Identification of goose,mule duck,chicken, turkey,and swine in foie gras by species-specific polymerase chain reaction

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.     

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.     

Identification by proteomic analysis of early post-mortem markers involved in the variability in fat loss during cooking of mule duck "foie gras"

Fat loss during cooking of duck "foie gras" is the main quality issue for both processors and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yield remains high and uncontrolled. To better understand the biological basis of this phenomenon, a proteomic study was conducted. To analyze the protein fraction soluble at low ionic strength (LIS), we used bidimensional electrophoresis and mass spectrometry for the identification of spots of interest. To analyze the protein fraction not soluble at low ionic strength (NS), we used the shotgun strategy. The analysis of data acquired from both protein fractions suggested that at the time of slaughter, livers with low fat loss during cooking were still in anabolic processes with regard to energy metabolism and protein synthesis, whereas livers with high fat loss during cooking developed cell protection mechanisms. The variability in the technological yield observed in processing plants could be explained by a different physiological stage of liver steatosis.     

实时荧光定量聚合酶链式反应快速鉴定三种鳕鱼

为鉴定常见的3种鳕科鳕鱼(大西洋鳕鱼、太平洋鳕鱼和黑线鳕),选用16S rDNA基因设计区分鳕科和非鳕科鱼类的引物,选用线粒体Cytb基因设计区分3种鳕鱼的物种特异性引物,建立实时荧光定量聚合酶链式反应(qPCR)体系,对其进行物种分析。结果表明,16S rDNA qPCR体系及3种鳕鱼品种特异性qPCR体系均具有良好的性能,结合标准曲线,可以对单一或混合样品中的目标鳕鱼进行定量检测。在模拟混合样品中目标鳕鱼的相对检测灵敏度可达0.01%。通过对13种市售样品的检测,本研究设计的qPCR体系可检测出原料及深加工产品中的鳕鱼成分。综上,所建立的qPCR体系具有很强的实用性,能满足日常检测的要求,并有望作为未来鳕鱼市场管理的检测方法。     

PCR技术在快生型大豆根瘤菌菌株鉴别上的应用

以基因外重复的回文因子（ｒｅｐｅｔｉｔｉｖｅｅｘｔｒａｇｅｎｉｃｐａｌｉｎｄｒｏｍｉｃ，ＲＥＰ）和肠杆菌基因间重复一致序列（ｅｎｔｅｒｏｂａｃｔｅｒｉａｌｒｅｐｅｔｉｔｉｖｅｉｎｔｅｒｇｅｎｉｃｃｏｎｓｅｎｓｕｓ，ＥＲＩＣ）的碱基顺序设计出的引物为引物，用ＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）技术分别扩增了８株快生型大豆根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ）的总ＤＮＡ，得出了具有菌株特异性的扩增产物电泳图谱，称ＲＥＰ－ＥＲＩＣＰＣＲ指纹图谱，将所得图谱进行性状编码后，用计算机数值分类系统进行平均连锁聚类分析，得出这８个菌株的聚类树状图。这一聚类结果与用其它方法聚类结果基本一致，说明ＲＥＰ－ＥＲＩＣＰＣＲ是一种鉴别快生型大豆根瘤菌菌株的经济、快速而又可靠的新方法。     

Polymerase Chain Reaction assay for the detection of Kudoa paniformis and Kudoa thyrsites in Pacific Hake (Merluccius productus)

Myoliquefaction of Pacific hake has been attributed to proteolytic action associated with parasitic infection. Among the two infecting species of Kudoa, Kudoa paniformis and Kudoa thyrsites, the former is reported to be more virulent for the "soft flesh" phenomenon in Pacific hake. The objective of this research was to develop a sensitive and specific polymerase chain reaction (PCR) assay to detect infection of hake by K. paniformis. Primers based on specific regions ( approximately 1562 bp) of the small subunit ribosomal DNA of K. paniformis successfully amplified the target DNA segments from both spore and muscle extracted DNA templates. DNA sequencing confirmed the veracity of this method to distinguish parasitic infection by K. paniformis versus K. thyrsites. The established PCR method was applied to investigate Kudoa infection in 44 Pacific hake samples using DNA extracted from muscle and/or spores, and the results were compared to infection evaluated by microscopic examination of extracted spores.     

