The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material

 The fate of ingested recombinant plant DNA in farm animals (cattle and chicken) being fed a diet containing conventional maize or recombinant Bacillus thuringiensis toxin-maize (Bt-maize) is described. The probability of the detection by polymerase chain reaction of chloroplast-specific gene fragments of different lengths (199 bp and 532 bp) and a Bt-maize-specific fragment [truncated version of CryIA(b)] is shown. First data indicated that only short DNA fragments (<200 bp) derived from plant chloroplasts could be detected in the blood lymphocytes of cows. In all other cattle organs investigated (muscle, liver, spleen, kidney) plant DNAs were not found, except for faint signals in milk. Furthermore, Bt-gene fragments possibly recording the uptake of recombinant maize, were not detected in any sample from cattle. However, in all chicken tissues (muscle, liver, spleen, kidney) the short maize chloroplast gene fragment was amplified. In contrast to this, no foreign plant DNA fragments were found in eggs. Bt-gene specific constructs originating from recombinant Bt-maize were not detectable in any of these poultry samples either. Received: 23 February 2000 / Revised version: 20 March 2000     

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.     

Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize

The aim of this study was to trace selected nucleic acid and protein components of isogene versus Bt transgene maize within the bovine gastrointestinal tract (GIT). After feeding 22 cattle for 4 weeks with Bt176 maize, different plant genes and the recombinant protein CryIAb were quantified during digestion. Furthermore, a first initial characterization of rumen bacteria was approached, using 16rDNA gene sequencing comparing isogene- against transgene-fed animals. Ingesta samples of different GIT sections (rumen, abomasum, jejunum, colon) were analysed for chloroplast, maize invertase, zein and Bt toxin (CryIAb) gene fragments using quantitative real-time PCR. First, the initial gene dose of these maize genes was detected in maize silage. During digestion, a significant reduction of high-to-medium abundant plant gene fragments was shown depending on the dwell-time and the initial gene copy number. Immunoreactive CryIAb protein was quantified by ELISA in intestinal samples indicating a significant loss of that protein. Remarkable amounts of Bt toxin were found in all contents of the GIT and the protein was still present in faeces. For the first time, the influence of CryIAb transgene maize on rumen bacterial microflora was investigated compared to isogene material through analysis of 497 individual bacterial 16S rDNA sequences. In principle, specific bacterial leader-species could be identified in all bovine rumen extracts, but no significant influence of Bt176 maize feed was found on the composition of the microbial population. This investigation provides supplementing data to further evaluate the fate of novel recombinant material originating from transgene feed or food within the mammalian GIT.     

Detection of feed-derived maize DNA in goat milk and evaluation of the potential of horizontal transfer to bacteria

The presence and the transforming capacity of feed-derived DNA in milk obtained from eight lactating goats fed on maize E176 silage were evaluated. The presence of single- and multi-copy maize genes was monitored by real-time PCR and conventional PCR. Chromosomal and plastid DNA extracted directly from maize flour and silage were readily amplifiable by conventional PCR, however, only chloroplast-specific gene fragments of 199 and 532 bp were detected in about 60 and 20%, respectively, of the milk samples analysed. Quantification by real time PCR yielded 9.5 (±6.7) × 10² plant gene copies/mL of milk sediment. In contrast, all milk samples were negative for the chromosomally located maize zein gene or the E176 specific cry1Ab transgene. The minimum concentration of plant DNA required for detection was 0.01 ng/mL raw milk for the chloroplast-specific fragment and 1 ng/mL for the cry1Ab transgene. The detection limit was determined by spiking milk samples with plant DNA prior to DNA extraction. The transformation capability of DNA in milk was evaluated after constructing a marker rescue system in Acinetobacter baylyi strain BD413 based on recombinational repair of the bla _TEM gene. Two systems were developed that allowed the plant marker gene to recombine with the bacterial chromosome [A. baylyi BD413 (pUC-bla)] or plasmids [A. baylyi BD413 (pBBR1MCS-2Φ)]. The two systems showed the same efficiency of transformation, yielding 10⁻⁵ transformants per recipient cell (t/r) using plasmid pUC18 or a 1,873 bp fragment as donor DNA, and 3.5 × 10⁻¹¹ t/r using DNA isolated from flour (E176). No transformants were detected when exposing the recipient bacterium to DNA extracted from maize (E176) silage or from milk obtained from goats feed maize (E176).     

