Evaluation of a PCR kit for the detection ofErwinia carotovora subsp.atroseptica on potato tubers

Summary A PCR-based kit, Probelia^TM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3×10² to 1.5×10³ cells ml⁻¹). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0×10¹ to 6.2×10³ cells ml⁻¹ peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.     

Development of real-time PCR systems based on SYBR® Green I,Amplifluor™ and TaqMan® technologies for specific quantitative detection of the transgenic maize event GA21

Maize GA21 line has integrated several tandemly repeated copies of the r-act 5-enol-pyruvylshikimate-3-phosphate synthase construct used for plant transformation. We were able to amplify a nucleotide sequence corresponding to the polylinker plasmid vector flanked by the r-act promoter and nopaline synthase 3′-terminator. A method for specific detection and quantification of Roundup Ready^® transgenic maize line GA21 DNA using conventional and real-time PCR and based on this transgenic sequence is described. GA21 specific primers and probe were designed targeting the vector–promoter junction region and amplifying a 72-bp DNA fragment. Quantification methods were optimized through three different real-time PCR chemistries, i.e. SYBR^® Green I, Amplifluor™ and TaqMan^®. All three methods proved to be specific, highly sensitive and reliable for both identification and quantification of GA21 DNA.Plasmid pGAivr containing single copies of the GA21 and invertase amplicons was constructed for use as external standard in calibration curves. Using pGAivr, a TaqMan^® based real-time PCR assay was optimized in duplex format targeting the maize species-specific ivr1 gene and the GA21 junction region. The detection limit of the method was 0.01% GA21, which is far below the established threshold for accidental presence of genetically modified organisms (GMO), this method therefore being suitable for use in routine GMO analysis.     

Evaluation of spray programs containing famoxadone plus cymoxanil, acibenzolar-S-methyl, and Bacillus subtilis compared to copper sprays for management of bacterial spot on tomato

Bacterial spot caused by Xanthomonas euvesicatoria Jones et al. and Xanthomonas perforans Jones et al. is a major disease on fresh market commercial tomato in Florida. Fourteen field trials were conducted between 1999 and 2005 (10 in south Florida and four in north Florida) testing famoxadone plus cymoxanil (Tanos 50DF^®, E.I. du Pont de Nemours and Company, Wilmington, DE), Bacillus subtilis strain QST 713 (B. subtilis) (Serenade WPO^® or Serenade Max^®, AgraQuest, Inc., Davis, CA), and acibenzolar-S-methyl (ASM) (Actigard 50WG^®, Syngenta Crop Protection, Greensboro, NC) at different rates and in various application programs that were combined and rotated with copper hydroxide and mancozeb for management of bacterial spot. In field applied spray treatments containing famoxadone as a component, all of the programs significantly reduced bacterial spot severity on plants compared to the untreated control plants (UTC) and 97% of the programs were equal for disease suppression conferred by the copper-mancozeb standard. In spray programs containing ASM or B. subtilis plus copper hydroxide, treated plants had significantly reduced disease compared to the UTC plants and were not different from the plants treated with the copper-mancozeb standard. Yield data from small plots was unaffected. Several of the programs which used these compounds in rotation with copper-mancozeb provided similar levels of reduction in the disease severity for bacterial spot while reducing by 50% the amount of copper applied to plants. The effect of famoxadone plus cymoxanil on the survival of Xanthomonas in vitro did not cause a significant reduction in the bacterial population and was not determined to be directly bactericidal. However, greenhouse and field testing supports disease reduction of bacterial spot on plants treated with these compounds. The products tested in these trials may be useful, alternative tools for use in an integrated management program for bacterial spot on tomato.     

Combination of On-Line Solid-Phase Extraction with LC-MS for the Determination of Potentially Hazardous Glycoalkaloids in Potato Products

