Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.     

Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.     

Microwave digestion of plant samples for sulfur analysis

Abstract

The goal was to develop an easy, fast, and economical procedure to produce plant digests that can be accurately analyzed by plasma emission spectrometry for foliar sulfur content. The closed vessel microwave procedure described requires less than 2 hours to produce 12 digested 0.5 gram samples from the point of weighing the samples to applying them to plasma emission spectrometry analysis. The procedure utilizes nitric acid and hydrogen peroxide as a predigestion. The predigested samples are then each microwaved for 2.25 minutes. With this procedure, sulfur levels are within an average of 1.27% of that reported for the NBS Citrus Standard, are within 102.25% to 99.44% of the values determined for wet ash digested materials and display recovery rates of 96 to 98% for known levels of sulfur in the range expected in plant foliage.     

Rapid preparation of plant samples for cation analysis

Abstract

In the dry ashing of plant material for chemical analysis, the hot digestion on the steam bath of the ashed sample was replaced by the quantitative addition of dilute HCl and stirring very briefly to dissolve the ash. By waiting a few minutes for the particles to settle, there was no need to filter the solution prior to dilution with an automatic diluter and the determination of K, Na, Ca and Mg by atomic absorption spectrophotometry. The new procedure permits the handling of large batches of samples conveniently, eliminates the use of pipettes and volumetric flasks and decreases the risk of inadvertent contamination by sodium and therefore enhances accuracy. Precision was at least as good as with the standard procedure and the method is expected to be more suitable for use in conjunction with automated analytical techniques.     

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).     

A single plant tissue digestion for macronutrient analysis

Abstract

A method was tested for determinations of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) in one single digestion of plant tissue. Traditionally, for determinations of these five elements in plant tissue, at least two digestion or ashing procedures are used, totaling about 6 hours of tissue mineralization time. The method tested involved using a digesting medium of concentrated H₂SO₄ and a mixed catalyst of Na₂SO₄ and CuSO₄. The total digestion time was about 60 minutes on a hot plate. After tissue digestion, determinations were by volumetric analysis (N), colorimetry (P), and atomic absorption (Ca, Mg) or emission spectrophotometry (K). Leaves of eight different species, including one standard tomato leaf sample and one internal quality‐control apple leaf sample were analyzed. High correlations occurred between the results obtained with the new procedure and with the standard digesting methods (furnace ashing and Kjeldahl digestion). The new method used much less reagents, electrical energy, and labor than standard methods. The new method appears acceptable for routine macronutrient analysis.     

宏基因组漆酶基因片段的克隆与分析

漆酶(Laccase,EC1.10.3.2)是一种含铜的多酚氧化酶,能够有效降解木质素物质,在环境碳素循环中占有重要的地位。近年来以环境宏基因组DNA为模板,设计简并引物及PCR扩增方法研究环境微生物基因来监测环境微生物已成为土壤微生物多样性研究的新方法[1]。如Luis等[2]通过对森林土壤微生物中漆酶基因的研究来监测该地区的微生物多样性;Lyons等[3]通过对高盐度沼泽地土壤微生物中漆酶基因的研究来监测该地区的微生物多样性;张桂敏等[4]通过对土壤微生物中木聚糖酶基因的研究来监测该地区的微生物多样性。但是,国内尚无研究环境微生物漆酶基因多样性及监测环境微生物功能的报道。同时漆酶在化工染料     

Preparation of plant samples for high precision nitrogen isotope ratio analysis

Abstract

Methodologies for preparation of plant samples for high precision nitrogen isotope ratio analysis are described, including detailed attention to Kjeldhal digestion, steam distillation, concentration of distillates, and generation of nitrogen gas (N₂) from ammonium sulfate [(NH₄)₂SO₄], Apparatus developed in the authors laboratory is illustrated. Using a systematic approach to the assessment of errors associated with each of the steps in preparation and analysis procedures, the methods outlined in this paper are shown capable of determining 8¹⁵N in plant material with a precision of ±0.2°/oo.     

