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.     

Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction

A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.     

Establishment and application of a fluorescent polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying porcine,caprine, and bovine meats

A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the species-specific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy.     

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.     

不同动物部分组织基因组甲基化程度的差异分析

应用甲基敏感扩增多态性(MSAP)技术,检测和分析了猪、牛、羊、小鼠、鸡和鸭基因组的甲基化程度。结果表明,对CCGG位点,实验中检测的几种动物的甲基化程度多数在0.40￣0.50之间(不包括牛);不同动物来源相同组织基因组的甲基化程度不同,相同动物不同组织基因组的甲基化模式具有特异性;同一种动物,组织基因组的甲基化程度一般都高于血液基因组;另外,哺乳动物与禽类基因组甲基化程度未发现较大的差别,但哺乳动物基因组全甲基化位点较多,半甲基化位点较少。     

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.     

Liquid chromatographic identification of meats

Raw beef, pork, veal, lamb, chicken, turkey, and duck have been identified with a liquid chromatographic (LC) method. Meat samples are blended in water, and soluble proteins in the aqueous blends are separated by the LC method. Meat cuts and parts from same species had similar chromatographic profiles and differed only quantitatively. However, meat cuts or parts from different species resulted in different chromatographic profiles. The qualitative and quantitative chromatographic differences among meat species were used for their identification. The LC method applies only to fresh and frozen meats. It is simple, rapid, and reliable, and can be used for quantitative detection of meat species in unheated meat blends.     

Identification of the clam species Ruditapes decussatus (Grooved carpet shell), Venerupis pullastra (Pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell)by PCR-RFLP

PCR-RFLP analysis has been applied to the identification of three clam species: Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell). PCR amplification was carried out using a set of primers designed from the DNA nucleotide sequences reported for alpha-actins from humans and various animals. Restriction endonuclease analysis based on sequence data of the PCR products of each clam species revealed the presence of species-specific polymorphic sites for MaeIII and RsaI endonucleases. Electrophoretic analysis of the amplicons digested with MaeIII and RsaI produced species-specific profiles that allowed the genetic identification of the three clam species.     

Quantitative thin layer chromatographic multi-sulfonamide screening procedure: collaborative study

A thin layer chromatographic procedure suitable for detection of multiple sulfonamides at 0.1 ppm was studied in an interlaboratory collaborative study. Sulfamethazine, sulfadimethoxine, and sulfaquinoxaline were variously analyzed in liver and muscle tissues from swine, turkey, and duck. The average recovery for all drugs across all tissues was 95%. The corresponding repeatability and reproducibility were 7.7% and 10.5%, respectively.     

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.     

Identification of species-specific DNA in feedstuffs

Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.     

Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets by polyclonal antibody-based enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the species identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. The assay was performed in two different formats, microtiter plates and immunostick tubes, and uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of grouper (anti-GSP), wreck fish (anti-WSP), and Nile perch (anti-PSP). The antibodies were made species-specific by blocking them with the heterologous soluble muscle proteins. Immunorecognition of polyclonal antibodies adsorbed to their specific fish samples was made with swine antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of the species studied.     

Wildfowl population trends in Mexico, 1961-2000: a basis for conservation planning

We analysed population trends of 24 wildfowl species in Mexico. Wildfowl numbers peaked during the early 1980s; lowest counts were recorded in 1997. Total wildfowl numbers (all species combined) and duck numbers (duck species combined) showed significant short-term (1981-2000) declines, while geese (goose species combined) showed a significant long-term (1961-2000) increase. Six wildfowl species suffered significant long-term declines, while four showed increases. During 1981-2000, 11 species declined, but none had significant increases. Redhead (Aythya americana), Mexican duck (Anas diazi), northern pintail (A. acuta) and black brant (Branta bernicla nigricans) should be given high conservation priority because of the high proportions of their North American populations in Mexico. Declining numbers of the later two species should trigger further investigation into the possibility of assigning them legal protection status. Other species with apparent declines in numbers should also be more closely monitored. For setting hunting limits in the country, the population status of each species should be accounted for, as well as the condition of breeding populations the previous spring. Other species with poor data or combined counts should be targeted for basic population studies. We suggest that the mid-winter counts be expanded to cover non-surveyed areas and conducted every year to more precisely detect wildfowl population change. Integrated to a site-selection analysis, the information presented here can provide the basis for a wildfowl conservation strategy in Mexico.     

番鸭源小鹅瘟病毒PT株VP基因的克隆与序列分析

为了获得番鸭(Cairina moschata)源小鹅瘟病毒(Goose parvovirus,GPV)结构蛋白VP基因的相关信息,根据国内外已发表鹅源GPV与番鸭细小病毒(Muscovy duck parvovirus,MDPV)全基因序列,应用DNAStar分子生物学软件设计一对引物,应用高保真PCR技术扩增番鸭源小鹅瘟病毒PT株(GPV PT株)VP全基因序列.将扩增得到的VP全基因克隆到pMD 18-T载体上,获得的重组质粒经PCR鉴定后进行序列测定.结果表明,PT株VP全基因大小为2 199 bp,编码732个氨基酸(GenBank登录号:JF926695),与番鸭源小鹅瘟病毒GPV DY株核苷酸及其推导氨基酸同源性分别为98.8％和98.8％,高于鹅源GPV与MDPV参考毒株.在国内外首次发现番鸭源GPV VP基因VP1独特区具有MDPV核苷酸序列特征,而VP2基因具有鹅源GPV核苷酸序列特征.本研究从番鸭源GPV PT株成功克隆到结构蛋白全基因序列,为深入研究番鸭源GPV的起源及水禽细小病毒遗传衍化提供参考.     