Quantification of Common Wheat Adulteration of Durum Wheat Pasta Using Real‐Time Quantitative Polymerase Chain Reaction (PCR)

Common wheat adulteration of durum wheat pasta was quantified using real‐time duplex polymerase chain reaction (PCR). The total DNA content of pasta was determined by amplifying part of a wheat gene encoding a lipid transfer protein, and common wheat DNA was quantified by amplifying part of the puroindoline‐b gene. Under the conditions defined by this study, for pasta with a theoretical adulteration of 3%, the experimentally determined mean value was 2.6–3.4%, depending on drying temperature. Pure durum wheat pastas were distinguished from adulterated pastas without ambiguity. This study demonstrates the feasibility of using real‐time duplex PCR to quantify common wheat adulteration of pasta dried at high temperature, quantification that was impossible with the French official peroxidase‐marker method.     

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.     

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).     

苹果锈果类病毒在新疆的发生检测及其序列分析

苹果锈果病一度被认为是病毒病害，直到日本学者Hashimoto等首次报道了ASSVd全序列，才确认为类病毒病害 [4]。苹果锈果类病毒（Apple scar skin viroid，ASSVd）属于Pospiviroidae科，主要侵染苹果和梨[1]，是我国苹果产区的一种重要病害，果树感染后症状主要表现在果实上，随品种和感病阶段不同，有花脸、锈果、裂果、果实变小皱缩等，严重时，整树的果实都不堪食用，失去经济价值[2]。国内早在三十年代对苹果锈果病有报道，但病原一直未定。早在上个世纪五十年代，刘福昌先生对苹果锈果病和梨树的关系[3]，进行了大量的研究，八十年代陈炜等做了大量的研究工作[1，2]，。迄今，苹果锈果病在辽宁、陕西等不同苹果主产区已有报道,但在新疆地区尚未报道，另外，不同地区的锈果类病毒是否同源，也未有相关研究报道。本研究首次对新疆地区苹果锈果=病进行鉴定和序列分析，为该类病毒的同源性分析和防治策略提供依据。     

Quantitation of total folate in whole blood using LC-MS/MS

An accurate method for measuring whole blood total folate using liquid chromatography with tandem mass spectrometry is described and compared to GC/MS and a chemiluminescence assay. Whole blood from normal adults (n = 15) was fortified with a [(13)C(6)]para-aminobenzoic acid (pABA) internal standard and treated with 12.1 N hydrochloric acid at 110 degrees C for 4 h to hydrolyze all folates to pABA. Contaminants in the hydrolysate were adsorbed onto a C18 SPE cartridge. The eluate containing the folate catabolite pABA was partitioned into ethyl acetate and methylesterified with trimethylsilyldiazomethane. The methyl-pABA derivatives were quantified by positive-ion atmospheric pressure chemical ionization (APCI)LC-MS/MS. An isocratic mobile phase of acetonitrile-water (70:30) (v/v) on a C18 analytical column was used with a postcolumn reagent of 0.025% formic acid. The limit of quantitation for folate was 56.6 nmol/L RBC, and the limit of detection was 22.6 nmol/L RBC. Folate levels as determined by LC-MS/MS correlated well with the chemiluminescence assay and a GC/MS method. This new LC-MS/MS method provides enhanced sample throughput (n = 36 per day) as compared to GC/MS methods. LC-MS/MS will enable accurate measurements of red blood cell (RBC) folate in nutrition surveys and clinical trials.     

Quantitation of added water in milk by using vapor pressure osmometry

A vapor pressure osmometer (VPO) was successfully studied in 1975. An improved osmometer with greater range and temperature stability was evaluated. Repeatability on 290 mOs/kg standard solution was 289.5 +/- 1.08. The mean and standard deviation on 36 water-free milk samples was 280.5 +/- 4 compared to 280.1 +/- 3 in the previous study. Instrument data from 2 osmometers and 2 cryoscopes produced acceptable chi-square values, and the correlation coefficients between methods were better than 0.99 for samples containing 1--21% added water. In a survey involving 760 samples, more than 37% of producer samples in one cheese plant contained greater than 3% added water. Additional evidence of added water in market milk samples suggests the immediate application of the VPO to help prevent milk adulteration. The vapor pressure osomometer method for quantitating added water in milk has been adopted as official first action.     

Determination of 4,4'-dinitrocarbanilide (DNC), the active component of the antifertility agent nicarbazin, in chicken, duck, and goose plasma

4,4'-Dinitrocarbanilide (DNC) was extracted from chicken, duck, and goose plasma and isolated by reversed-phase high-performance liquid chromatography. DNC was detected by ultraviolet absorbance at 347 nm and quantified by comparison to a calibration standard. Recovery data were determined by analyzing DNC-fortified control plasma. The mean recovery of DNC in fortified chicken plasma samples was 99.7 +/- 1.9% for 0.18 and 9.1 ppm DNC, and in fortified duck and goose plasma samples was 99.5 +/- 4.9% and 101.4 +/- 4.5%, respectively, for 0.18, 9.1, and 18 ppm DNC.     