A simple and rapid method for the determination of naturally occurring, potentially hazardous glycoalkaloids (GAs) in potatoes and their products has been developed. The procedure is based on the on-line solid-phase extraction of the acetic acid extracts from potato products and combined with liquid chromatography (LC)-mass spectrometry (MS) in a fully automated system (Symbiosis™, Spark Holland Instruments, Emmen, The Netherlands). As sorbent material HySphere™ 18HD was used for alkaloid enrichment. GAs were eluted with the LC gradient and directly analysed by MS. Detection of the analytes was achieved in the sensitive multiple reaction monitoring mode using two characteristic ions (m/z 98 as a qualifier for GAs and m/z 868.3 as a quantifier for α-solanine or m/z 852.4 for α-chaconine). Typical validation data for method precision (v _k α-solanine = 5.3–6.5, v _k α-chaconine = 3.4–15.4), accuracy (average recovery of α-solanine = 84%, average recovery of α-chaconine = 87%) and linearity over the range from 1 to 1,000 ng ml⁻¹ (R ² = 0.9915 for α-solanine, R ² = 0.9939 for α-chaconine) with detection limits of 0.3 ng ml⁻¹ for α-chaconine and 0.5 ng ml⁻¹ for α-solanine were obtained. GA contents of commercial potato products were determined by the new on-line method and afterwards compared with those obtained with an established high-performance LC routine procedure. Better performance of the on-line procedure was obvious from the standard deviations of both methods. Other advantages included a strong reduction of overall analysis time, human intervention and solvent consumption as well as waste production. The time required for the on-line analysis was 5 min, which would allow processing of almost 100 samples in 8 h.     

Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene,bar

Screening of gene manipulation events (transgenic, mutation/genome editing, etc.) is a cost/labor-intensive and time-consuming process in plant science research. While polymerase chain reaction (PCR) is the most commonly used method for screening, the process still requires efficient DNA extraction and subsequent confirmation. However, PCR cannot predict gene expression. To screen a larger number of transgenic plants, it would be ideal to develop a quick and reliable screening procedure. We have applied a Liberty^® leaf-painting method (against bar gene under 4x35S promoter) to screen transgenic maize (Zea mays L.) plants and validated the results through PCR and quantitative real-time PCR (qRT-PCR). Liberty leaf painting at 500 mg L^?1a.i. was > 95% accurate in identifying transgenic events that agreed with the PCR results. Further investigation of bar gene expression in sensitive lines that were PCR positive shows very low expression of the bar gene. We have provided a simple, and rapid assay to determine the transgene expression potential of maize plants expressing the bar gene. The herbicide can be applied to a fully expanded leaf and evaluated one week after application. Green or partially green leaf blades indicate high or moderately high expression of the bar gene and a total yellowing indicates absence or extremely low expression of the bar gene in the transgenic plants. A small volume of Liberty solution is adequate to test hundreds of maize plants, and the assay is reproducible with a high frequency (> 95%) and also displays good correlation with gene expression in planta.     

小麦TILLING突变体检测中基因组DNA提取方法的优化

为了优化小麦高通量TILLING突变体扫描所需基因组DNA提取的方法,对利用SDS法、改良SDS法、CTAB法、改良CTAB法、AxyPrep DNA提取试剂盒、Qiagen DNeasy Plant Mini Kit试剂盒以及再生Qiagen硅胶柱方法提取的小麦基因组DNA进行了系统比较。结果表明,尽管7种方法所提取DNA的纯度及琼脂糖凝胶电泳检测结果均良好,但再生Qiagen硅胶柱方法、CTAB法和改良SDS法的DNA浓度显著高于其他方法。根据小麦细胞分裂素氧化酶基因1(TaCKX1)的DNA序列设计引物,以新鲜DNA样品用于PCR扩增大于1 000bp的片段时,后4种方法扩增效果优于前3种方法;DNA在-20℃下保存100d后重新进行PCR扩增时,则只有Qiagen试剂盒和再生Qiagen硅胶柱2种方法提取的DNA能扩增出大于1 000bp的片段。采用本实验室与新西兰坎特伯雷大学合作建立的再生硅胶柱与自配提取液提取小麦基因组DNA,既保证了DNA的高质量,又降低了使用试剂盒的成本,为小麦TILLING库的构建及其他相关试验提供了一种经济有效的DNA提取方法。     

Differentiating PVYNTN by unique single-restriction cleavage of PCR products

Summary A procedure for differentiating PVY^NTN from PVY^N is described and is based on the unique cleavage of their respective PCR products with strain specific restriction endonucleases. The PCR products corresponding to the 5′ end of the N and NTN strains of PVY were cloned and sequenced, and a restriction map was constructed which included common enzymes that were used for the differentiation of PVY^NTN. Unique, single cleavage of PCR products derived from the 5′ end of the PVY^NTN genome by Nco I, and that of the N-strain of PVY by Bgl II restriction endonuclease were demonstrated. The specific digestion patterns in polyacrylamide gel were used for the unequivocal differentiation between the N and NTN strains of the virus. Both single and mixed infections were detected in field samples of potatoes using this procedure.     

