet, the PP diet significantly reduced plasma glutamic-pyruvate transaminase activity, with accumulation of β-cryptoxanthin in the liver. DNA microarray analysis revealed that the PP diet altered hepatic gene expression profiles. In particular, expression of insulin signaling pathway-related genes was significantly enriched in differentially expressed gene sets. Moreover, Western blotting analysis showed an increase in insulin receptor beta tyrosine phosphorylation in rats fed the PP diet. These data suggest that the PP diet improves insulin resistance in GK rats.     

Genetic methods for the detection of microbial pathogens. Identification of enterotoxigenic Escherichia coli by DNA colony hybridization: collaborative study

Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures of E. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-1 adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.     

Furosine: a suitable marker for assessing the freshness of royal jelly

Fifteen commercial samples of royal jelly, consisting of 10 imported samples, and 5 samples of known origin obtained freshly harvested from beekeepers, were analyzed for protein, lysine, and furosine content. In addition, a commercial sample of royal jelly, at the beginning of its commercial shelf life, was stored for 10 months both at 4 degrees C and at room temperature in order to assess the development of the Maillard reaction (furosine) and relative nutritional damage (blocked lysine). The commercial royal jelly products contained different amounts of furosine, ranging from 37.1 to 113.3 mg/100 g protein, evidence of different storage times and conditions. The average furosine content of the royal jelly samples of known origin and harvesting was significantly lower than that of the imported samples (41.7 versus 73.6 mg/100 g protein, respectively). With regard to shelf life, furosine content increased significantly from 72.0 mg/100 g protein to 500.8 mg/100 g protein after 10 months of storage at room temperature, while it increased to a much lower level (100.5 mg/100 g protein) when the royal jelly was stored at 4 degrees C. However, nutritional damage, expressed as blocked lysine (calculated indirectly from the furosine content), was minor or negligible, 11.9 and 2.3% of total lysine, in samples stored at room temperature and at 4 degrees C, respectively. Lysine was determined by an innovative procedure based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that furosine is a suitable index for assessing the quality and freshness of royal jelly.