Quantitation of odor-active compounds in rye flour and rye sourdough using stable isotope dilution assays

Application of the aroma extract dilution analysis on a flavor distillate prepared from freshly ground rye flour (type 1150) revealed 1-octen-3-one (mushroom-like), methional (cooked potato), and (E)-2-nonenal (fatty, green) with the highest flavor dilution (FD) factors among the 26 odor-active volatiles identified. Quantitative measurements performed by stable isotope dilution assays and a comparison to the odor thresholds of selected odorants in starch suggested methional, (E)-2-nonenal, and hexanal as contributors to the flour aroma, because their concentrations exceeded their odor thresholds by factors >100. Application of the same approach on a rye sourdough prepared from the same batch of flour revealed 3-methylbutanal, vanillin, 3-methylbutanoic acid, methional, (E,E)-2,4-decadienal, 2,3-butanedione, and acetic acid as important odorants; their concentrations exceeded their odor thresholds in water and starch by factors >100. A comparison of the concentrations of 20 odorants in rye flour and the sourdough made therefrom indicated that flour, besides the fermentation process, is an important source of aroma compounds in dough. However, 3-methylbutanol, acetic acid, and 2,3-butanedione were much increased during fermentation, whereas (E,E)-2,4-decadienal and 2-methylbutanal were decreased. Similar results were obtained for five different flours and sourdoughs, respectively, although the amounts of some odorants in the flour and the sourdough differed significantly within batches.     

Evaluating homogeneity of LL601 rice in commercial lots using quantitative real-time PCR

Homogeneity analysis was performed on four distinctive commercial lots, derived from the 2006 rice harvest in the United States. Lots that had previously been tested and suspected to have some level of LL601 were selected to determine lot homogeneity. LL601 infiltration in the lots was low and estimated to contain <0.01% (sigma = 0.026), 0.014% (sigma = 0.020), 0.054% (sigma = 0.043), and 0.074% (sigma = 0.031) LL601. Lots were analyzed statistically as a one-way classification, or one-factor experiment, to assess the presence of strata within the lot. A p value of 0.05 or lower is needed to declare statistical significance and would suggest significant differences among the samples. The data revealed p values ranging between 0.105 and 0.607. The calculated p values for all lots were greater than the critical value of 0.05. Samples taken from different locations throughout these four commercial lots did not show statistically significant stratifications within the lot.     

Quantitation of clovamide-type phenylpropenoic acid amides in cells and plasma using high-performance liquid chromatography with a coulometric electrochemical detector

A high-performance liquid chromatography (HPLC) method was developed for measuring the concentrations of clovamide-type phenylpropenoic acid amides (N-caffeoyldopamine and N-caffeoyltyramine) in cell and plasma samples. The separation was performed on a Nova-Pak C18 column using an isocratic buffer with a coulometric electrochemical detector with four electrode channels. Using the HPLC method, N-caffeoyldopamine and N-caffeoyltyramine could be detected with good peak resolutions at respective retention times (4 and 6.4 min). The calibration curves were linear over the ranges (0.1 and 100 microM), and their lower limit of detection was as little as 100 fmol. For quantifying N-caffeoyldopamine and N-caffeoyltyramine in cell and plasma samples, the samples were extracted by extraction methods with more than 95% recoveries. After extraction, the amides were detected with the same sensitivity, peak resolutions, and retention times. Using this method, plasma concentrations of N-caffeoyltyramine were determined in blood samples collected at 12, 24, 30, 36, 48, 60, and 75 min after the oral administrations of N-caffeoyltyramine (0.5 mg and 2 mg/30 g body weight). This HPLC method with an electrochemical detector is the first reported method able to quantify N-caffeoyldopamine and N-caffeoyltyramine in biological samples with excellent detection limits, peak resolutions, discrete retention times, and consistent reproducibility.     

Quantitation of toxic arsenic species and arsenobetaine in Pacific oysters using an off-line process with hydride generation-atomic absorption spectroscopy