Digestion of plant tissue for analysis by ICP emission spectroscopy

Abstract

A method of digesting plant tissue samples for multi‐element analysis by inductively‐coupled plasma (ICP) emission spectroscopy was developed which does not require the use of HclO₄. A substitute oxidant, H₂O₂, eliminated the hazards associated with hot, concentrated HClO₄. Optimal conditions for pre‐digestion and digestion were investigated.

The procedure adopted consists of a pre‐digestion in concentrated HNO₃ from room temperature to 60°C in 30 min followed by digestion at 90°C for 90 min with the addition of 30% H₂O₂ Analysis of NBS standard tissue samples by the proposed method gave results in good agreement with analysis following digestion in HNO₃,‐HClO₄ and with certified values, except for Fe.     

An automated procedure for the analysis of boron in plant tissue

Abstract

Boron analysis of plant tissue by the quinallzarin procedure has been adapted to the Technicon AutoAnalyzer system. Results obtained are comparable to those by the curcumin method for evaluating the boron status of the plant. Details of the mechanical assembly are presented and precautions to be observed are discussed. The suggested procedure permits the analysis of 30 samples of plant ash extract per hour.     

Automated instrumental analysis of carbon and nitrogen in plant and soil samples

Abstract

An automated CHN Analyzer was compared with the Walkley‐Black and Kjeldahl methods for organic carbon (C) and nitrogen (N). Four organic compounds, twenty nine plant materials and five soils were tested. The CHN Analyzer gave C and N values that were not significantly different (P<0.05) to the theoretical weight percents of the organic compounds. The Walkley Black method gave soil C values significantly lower (P<0.05) than those obtained with the CHN Analyzer. The Kjeldahl method gave soil N values significantly lower (P<0.05) than the CHN Analyzer on three of five soils tested. The discrepancies observed between methods appear to be due to different oxidation efficiencies. CHN Analyzer and Kjeldahl N analyses were not significantly different (P<0.05) for the plant materials except where samples contained greater than 0.7% NO3‐N. Potassium nitrate was also added as a spike to a tall fescue sample. Based on recovery of the spiked NO3‐N, the Kjeldahl method was a poor measure of total N for plant materials containing greater than 0.7% NO₃‐N. The findings suggest the CHN Analyzer can be used for the rapid, accurate and simultaneous determination of C and N in plant and soil samples.     

Microscopic observation of charred plant fragments isolated from several Ando soil samples by the specific gravity method

Abstract

Charred plant fragments were isolated from 5 Ando soil samples, containing Type A humic acids, by the specific gravity (s.g.) method using a sodium polytungstate solution as heavy solution. Microscopic observation indicated that the charred plant fragments, which are black or blackish brown, were the main components in both fractions of less than s.g. 1.6 Mg m^?3 that had been isolated before and after HCl-HF treatment of the soil samples. Furthermore, it was suggested that most of the fragments in the < 1.6 fractions isolated before the treatment originated from woody plants. On the other hand, the fragments in the < 1.6 fractions isolated after the treatment were mainly amorphous, although the shape of several fragments was similar to that of vascular tissues of woody and herbaceous plants. The sum of the organic-C contents of both < 1.6 fractions ranged from 4.08 to 47.8 g kg^? whole soil, and 3.8–32.7% of total organic-C of the whole soil originated from these fractions. No constant relationship was observed between the organic-C contents of both fractions and the morphological characteristics of the charred plant fragments.     

