Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Detection of Colletotrichum coccodes and Helminthosporium solani in soils by bioassay

The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.     

行走式自动点灌机的设计分析及应用

从改变点灌机整机的传动原理入手，使整机结构简单、布置合理、造价低廉；将所需要的灌水排量变为可调式，使水能及时、准确地注入穴位；简化了直流永磁电机的制作工艺，从上述诸方面阐述了行走式自动点灌机结构的设计特点，简述了该机的效益及应用前景，并对今后进一步的研究提出了建议。     

基于MSP430F149芯片的便携式发动机窜气量测量仪的研制

介绍了在不解体车用发动机的情况下,通过测试曲轴箱窜气量的大小,即可检测活塞环与气缸壁的磨损程度的一种专用检测仪器,并以EQ6100发动机的结构特点为例,设计了专用测试用传感器,测出了其特性曲线,用低功耗单片机MSP430作为微控制器核心,构造了该测量仪器。测试结果表明:该仪器抗扰能力强,测试精度高,测试简单、方便,为发动机故障诊断提供了依据,用户可把仪器的结构、参数稍加改动即可应用于其它类型发动机的不解体故障诊断,具有一定的通用性。     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

线粒体nad 6基因在油菜种子发育中的特异性表达

以油菜为材料，研究线粒体ｎａｄ６基因在种子发育中的表达。结果表明，ｎａｄ６基因在叶片中的表达很低，在种子发育过程中各阶段均有较高表达，并随种子发育进程而有加强的趋势。推测线粒体ｎａｄ６基因可能除与呼吸作用有关外还有其他功能     

基于纳米材料的免疫层析试纸在兽药残留检测中的应用

兽药残留是食品安全的重要影响因素,开发兽药残留检测方法是保证食品安全的重要手段。免疫层析试纸是一种操作简单、生产成本低且具有良好灵敏度的检测方法,适用于现场检测、大规模食品筛选,在兽药残留检测中具有巨大的应用价值。纳米材料可提供特殊的检测信号,是影响免疫层析试纸检测灵敏度和定量分析的关键因素。文章主要对胶体金、磁性纳米材料、量子点以及时间分辨荧光微球这几种纳米标记材料进行介绍,指出各自的优缺点及应用情况,并对该技术在兽药残留检测中的应用趋势进行展望。     

紫荆光合日进程及光响应

自然条件下使用LCA-4型便携式光合测定系统研究了紫荆连体叶片的光合日变化及光响应。结果表明:(1)净光合速率的日进程曲线呈“双峰”型,胞间CO2浓度的日进程基本与净光合速率相反,中午胞间CO2浓度增高,表明净光合速率午间降低主要受非气孔限制因素的影响。暗呼吸速率日进程为单峰曲线,中午最高。气孔阻力的日变化曲线是单谷型,午间最低。(2)一天分时段光响应试验表明,饱和光强和表观量子效率上午最高,中午最低,下午有所上升。下午的光补偿点是上午的2倍,中午则是上午的近3倍。不同时段遮光都使得气孔阻力和胞间CO2浓度升高。