防治大豆胞囊线虫病的土壤真菌的筛选

以辽宁省大豆田土壤中分离筛选获得的39株真菌菌株为研究对象,通过测定不同真菌菌株代谢产物浸出液在不同处理时间对大豆胞囊线虫二龄幼虫(J2)的抑制作用,评价不同菌株对大豆胞囊线虫的生防效果,从而进一步筛选出生防效果较好的菌株。结果表明,与对照相比不同菌株对大豆胞囊线虫的抑制作用具有显著差异。39株被测菌株中有7株表现出较强的抑制作用。其中真菌snf 2455-2的浸出液对大豆胞囊线虫有较强的致死作用。随着处理时间的延长,大多数菌株的抑制效果增强,同时具有抑制效果的菌株也增多。在处理72 h时,与对照相比有17株菌株抑制效果达到了显著水平。     

对根结线虫抗病及感病烟草品种的RAPD分析

对12个烟草品种分别提取DNA并用OPA、OPG、OPJ、OPK、OPL、OPM、OPN共7组140个引物进行扩增,其中113个引物能有效扩增,113个引物共产生788条不同分子量的谱带,但不同烟草品种间,甚至烤烟与晾晒品种间表现出的多态民生水平低（小于1％）。表明尽管这些烟草品种间形态上存在明显的差异,但基因组相似度很高,亲缘关系很近,它们在形态上的差异可能是少量基因表达差异引起,用113个能有效扩增的引物对12个烟草品种进行反复实验,发现OPJ13能够在包括晾晒烟在内的所有感病品种中扩增出一条780bp的DNA片段,而所有抗病品种的扩增产物中均无该片段,由于目前抗根结线虫烤烟品种的抗源均来自原始抗病亲本TI1706,RAPD标记J13－780极有可能是与烟草的抗、感病基因相连锁的。     

大豆疫霉根腐病菌诱导下SSH文库构建及初步分析

大豆疫霉根腐病是危害大豆生产的毁灭性病害之一,研究利用抑制性消减杂交技术（Suppression Subtractive Hybridization,SSH）成功构建了疫霉菌诱导的大豆抗病品种绥农10差异表达的cDNA消减文库,从文库中共筛选到2067个阳性克隆,PCR鉴定插入片段大部分集中在100—800bp之间,利用BLAST在GenBank数据库进行序列相似性比对,获得有功能的EST375个,分析表明这些EST功能涉及大豆的抑制病原菌生长、细胞自身保护、信号传导、系统获得抗性、蛋白质合成、呼吸作用等。     

施用农用化学品及生防制剂的大豆胞囊线虫田根际真菌区系动态

采用除草剂、尿素和线虫生防制剂豆丰1号颗粒剂处理大豆胞囊线虫田土壤,定期取样、分离和鉴定,研究不同处理根际土壤真菌区系的动态变化。试验表明：施用尿素有利于协调根际真菌区系平衡;除草剂施有后对根际真菌区系有抑制作用,但通过对除草剂有降解作用的真菌的产生可恢复根际真工力区系平衡;而颗粒剂的施用有利于有益真菌类群的增加,可通过向颗粒剂中添加多种大豆胞囊线虫寄生真菌如镰孢属（Fusarium)、粘帚霉属（Gliocladium)和拟青霉属（Paecilmyces）来进一步稳定和提高生防制剂的防治效果。     

Simulation and experimental validation of a SU-8 based PCR thermocycler chip with integrated heaters and temperature sensor

We present a SU-8 based polymerase chain reaction (PCR) chip with integrated platinum thin film heaters and temperature sensor. The device is fabricated in SU-8 on a glass substrate. The use of SU-8 provides a simple microfabrication process for the PCR chamber, controllable surface properties and can allow on chip integration to other SU-8 based functional elements. Finite element modeling (FEM) and experiments show that the temperature distribution in the PCR chamber is homogeneous and that the chip is capable of fast thermal cycling. With heating and cooling rates of up to 50 and 30 °C/s, respectively, the performance of the chip is comparable with the best silicon micromachined PCR chips presented in the literature. The SU-8 chamber surface was found to be PCR compatible by amplification of yeast gene ribosomal protein S3 and Campylobacter gene cadF. The PCR compatibility of the chamber surfaces was enhanced by silanization.     