The use ofStreptococcus zymogenes for estimating tryptophan and methionine bioavailability in 17 foods

As part of a cooperative study assessing amino acid bioavailability and/or protein quality, the provisional method of Boyneet al. (Brit J Nutr 21: 181–206) was used to assay 17 protein sources for methionine and tryptophan availability withS. zymogenes. Pronase was used as the predigesting enzyme. Product composition was found to affect reproducibility. The microbial assay results correlated positively with results from rat growth studies on the same foods (p=0.05), and were generally accurate in identifying products of lower protein quality. Defatting four high-fat products increased microbial values in the methionine assay, but only the chicken franks and the sausage values in the tryptophan assay. Heating non-fat milk increased methionine values slightly. Low values for rolled oats were further reduced by finer grinding.     

玉米ZmCOL3pro217启动子的克隆及功能分析

ZmCOL3是玉米开花期光周期调控网络中一个重要的功能基因,研究发现自然群体中该基因的启动子存在遗传变异,推测这些变异可能参与玉米开花调控。研究从热带血缘玉米材料CIMBL119中克隆了ZmCOL3启动子,命名为ZmCOL3pro217。序列比对发现,该启动子序列与温带血缘玉米B73序列存在1个217 bp大片段置换。生物信息学分析表明,ZmCOL3pro217含有分生组织特异性、光响应、胁迫响应等多个顺势作用元件,预测ZmCOL3pro217可能具有组织特异性,并通过响应光和胁迫反应参与玉米开花调控。进一步构建ZmCOL3pro217驱动gus报告基因表达载体,转化玉米,实时荧光定量PCR、酶联免疫吸附剂测定、GUS组织化学染色等研究结果发现,转基因植株中ZmCOL3pro217驱动gus基因在根和叶器官中高表达,而在茎、花丝和子粒中几乎不表达,证实ZmCOL3pro217是1个新的组织特异性表达启动子。     

Antioxidative and Anticancer Activity of Extracts of Cherry (<Emphasis Type="Italic">Prunus serrulata</Emphasis> var. <Emphasis Type="Italic">spontanea</Emphasis>) Blossoms

Organic solvent (methanol, ethanol, and acetone) extracts and water extracts of cherry (Prunus serrulata var. spontanea) blossoms were prepared, and antioxidant activities of the extracts were evaluated. Methanolic CBE (100 μg/ml) showed the highest total phenol content (104.30 μM), radical scavenging activity (34.2%), and reducing power (0.391). The effect of CBE on DNA damage induced by H₂O₂ in human leukocytes was evaluated by Comet assay. All CBE was a potent dose dependent inhibitor of DNA damage induced by 200 μM of H₂O₂, methanolic CBE showed the most strong inhibition activity. The methanolic CBE of 500 μg/ml showed 38.8% inhibition against growth of human colon cancer cell line HT-29. These results indicated that cherry blossoms could provide valuable bioactive materials.     

玉米PRC2基因在子粒发育过程中的表达分析

以玉米自交系昌7-2授粉后不同天数的玉米子粒为研究对象,通过实时荧光定量PCR技术,对玉米中3个Polycomb Repressive Complex 2基因(PRC2)Mez1(Maize enhancer of zeste 1)、Fie1(Fertilization independent endosperm 1)和Vef101(VEF family protein 101)在授粉后不同天数的表达情况进行研究。结果表明,Mez1、Fie1和Vef101在授粉后子粒发育不同阶段均有表达,Fie1在子粒发育过程中呈先上升后下降的趋势,授粉后10 d表达量最高,表明Fie1可能在子粒由胚胎形成到干物质积累的过渡时期发挥调控作用;Mez1在子粒发育过程中呈先下降后上升的趋势,授粉后10 d表达量最低,子粒发育后期表达量明显上升,表明Mez1可能在子粒淀粉积累过程中发挥调控作用;Vef101只在授粉后5 d的子粒中呈现高表达,其他时期表达量均较低,表明Vef101可能在玉米子粒发育早期胚胎形成过程中发挥调控作用。     

QuantifyingVerticillium dahliae in soils collected from potato fields using a competitive PCR assay