Species-specific myoglobin oxidation

The effect of the lipid oxidation product, 4-hydroxy-2-nonenal (HNE), on oxidation of oxymyoglobin (OxyMb) from seven different meat-producing species was investigated. Relative to controls, HNE increased OxyMb oxidation within all species (p < 0.05) at both 25 and 4 °C, pH 5.6. The relative effect of HNE was greater for myoglobins (Mbs) that contained 12 ± 1 histidine (His) residues than for those that contained 9 His residues (p < 0.05); HNE efficacy in all species except chicken and turkey decreased with time. Mono-HNE adducts were detected in all species except chicken and turkey. In general, HNE alkylation increased the Mbs' ability to accelerate lipid oxidation in a microsome model. However, neither an HNE nor a Mb species dependent effect was observed. Results suggested that microsome model system associated lipid oxidation overshadowed HNE and species effects on OxyMb oxidation observed in lipid-free systems.     

Identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR amplification of the 5S rDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene, has been used for the identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Two species-specific primers were designed to amplify specific fragments of the 5S rDNA gene in each species. The remarkably different size of the amplicons obtained gives, by simple agarose gel electrophoresis, two distinguishable band patterns for both flatfish species. This genetic marker can be very useful for the accurate identification of S. solea and Greenland halibut, to enforce labeling regulations.     

Detection of poultry and pork in cooked and canned meat foods by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISA) are described for the detection of poultry and pork in cooked and canned meat foods. These assays are based on species-specific, polyclonal antibodies raised against heat-resistant antigens. The heat-resistant antigens were isolated from raw skeletal muscle tissue of pork and chicken and were found to be immunoreactive even after heating to 120 degrees C for 15 min. The poultry ELISA could detect chicken or turkey at the 126 ppm level, and the pork ELISA could detect pork at the 250 ppm level. Samples of frankfurters, bolognas, pressed meats, canned baby foods, and canned spreads were prepared by simple aqueous extractions.     

PCR-SSCP: a simple method for the authentication of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets

A method of DNA analysis has been developed to verify the authenticity of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. A short fragment (208 bp) of the mitochondrial 12S rRNA gene was amplified by the polymerase chain reaction and analyzed by single-strand conformation polymorphism to get species-specific patterns of single-stranded DNA (ssDNA). DNA strands were separated by native polyacrylamide gel electrophoresis and visualized by silver staining. Discrimination among the three fish species studied was possible, because each one expressed a specific ssDNA pattern.     

The incidence and significance of ingested lead pellet poisoning in British Wildfowl

The objective of this study was to assess the extent of lead pellet ingestion by British wildfowl, particularly ducks and geese, and to examine regional variations. The gizzard contents of 2445 shot and 238 found-dead birds were examined, and lead concentrations were determined for 1620 liver and 1841 wing bone samples. In addition, X-ray photographs and blood samples were taken from live-caught birds. Ingested lead pellets were found in 3·2% of the birds examined. For a range of species, including pink-footed goose, white-fronted goose, barnacle goose, wigeon, teal, pintail, shoveler, scaup and moorhen, recorded incidences were either very low or zero. Relatively high incidences were noted for swans, greylag goose (7·1% of shot birds), gadwall (11·8%), mallard (4·2%), pochard (10·9%), tufted duck (11·7%) and goldeneye (6·7%). Most ingested pellets originated from shotguns, though anglers' split shot were found in one pochard and four mute swans. A marked seasonal variation in the extent of pellet ingestion was noted for mallard, with a peak in September, and evidence of high levels immediately before and after the shooting season. Pellet ingestion by mallard was found to be of widespread occurrence, though with considerable variation in recorded incidence from place to place. Inland areas tended to be worse, with the highest incidences recorded for birds shot at flight ponds and other freshwater bodies. Six per cent of mallard shot at inland sites contained ingested pellets, compared with 2·6% of those collected from coastal areas. The observed extent of pellet ingestion in British mallard is calculated directly to cause the death of at least 8000 each winter. Some measures that could be taken to alleviate the lead-poisoning problem in Britain are discussed, and progress of the USA's non-toxic pellet programme is examined.     

黑番鸭血管活性肠肽受体-1基因(VIPR-1)的cDNA克隆及其组织表达特性分析

最近的研究表明,血管活性肠肽(VIP)与家禽的就巢习性密切相关。本研究采用反转录PCR方法从黒番鸭(Cairina moschata)母鸭下丘脑组织中克隆了血管活性肠肽受体(VIPR-1)基因的cDNA序列,长度为1125bp,编码355个氨基酸(GenBank登录号:JN625215)。序列比对结果表明,该序列与家禽(鸡、火鸡、鹌鹑)和哺乳动物(人、小鼠、猪、牛)的基因序列分别有93%~94%和69%~71%的同源性,而相应氨基酸序列的同源性分别为96%和70%~72%。荧光定量PCR结果发现,黒番鸭VIPR-1基因的表达量在产蛋期、就巢期和休产期差异显著(P<0.05)或极显著(P<0.01),就巢期表达量最高,休产期次之,产蛋期表达量最低,表明VIPR-1基因与繁殖阶段变化密切相关;对不同组织VIPR-1的表达量分析发现,VIPR-1在垂体、下丘脑和卵巢中均有表达,其中垂体最多,其次是下丘脑,卵巢中的表达量最低,差异极显著(P<0.01)。研究结果提示,VIPR-1基因具有高度保守性,参与下丘脑-垂体-卵巢轴(特别是垂体)对黑番鸭就巢行为的调控。