An off-line process-based speciation technique was devised here to quantitatively determine toxic inorganic arsenic (iAs), methylarsonic acid (MA), dimethylarsinic acid (DMA), and the dominant, albeit virtually nontoxic, arsenobetaine (AB) in Pacific oysters (Crassostrea gigas). Oysters were extracted with fresh methanol-water (8+2), and this was replicated three times. They were then evaporated to near dryness and subsequently redissolved in pure water; defatting was then performed with a C18 cartridge. The trace hydride active arsenic species, that is, iAs, MA, and DMA, in the defatted solutions were determined with a sensitive hydride generation-packed coldfinger trap-atomic absorption spectrometric (HG-PCFT-AAS) coupled system. The arsenicals that were desorbed from the cation-exchange resin (Dowex 50W-X8) in the washings of 4 M NH3 were categorized on the basis of AB + DMA. The total quantity of arsenic in the recovered AB + DMA was determined with a commercial hydride generation-atomic absorption spectrometric (HG-AAS) system, and finally, AB was calculated from (AB + DMA) - DMA. The average concentrations of iAs, MA, DMA, AB, and total arsenic (TAs) in the oysters collected from six aquacultural sites along the west coast of Taiwan were, respectively, 0.15, 0.06, 0.64, 6.93, and 13.74 mg kg(-1) of dry weight. AB was the major species, whereas iAs (arsenite + arsenate) were the most toxic species, although the iAs made up only approximately 1% of the TAs in the oysters. The lifetime target cancer risk, as determined by the concentration of iAs on a fresh weight basis in the oysters, was well below the ordinary health protection criteria (10(-6)).     

Quantification of ammonia-oxidising bacteria in limed and non-limed acidic coniferous forest soil using real-time PCR

Ammonia-oxidising bacteria (AOB) in limed and non-limed acidic coniferous forest soil were investigated using real-time PCR. Two sites in southern Sweden were studied, 244 Åled and Oxafällan. The primers and probe used earlier appeared to be specific to the 16S rRNA gene of AOB belonging to the β-subgroup of the Proteobacteria [Appl. Environ. Microbiol. 67 (2001) 972]. Plots treated with two different doses of lime, 3 or 6 t ha⁻¹, were compared with non-limed control plots on two occasions during a single growing season. Three different soil depths were analysed to elucidate possible differences in the density of their AOB communities. The only clear effect of liming on the AOB was recorded in the beginning of the growing season at 244 Åled. In samples taken in April from this site, the numbers of AOB were higher in the limed plots than in the control plots. At the end of the growing season the AOB communities were all of a similar size in the different plots at both sites, irrespective of liming. The number of AOB, determined using real-time PCR, ranged between 6×10⁶ and 1×10⁹ cells g⁻¹ soil (dw) at the two sites, and generally decreased with increasing soil depth. The results showed no correlation between community density and potential nitrification. This may indicate a partly inactive AOB community. Furthermore, more than 10⁷ cells g⁻¹ soil (dw) were recorded using real-time PCR in the control plot at 244 Åled, although Bäckman et al. [Soil Biol. Biochem. 35 (2003) 1337] detected no AOB like sequences in the same plots using PCR followed by DGGE. Taken together our results strongly suggest that the primers and probe set used are not well suited for quantifying AOB in acidic forest soils, which is probably due to an insufficient specificity. This shows that it is extremely important to re-evaluate any primers and probe set when used in a new environment. Consideration should be given to the specificity and sensitivity, both empirically and using bioinformatic tools.     

Determination of 4,4'-dinitrocarbanilide (DNC), a component of Nicarbazin,in Canada goose (Branta canadensis) eggshells using high-performance liquid chromatography

A method was developed using high-performance liquid chromatography to assay 4,4'-dinitrocarbanilide (DNC), the active ingredient in Nicarbazin, in eggshells collected from Canada geese fed a formulated feed fortified with Nicarbazin at doses of 0, 125, 250, and 500 microg/g. The method was developed using chicken eggshells fortified with DNC. The method was used to quantify DNC in both the shell-associated membranes and the calcified shell extracellular matrix. These values were compared to those obtained for a composite sample consisting of both the membranes and the calcified shell extracellular matrix. The validated method was used to quantify DNC in eggshells from geese fed fortified feed to ascertain the effect of Nicarbazin feed concentration on shell DNC concentration. DNC levels in the eggshells were highly correlated with feed dose.     

利用马尔柯夫过程预测蒙阴县土地利用/覆被格局变化

为了解蒙阴县土地利用/覆被的变化规律及其未来变化,由蒙阴县1997年和2007年2个时期的遥感图像获得的土地利用/覆被数据,确定土地利用/覆被的初始状态矩阵和转移概率矩阵,据此用马尔柯夫模型对研究区的土地利用/覆被格局的未来变化趋势进行了预测。结果表明:蒙阴县土地利用/覆被格局正处在一种变化状态,耕地、草地、水域及水利设施用地、未利用地面积在今后一段时期内仍呈减少趋势,到2017年耕地面积比例较2007年减少3.22%,林地、城镇村及工矿交通用地仍呈增加趋势,其中以林地增长幅度较大,到2017年其占比将达到43.45%。根据模拟结果可调整各类土地利用/覆被格局,为宏观决策提供科学依据。