A quantitative PCR assay based on the competitive PCR technique was compared to the classical soil dilution (SD) method for its ability to estimateV. dahliae propagules directly in soils collected from fields under potato production. A strong correlation (r = 0.97) was observed betweenV. dahliae propagules estimated using the quantitative PCR assay and those using the SD method. Coamplification ofV. dahliae DNA with competitor DNA provided accurate quantification in the range of 10² to 10⁷ spores and 1 to 100 microsclerotia/g of soil. The number ofV. dahliae propagules detected in PEI soils ranged from 4.9 to 15.6 and 0.06 to 0.5 microsclerotia/g of soil for PCR assay and SD method, respectively. The strong correlation between PCR assay and SD method and the non significant differences between replications of PCR estimates ofV. dahliae propagules in soils (P< 0.05) show that the PCR assay is reliable and reproducible, and comparable to the SD method. This method is fast, does not depend on the subjectiveness of the traditional plating method, and offers an improvement in speed and precision over currently used methods. In addition, it can be extended to estimateV. dahliae propagules in other pathosystems and finds immediate and practical use in epidemiological studies to determine the effects of various crop management strategies on the dynamics and level of fungal propagules in the soil in order to establish threshold levels for assessing disease risks and develop disease prediction systems.     

Screening of essential oils from wild-growing plants in Tunisia for their yield and toxicity to the poultry red mite, Dermanyssus gallinae

A laboratory experiment was conducted with the poultry red mite Dermanysuss gallinae (De Geer) to assess the toxicity of a range of essential oils obtained from wild-growing plants in Tunisia to this pest. Details of the percentage essential oil yield from these plants were also recorded. For comparison, commercially sourced essential oil from Thymus vulgaris (L.) was also tested against D. gallinae after work elsewhere found this product to be acaricidal. Recently fed adult female D. gallinae were exposed to the essential oils at 0.1 mg oil/cm² in Petri-dishes at 22 °C over a period of 24 h.Results showed that the yield of essential oil varied considerably depending upon the source plant. Whilst maximum yields of 0.5% were achieved, three of the seven wild plants selected provided yields of less than 0.1%. Similar variability was recorded with respect to the toxicity of the essential oils to D. gallinae. Three of the essential oils tested did not cause significant D. gallinae mortality (in comparison to the control). However, all other selected oils provided mortality levels statistically similar to the 90% mortality achieved with commercial T. vulgaris essential oil, with the oil from Pelargonium graveolens (L’Hér.) killing 100% of D. gallinae exposed to it. Essential oil from P. graveolens in particular may be suitable for further development as a D. gallinae acaricide alongside or in place of commercial thyme essential oil.     

Antioxidative and Antigenotoxic Activity of Extracts from Cosmos (Cosmos bipinnatus) Flowers

The Cosmos bipinnatus has been used in a traditional herbal remedy for various diseases such as jaundice, intermittent fever, and splenomegaly. The present study describes the preliminary evaluation of antioxidant activities and antigenotoxic effect of Cosmos bipinnatus flowers according to four different colors (white, pink, orange, and violet). The antioxidants properties were evaluated by determining TPC, DPPH RSA, ABTS RSA, and RP. The highest TPC of methanolic CFE (at concentration of 1 mg/ml) showed in violet colored CF (1,013 μM), and IC₅₀ of DPPH RSA, ABTS RSA, and RP were also the lowest in violet colored CFE with values of 0.61, 1.48, and 0.82 mg/ml, respectively. The antigenotoxic effect of the CFE on DNA damage induced by H₂O₂ in human leukocytes was evaluated by Comet assay. Pretreatments with CFE produced significant reductions in oxidative DNA damage at the concentration of 500 μg/ml, except for violet colored CFE. The ED₅₀ value of white colored CFE has shown the highest inhibition (0.40 mg/ml) on H₂O₂ induced DNA damage, followed by orange > pink > violet color. These results suggested that Cosmos bipinnatus has significant antioxidant activity and protective effect against oxidative DNA damage.     

Detection and quantification ofSpongospora subterranea f. sp.subterranea by PCR in host tissue and naturally infested soils

A polymerase chain reaction (PCR) assay using primers SsF and SsR designed from the internal transcribed spacer (ITS) regions ofSpongospora subterranea f. sp.subterranea was developed for the specific identification and quantification ofS. subterranea. These primers amplified a 434 bp product from DNA ofS. subterranea spore balls, but not from DNA of healthy potato, common scab tuber, and taxonomically related plasmodiophorids. This PCR assay was successfully used for the detection ofS. subterranea in naturally infected symptomatic and asymptomatic potato tubers.Spongospora subterranea in other infected symptomless host plants was detected by PCR. The PCR assay was modified with improved soil DNA extraction methods to detectS. subterranea in soil. The assay was sensitive, and one spore ball per gram of soil could be detected. Following the design of a heterologous competitor DNA template from the sequence of λDNA, a competitive PCR assay for the quantification ofS. subterranea in soil was developed and provided accurate quantification in the range of 1 to 10⁴ spore balls per 0.25 g of soil. In a preliminary survey of naturally infested field soil samples, spore ball concentrations were estimated to vary from ca 0 to 3600 spore balls per 0.25-g soil sample by this competitive PCR assay. The spore ball levels were compared with the powdery scab disease incidence of potatoes in these fields, and a correlationship between spore ball levels and subsequent disease incidence was found. The PCR assays developed in this investigation can be routinely used to detect and quantifyS. subterranea in diseased plant tissue, asymptomatic plant tissue, and infested soil.     

