Quantification of transgenic plant marker gene persistence in the field

Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, complementary to 20-bp sequences of the nopaline synthase promoter in a transgenic tobacco and the cauliflower mosaic virus 35S promoter in a transgenic potato. The PCR reverse primer was complementary to a 20-bp sequence of the N-terminal NPT-II coding region. The PCR protocol allowed for quantification of as few as 10 rNPT-II genes per reaction. We analysed rNPT-II marker gene amounts in samples obtained from two field experiments performed at different locations in Oregon. In transgenic tobacco leaves, buried at 10 cm depth in a field plot in Corvallis, marker DNA amount dropped to 0.36% during the first 14 days and was detectable for 77 days at a final level of 0.06% of the initial amount. Monitoring of residual potato plant litter, from the soil surface of a test field in Hermiston, was performed for 137 days. After 84 days marker gene amounts dropped to 2.74% (leaf and stem) and 0.50% (tuber) of the initially detected amount. At the final sample date 1.98% (leaf and stem) and 0.19% (tuber) were detectable. These results represent the first quantitative analysis of plant DNA stability under field conditions and indicate that a proportion of the plant genomic DNA may persist in the field for several months.     

Development of a rapid,reliable and simple multiplex PCR assay for early detection of transgenic plant materials

Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.     

番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。     

Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms

The aim of this work was to test the feasibility ofintroducing an anaerobic microbial reductivedechlorination activity into non sterile soil slurrymicrocosms by inoculation with the pure anaerobicbacterial strain Desulfomonile tiedjei, which iscapable of dechlorinating 3-chlorobenzoate tobenzoate. To show that the bacterium was establishedin the microcosms we followed the expression of thereductive dechlorination activity and a molecularprobe based on PCR amplification of the 16S rDNA genewas developed. However, the success of PCRamplification of the 16S rDNA gene depends on the DNAextraction and purification methodologies applied, asshown through the use of several protocols. In thisstudy we report a DNA extraction and purificationmethod which generates sufficient and very clean DNAsuitable for PCR amplification of the D. tiedjei16S rDNA gene. The threshold of detection was about5.10³ bacteria per gram of soil slurry.Introduction of D. tiedjei in soil slurrymicrocosms proved successful since 3-chlorobenzoatedechlorination activity was established with thisbacterium in microcosms normally devoid of thisdechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetateplus formate as cosubstrate, at their respectiveconcentrations of 5 and 6 mM, led to a totalbiotransformation of 2.5 mM of 3-chlorobenzoate within12 days. After complete 3-chlorobenzoatedechlorination, the 16S rDNA gene of this bacteriumwas specifically detected only in the inoculatedmicrocosms as shown by PCR amplification followed byrestriction mapping confirmation.     

Sensitive and selective colorimetric detection of Staphylococcus aureus-SPA gene by engineered gold nanosensor

Staphylococcal protein A (SPA) is an important virulence factor that enables Staphylococcus aureus to evade host immune responses. The current work aims to detect the S. aureus SPA gene by a colorimetric method based on gold nanoparticles (AuNPs). For this purpose, the chromosomal DNA of S. aureus was extracted. Thereafter, primers and thiolated oligonucleotide probe were designed based on protein A sequence data in the gene bank. PCR analysis was performed, and the PCR product was electrophoresed on 2 % agarose gel. Gold nanosensor (Au-Ns) was synthesized by the reaction between AuNPs and the thiolated oligonucleotide probe. The physicochemical properties of AuNPs and Au-Ns were characterized. The detection of the SPA gene was performed based on color change detected by the naked eye and UV–vis spectrophotometry. Finally, the described method was optimized and validated for standard, clinical, and food samples. The PCR analysis showed a characteristic fragment of the SPA gene with a molecular size of 545 base pairs (bp) and a detection limit of 60 pg/ µL. The physicochemical analyses illustrated Au-Ns’ correct preparation with a zeta potential of ?13.42 mV and particle size range 6–11 nm. Moreover, Au-Ns showed 100 % specificity with a detection limit (DL) of 6 fg/ µL. The proposed method was well described to be applied in clinical and research laboratories.     

The persistence of bifidobacteria populations in a river measured by molecular and culture techniques

Aims:  To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR).
Methods and Results:  Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T ₉₀ values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB.
Conclusions:  Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T ₉₀ when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells.
Significance and Impact of the Study:  The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST).     

Residual eDNA detection sensitivity assessed by quantitative real‐time PCR in a river ecosystem

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real‐time polymerase chain reaction (qRT–PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over 2 years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 h after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 h after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT–PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species.     

A simple method of DNA extraction from soil for detection of composite transgenic plants by PCR

This new and simple method of DNA extraction from composite soil allows the isolation of plant DNA with high efficiency, quality and reproductivity. The method is based on a simple CaCl₂-precipitation step and requires no additional purification steps to eliminate humic acids. The extracted DNA was obtained in sufficient purity and quantity to allow direct detection of transgenes by PCR. Furthermore, the simple procedure allows the assay of many samples at the same time.     