植物源食品DNA制备方法的比较研究

目的：探索可用作PCR方法检测和鉴定植物源转基因食品模板的DNA抽提方法。方法：分别用改良CTAB法、SDS法制备黄豆、玉米、土豆和番茄及其加工产品的DNA，PCR扩增叶绿体基因片段及黄豆、玉米的内参照基因lectin和zein10。结果：改良CTAB法所得DNA作为PCR扩增模板，叶绿体基因片段及lectin和zein10均呈阳性，SDS法所得DNA作为模板时，部分样品内参照基因lectin或zein10扩增阴性。结论：PCR方法检测转基因食品时，应用改良CTAB法制备DNA模板。     

Effect of Thermal Treatment on the Amplification and Quantification of Transgenic and Non-transgenic Soybean and Maize DNA

Processing of raw plant materials causes occurrence of degraded DNA in foods. The effect of DNA degradation on amplification and quantification of transgenic and non-transgenic DNA in raw and experimentally thermally processed foods was studied. The degree of DNA degradation was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). Cetyl trimethyl ammonium bromide method yielded DNA of a better quality, while Genespin and Wizard were less appropriate. Baking at 220 °C considerably reduced the size of DNA fragments. In order to measure the length of amplifiable DNA, primers for soybean and maize genes were used. Small DNA fragments ranging from 100 to 200 bp were amplified in all samples. DNA fragments over 1 kbp were amplified only if heating at 220 °C lasted less than 30 min. Baking of flour (220 °C) reduced the size of extracted DNA fragments so that 1,100 bp amplicon was no longer amplifiable, while the amplicons of 913 and 1,100 bp were obtained from the baked bread. When PCR assays targeting maize high mobility group and zein genes were used under the same conditions, analogous results were achieved. Quantification of genetically modified organism content was not influenced by baking.     

Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies

The passage and fate of ingested DNA in 48 pigs fed with diets containing (n=12) parental or (n=36) transgenic (Bt) maize were examined. Pigs were fattened from an initial live weight of 24 kg to approximately 108 kg. Animals fed transgenic maize were slaughtered in groups (n=6) 4, 8, 12, 24, 48 and 72 h after feeding the last maize-containing diet. Those slaughtered at up to 12 h received no further feed, while those held for longer prior to slaughter received a diet in which maize was replaced by barley and wheat. Control animals were slaughtered at 4 and 8 h. DNA extracted from tissues and gut contents was examined by PCR for the presence of plant DNA and for any transgenic material. Recombinant DNA was detectable in the intestinal contents up to 48 h after the last feeding of a diet containing the transgenic maize. PCR amplification of plant gene spacers produced fragments of different sizes, dependent on feed source. The feed source of rectum samples depended on individual passage rate in the groups and their restriction analysis showed grain species-specific patterns. Recombinant or maize-specific DNA was not detectable in tissue samples of pigs. In contrast, plant DNA fragments were detectable in the investigated pig tissues.     

转基因水稻潮霉素标记基因PCR检测方法的建立及其在基因水平转移研究中的应用

为建立用于基因水平转移研究, 尤其是DNA经加工和消化后稳定性研究的针对转基因水稻潮霉素标记基因hpt（hygromycin phosphotransferase）的定性和实时定量PCR体系,设计针对hpt的上游通用引物多个片段定性PCR扩增体系,以植物叶绿体基因rbcl为内对照,PCR扩增产物经测序验证.将定性PCR中最小片段（236 bp）连接到质粒载体pUC18-pMD T载体上,提取质粒经验证后做外标.应用TaqMan-MGB荧光探针和引物,建立定量的外标校正曲线法,并评价方法的精密度.建立的定性PCR体系能稳定扩增出236 bp～910 bp不同大小的5个hpt片段,并经测序验证.实时定量PCR的线性范围为105～10拷贝（R^2=0.998）,最低能检出10拷贝,重复性好.本研究已成功建立了用于转基因水稻标记基因hpt基因水平转移研究的定性和定量PCR系统。     

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

Methods for the detection of beef and pork in foods using real-time polymerase chain reaction

Summary Two TaqMan?‐polymerase chain reaction (PCR) systems have been developed which permit the detection of even minute amounts of beef and pork in processed foods. In these methods cattle‐specific primers amplified a fragment from the phosphodiesterase gene having a length of 104 base pairs (bp), and the swine‐specific primers amplified a fragment from the ryanodin gene having a length of 108 bp. Beyond this, a third TaqMan?‐PCR system, developed on the basis of the detection of the myostatin gene, permits a reliable exclusion of false‐negative results by detecting meat from a variety of mammals or poultry in the material to be tested.     