高压液相色谱法同时测定植物组织中六种细胞分裂素组分和生长素

利用高效液相色谱研究了植物组织中六种细胞分裂素组分和生长素含量的测定方法。结果表明,采用反相色谱柱Hibar.RT250-4.6,在45℃恒温下以甲醇1%乙酸(40/60,v/v)溶液为流动相0、.6.mL/min等度洗脱,在=269.nm处能准确检测出植物组织中六种内源细胞分裂素组分和生长素的含量,检测限低至0.001.mg/L。本研究对样品的提取和纯化过程也做了改进,排除了杂质和色素对样品的干扰,为研究植物体内源激素对环境响应特征提供了有效的测定方法。     

Rapid perchloric acid digest methods for analysis of major elements in plant tissue

Abstract

The nutrient element composition of tissue from a plant species is not fixed. Composition in the plant varies among different plant parts at the same or different physiological stages of maturity, as soil type changes, from day to day, even hour to hour on the same day due to past and current environmental conditions. These observations seem to undermine the general trend in plant tissue analysis to develop quantitative procedures that are time consuming and, therefore, foster insufficient nutrient status monitoring within and/or between different physiological stages of maturity of like or different plant parts. Therefore, until tissue sampling becomes more definitive, a digest procedure with 90–100% recovery should be more than adequate for major elemental analysis. The HClO₄ digestion procedures described here meet that requirement and take only 15 to 30 min to complete. N recovery was greater than 100% with the H₂O₂‐HClO₄ digestion procedure. However, when HNO₃ was combined with H₂O₂ and HClO₄, N was lost, although even then N volatilization occured only when digestion was extended past clearing.     

Sensitive trapping and gas chromatographic method for vinyl chloride in air samples.

A simple and sensitive method is described for determining vinyl chloride monomer in air. Vinyl chloride monomer from a sample of air is trapped onto a Tenax GC trap cooled with Dry Ice. The trap is installed on a gas chromatograph by a simple valve arrangement. The trap is heated, and the entire sample is flushed onto the gas chromatographic column. Vinyl chloride is determined at 6 and 60 ppb (v/v) levels with recoveries ranging from 79 to 104%.     

Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products

Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.     

Comparison of ion chromatography and a continuous‐flow technique for analysis of chloride and sulfate in plant samples

Abstract

A comparison of ion chromatography and continuous‐flow colorimetry for the determination of chloride and sulfate content in water extracts of plants is presented. Results of the two methods correlate well. The relationships are linear, with a slope of 0.72 for sulfate and 0.73 for chloride. The correlations indicate that the techniques and instruments used are compatible so that the regression equations are satisfactory for conversion of data.     

Real-time PCR技术用于中国大菱鲆虹彩病毒的组织敏感性检测及病毒流行情况调查

中国大菱鲆虹彩病毒(turbot reddish body iridovirus, TRBIV)是一种感染养殖大菱鲆的鱼类虹彩病毒,它可以引起大菱鲆病毒性红体病并导致养殖大菱鲆大量死亡。本研究利用TRBIV主要衣壳蛋白基因序列设计的一对引物,结合内嵌式核酸染料SYBR Green І,建立了TRBIV特异的Real-time PCR检测方法。实验结果表明,该对引物具有较高的灵敏度和较强的特异性,能够检测相当于102数量级的TRBIV基因组拷贝,而不与健康大菱鲆组织DNA、淋巴囊肿病毒DNA发生交叉反应。最后,应用建立的Real-time PCR检测方法,开展了TRBIV的组织敏感性检测和病毒流行情况调查。结果发现,大菱鲆的脾脏、肾脏、脑、鳃、心脏、肝脏、消化道、血液等组织中均可检测到TRBIV的存在,其中脾和肾是TRBIV的最主要的靶器官,每毫克组织的病毒含量分别高达5.23106个和2.18106个。分子流行病学调查结果显示,在山东半岛的多个大菱鲆养殖场中均存在TRBIV的感染和流行。     

Comparison of a microwave digestion system to other digestion methods for plant tissue analysis

Abstract

Most digestion procedures for the preparation for plant tissue for elemental analysis are often very laborious. Elemental contents of plant tissue prepared by microwave digestion were compared to those obtained by conventional dry ashing and sulfuric acid‐hydrogen peroxide wet digestion. All three digestion methods produced comparable quantitative values for the elements P, K, Ca, Mg, Fe, Mn, Zn, and Cu for all plant materials used. Thus, the microwave digestion technique was shown to be a viable rapid method for digesting relatively small numbers of samples prior to elemental analysis.     

Validation of a tomato-specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.