实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数

实时荧光定量PCR是近年新兴的一项技术,因其快速、方便、便宜,需要DNA样品量少,无需放射性检测等优点被广泛应用于基因的定量分析。该文就实时荧光定量PCR(TaqMan)技术的发展、基本原理及测定外源基因拷贝数的技术流程做一介绍。     

去除转基因植物标记基因的研究进展

得到转基因植物以后 ,标记基因就失去了筛选的作用。但它的存在引起公众对转基因植物的安全性以及环境效应的担心 ,所以在目的基因转入后 ,要去除标记基因。该文主要就利用共转化、转座子、同源重组、位点特异重组酶等去除标记基因的方法进行了总结 ,并对各种方法的优缺点进行了比较 ,对该技术未来的发展趋势也进行了展望。     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-1-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA ofp53 gene,N-ras gene andRb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

转基因植物环境监测进展

近20年来,转基因植物的商业化应用规模越来越大,而转基因生物安全问题依然是转基因植物产业进一步发展的最主要制约因素。转基因植物在商业化应用之前虽然预先进行了风险评估,但是,包括环境监测在内的风险管理措施是确保转基因植物安全应用的必要手段。在转基因作物大规模应用近20年之后,其在靶标生物抗性、对生物多样性的影响、基因漂移、在生态系统中的长期存留等方面产生的环境风险已经渐渐显现出来,表明风险评估无法为转基因植物应用提供足够的安全保障,还必须通过开展系统而长期的环境监测,明确转基因植物在生产应用后的实际环境影响。联合国环境规划署和欧盟等已经制定了转基因植物环境监测的法规和技术指南,一些国家实施了系统的转基因植物环境监测。对转基因植物所产生的环境风险以及环境监测应包括的内容进行了综述。     

转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

转Cry1Ab基因水稻Bt01为一种新型的转基因水稻,文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中,且为单拷贝,再利用TAIL-PCR方法获得了其插入位点信息,根据获得的Bt01的5′端插入位点序列,设计了相应的定性与定量PCR检测体系的引物及探针,实验结果显示,定性PCR检测体系的最低检测极限(LOD)为10个拷贝,定量PCR检测体系的LOD为5拷贝,最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性,利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品,定量结果分别为2.7%和0.47%。研究结果表明,该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性,为转基因水稻Bt01的身份识别和检测提供了有效的方法。     

转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

转Cry1Ab基因水稻Bt01为一种新型的转基因水稻, 文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中, 且为单拷贝, 再利用TAIL-PCR方法获得了其插入位点信息, 根据获得的Bt01的5′端插入位点序列, 设计了相应的定性与定量PCR检测体系的引物及探针, 实验结果显示, 定性PCR检测体系的最低检测极限(LOD)为10个拷贝, 定量PCR检测体系的LOD为5拷贝, 最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性, 利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品, 定量结果分别为2.7%和0.47%。研究结果表明, 该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性, 为转基因水稻Bt01的身份识别和检测提供了有效的方法。     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis

We investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.     

Effect of oxygen and temperature on the dynamic of the dominant bacterial populations of pig manure and on the persistence of pig-associated genetic markers, assessed in river water microcosms

Aims: The aim is to evaluate the dynamic of Bacteroides–Prevotella and Bacillus–Streptococcus–Lactobacillus populations originating from pig manure and the persistence of pig‐associated markers belonging to these groups according to temperature and oxygen. Methods and Results:  River water was inoculated with pig manure and incubated under microaerophilic and aerobic conditions, at 4 and 20°C over 43 days. The diversity of bacterial populations was analysed by capillary electrophoresis‐single‐strand conformation polymorphism. The persistence of the pig‐associated markers was measured by real‐time PCR and compared with the survival of Escherichia coli and enterococci. Decay was characterized by the estimation of the time needed to produce a 1‐log reduction (T90). The greatest changes were observed at 20°C under aerobic conditions, leading to a reduction in the diversity of the bacterial populations and in the concentrations of the Pig‐1‐Bac, Pig‐2‐Bac and Lactobacillus amylovorus markers with a T90 of 10·5, 8·1 and 17·2 days, respectively. Conclusions: Oxygen and temperature were found to have a combined effect on the persistence of the pig‐associated markers in river waters. Significance and Impact of the Study: The persistence profiles of the Pig‐1‐Bac, Pig‐2‐Bac and Lact. amylovorus markers in addition to their high specificity and sensitivity support their use as relevant markers to identify pig faecal contamination in river waters.     

基于植物rbcL基因测序对茶叶进行掺杂检验

茶叶是世界上最受人们欢迎的饮品之一,但茶叶中掺杂其他植物成分的现象时有发生。依靠传统的感官和理化检验方法难以准确判断茶叶中掺杂的植物种类。报道一种基于植物核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基(rbc L)基因序列进行茶叶掺杂定性检测的方法,包括rbc L基因片段的扩增、测序和序列分析等步骤。利用所建立的方法对7份茶叶样品进行分析,发现岳阳黄茶(黄茶)和信阳毛尖(绿茶)未掺杂其他植物成分,而正山小种(红茶)、铁观音(乌龙茶)、太姥银针(白茶)、六堡茶和普洱茶(黑茶)均一定程度上混杂有其他植物成分。所建立的检测方法对样品的需求量小,操作简便,检测结果可靠性高,能定性检测各类茶叶中是否掺杂及掺杂了何种植物成分。