玉米ZmKNOLLE基因的克隆及增强烟草耐盐性研究

从玉米基因组中克隆出ZmKNOLLE,对其蛋白的同源性和进化树进行分析,并在烟草中对其在盐胁迫中的功能进行验证。研究表明,SNARE蛋白参与植物抗逆性,研究描述了1个玉米SNARE蛋白ZmKNOLLE,其中,包含1个SNARE特定的结构域。它的c DNA为945bp,编码1个314个氨基酸的蛋白,预测分子量为34.54kD,等电点(p I)为7.02,在ZmKNOLLE蛋白C端有一个跨膜结构域。实时定量PCR结果显示,ZmKNOLLE能被盐和H_2O_2强烈诱导。通过农杆菌介导把ZmKNOLLE基因转化到烟草(W38)中,启动子为CaMV 35S。在盐胁迫下,与野生型相比,转基因烟草植株表现出较长的主根和较高的萌发率,相对含水量、POD、SOD活性较高,MDA含量较少,表明转基因烟草比野生型烟草对盐胁迫有更强的耐受性。     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

小麦miR5062靶基因的鉴定及功能研究

miRNA作为一种植物中重要的内源小分子,通过调控靶基因的表达来行使其功能.为了探索小麦(Triticum aestivum)中新发现的miRNA tae-miR5062的靶基因,利用生物信息学方法对tae-miR5062的靶基因进行预测和进一步分析,将属于小麦AGO2家族的TaAGO2确定为其靶基因;进一步构建重组表...     

Are traditional neem extract preparations as efficient as a commercial formulation of azadirachtin A?

Neem seeds contain many substances with insecticidal properties, the main insecticidal ingredient being azadirachtin A. In developing countries such as Mali, a neem seed water extract is prepared by soaking ground seeds in water for three or seven days. The aim of this study was to check the effectiveness of this extract in terms of azadirachtin A extraction yield and insecticidal activity. The yield of extraction was 0.19 g azadirachtin A/100 g seeds. The concentration of azadirachtin A in the seed extract was approximately 200 mg l⁻¹, eight times higher than the recommended concentration of commercial products (25 mg l⁻¹). A comparison of the extractive capacity of different solvents indicated that the best solvents were water and methanol. The azadirachtin A concentration declined in extracts stored for more than 3 days at a temperatures higher than 30 °C. Bioassays were performed on target insects (the leafhopper Macrosteles quadripunctulatus, the moth Spodoptera littoralis and the tobacco whitefly Bemisia tabaci) in order to compare the insecticidal activity of the neem extract with that of the commercial product Neemazal T/S and of a solution of pure azadirachtin A. The bioassays conducted on the leafhopper and the moth demonstrated that the neem extract at the recommended concentration (25 mg l⁻¹ active ingredient) was as effective as the azadirachtin-based commercial product at the same concentration, while for the control of the whitefly B. tabaci a higher concentration of the water extract was needed.     

Starch swelling power and amylose content of triticale and Triticum timopheevii germplasm

Triticale (× Triticosecale Whittmack) and Triticum timopheevii have undergone little selection relative to other grains for quality characters, including starch amylose content. Using starch swelling power (SSP) in water and spectrophotometric analysis of the iodine binding ratio, 247 lines of triticale and 20 lines of T. timopheevii were screened for amylose content. Following this, the expression of the starch-forming protein granule-bound starch synthase (GBSS) in triticale was investigated by SDS-PAGE in the eight highest and eight lowest SSP lines. A strong correlation (R² = 0.8174) was found between iodine binding and SSP. The SSP of T. timopheevii lines ranged from 13.7 to 16.7, indicating an approximate range of amylose content from 28.1 to 33.8%: a small range within typical results from commercial wheat cultivars. The SSP of triticale ranged from 12.5 to 23.6 suggesting amylose content ranged from 12.8 to 35.1%: a much wider range reflecting the contribution of both the wheat and rye genomes. It appeared that expression of GBSS-4A was down-regulated in low amylose lines. Therefore there is significant potential to select for amylose content in triticale to increase quality in both the animal and human feed markets.