Determination of mitochondrial cytochrome B gene sequence for red deer (Cervus elaphus) and the differentiation of closely related deer meats

The cytochrome b gene sequence for red deer was determined using the Dye Terminator Cycle Sequencing method and used for identification of deer meat in meat and meat products. Red deer showed a similarity of 94.1, 84.0, 81.1, 85.5 and 85.6% to sika deer (Cervus nippon), bovine, pigs, sheep and goats, respectively. To differentiate the deer meat, oligonucleotide primers RD-1(5′-TCATCGCAGCACTCGCTATAGTACACT-3′), RD-2(5′-ATCTCCAAGTAGGTCTGGTGCGAATAA-3′) were designed for the region of the cytochrome b gene of red deer. The PCR amplified 194 bp fragments from red and sika deer, but no fragments from bovine, pig, chicken, sheep, goat, horse and rabbit DNA. Although cooking the meats reduced the PCR products, red deer could still be detected in meat heated at 120 °C. To discriminate between red and sika deer, these PCR products were digested by a restriction enzyme (EcoRI,BamHI,ScaI) and analyzed by 4% agorose gel electrophoresis. As a result, the red deer fragment was digested by EcoRI to 67/127 bp fragments but not by BamHI and ScaI. The sika deer fragment was digested to 48/146 bp and 49/145 bp fragments with the two other enzymes, and thus it is possible to differentiate between the two kinds of deer from the digestion pattern of restriction enzymes.     

TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures

A rapid and highly specific real-time quantitative PCR, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA), has been developed for the quantitation of pork (Sus scrofa) in binary pork/beef muscle mixtures. The method combines the use of pork-specific primers, that amplify a 411bp fragment from pork DNA, and mammalian-specific primers amplifying a 425-428bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan), that hybridizes in the "pork-specific" and also in the "mammalian" DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C(t)) at which mammalian and pork-specific PCR products are first detected, in combination with the use of reference standards of known pork content, allows the determination of the percentage of pork in a mixed sample. Analysis of experimental pork/beef muscle binary mixtures demonstrated the specificity and sensitivity of the assay for detection and quantitation of pork in the range 0.5-5%.     

食品中若干植物源性成分的PCR检测

采用CTAB法提取豆奶粉、马铃薯、玉米、番茄及其制品样品中的总DNA,通过PCR方法检测大豆lectin、马铃薯patatin、玉米IVR、番茄PG等特异性内源基因,判断食品中是否含有大豆、马铃薯、玉米、番茄等植物源性成分。同时还进行了这些内源基因分别与核糖体18SrDNA或叶绿体rbcL基因之间的二重PCR分析。结果表明单PCR与二重PCR分析方法可靠稳定,可应用于食品中的植物源性成分的鉴定。     

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.     

Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

Polymerase chain reaction assay for detection of sheep and goat meats

Polymerase Chain Reaction (PCR) was applied to a qualitative differentiation between sheep, goat and bovine meats. Oligonucleotide primers were designed for the amplification of sheep satellite I DNA sequence. The PCR amplified 374 bp fragments from sheep and goat DNA, but no fragment from bovine, water buffalo, sika deer, pig, horse, rabbit and chicken DNA. Sheep DNA (10 pg) was detected by 4% agarose gel electrophoresis following PCR amplification. Althoug cooking of the sample meats reduced the PCR products, sheep DNA was detected in the meat heated at 120°C. In order to differentiate between sheep and goat meats, nucleotide sequences of the PCR products were determined directly by cycle sequencing. The sequence of PCR products showed 92% of homology between sheep and goat. They were differentiated by ApaI digestion of the PCR products because sheep had one ApaI site and goat had no site in the PCR products.     

一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一,目前已有多种基于PCR的肉类鉴定方法,但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标,利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种,扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段,每种PCR产物经SspI酶切后产生数量和大小不同的片段,可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应,DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明,羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比,该方法具有简便易行和高通量的优点,可以作为肉类掺假筛选检测的常规方法。     

利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism,T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记,从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记,上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记,下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳,片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼,而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱,能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同,结果具有可重复性。没有出现物种内多态的现象。大多数情况下,实际得到的末端片段长度与理论值非常接近,只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确,适用于鉴定动物种类。     

The effect of ensiling on PCR-based detection of genetically modified Bt maize

 The detection of the genetic modification in silage obtained from insect-resistant Bt maize by means of the polymerase chain reaction (PCR) is described. The detectability of the transgene was shown to be dependent on the length of the genomic target sequence chosen for amplication by the PCR. By amplifying a Bt-maize-specific DNA sequence of 211 bp the genetic modification was detected up to 7 months after ensilage. The effect of maize DNA degradation in the course of the ensilage on the detectability of target sequences was demonstrated in model experiments. Received: 17 December 1998