多级水脊线算法在二维医学超声图像中的应用

以肝囊肿超声图像为例,介绍一种新的医学超声图像的分割方法：多级水脊线分割算法在医学二维超声图像分割中的应用。由于斑点噪声和超声衰减产生的假象,二维和三维超声成像中的图像分割问题一直是公认的一大难点。本文采用由若干数学形态学辅助的水脊线方法提取超声图像中的肝囊肿边缘,取得了相当令人满意的结果,这为进一步的图像处理和化疗等治疗手段奠定了必要的基础。该方法自动选择并分割图像中的主要目标对象,从而克服了传统水脊线算法中常见的过分割和改进水脊线算法中半自动的缺陷。     

Inhibition of on-chip PCR using PDMS–glass hybrid microfluidic chips

In the course of developing a microfluidic analytical platform incorporating the polymerase chain reaction (PCR) and subsequent capillary electrophoresis (CE) analysis for a variety of bio-assays, we examined PCR inhibition through surface interactions with the chip materials. Our devices perform PCR in a three-layer chip, a glass–poly(dimethylsiloxane)–glass sandwich in which the poly(dimethylsiloxane) (PDMS, a silicone rubber) layer is used for pneumatic membrane pumping and valving of the PCR reagents. Initial on-chip PCR–CE tests of BK virus replicated in multiple uncoated chips showed variable results, usually yielding no detectable product at the target sample concentrations used. Subsequent “chip-flush” experiments, where water or reagents were flushed through a chip and subsequently incorporated in off-chip PCR, highlighted bovine serum albumin (BSA) amongst other pre-treatments, chip materials and PCR recipes as being effective in mitigating inhibition. When the BSA channel pre-coating was applied to on-chip PCR–CE experiments, a substantial improvement (10× to 40×) in signal-to-noise (S/N) of the CE product peak was conferred, and was shown with high confidence despite high S/N variability. This is the first study to quantitatively examine BSA’s ability to reduce inhibition of PCR performed on PDMS chips, and one of very few microfluidic PCR inhibition studies of any kind to use a large number of microfluidic chips (~400). The simplicity and effectiveness of our BSA coating suggest that passivating materials applied to microfluidic device channel networks may provide a viable pathway for development of bio-compatible devices with reduced complexity and cost.     

基于网格区域化DBSCAN聚类的数字PCR液滴分类方法

针对数字PCR系统的配套软件缺少多重实验液滴分类功能的不足,提出了一种网格区域化DBSCAN聚类算法.首先对数据进行网格映射,建立网格索引;然后计算网格间权值并进行深度搜索扩张;最后将网格空间的聚类结果映射回数据空间.人工数据集的仿真实验表明,所提算法能够有效识别簇边缘区域且具有优良的运行效率.在此基础上,提出了基于网格区域化DBSCAN聚类的数字PCR液滴分类方法,经由对比实验及有效性测试,结果表明所提方法能够便捷、准确地进行液滴分类.可见所提方法适用于数字PCR液滴分类.     

Fabrication and application of a new DNA biosensor based on on-substrate PCR and electrochemistry

DNA probes immobilized on a gold electrode (AuE) were employed as the primers of asymmetric PCR on the AuE. In the asymmetric PCR process, the DNA probes extended in the presence of target strands in the PCR solution. After PCR the dsDNAs were denaturalized and the target DNAs were eliminated and only the extended probes maintained on the AuE. At last the electrochemical indicator of methylene blue combined to the extended probes and the electrochemical signal of indicator was measured. This signal was higher than that of the AuE modified only by original probe. When there was no target in the PCR solution, the probe did not extend and the signal did not increase. The specific sequences of chitinase gene were detected successfully from four sorts of target with different length: oligonucleotide acid, PCR products, molecule cloning vector DNA and total genome DNA of transgenic capsicum, and the estimated detection limit were 7.3 × 10⁻¹², 3.2 × 10⁻¹¹, 5.4 × 10⁻¹¹ and 4.1 × 10⁻¹⁰ mol l⁻¹ respectively. The regeneration of the biosensor was also tested and the results indicated that its half life was 6 times.     

Primer design for multiplex PCR using a genetic algorithm

Multiplex Polymerase chain reaction (PCR) is the term used when more than one pair of primers is used in a polymerase chain reaction. The goal of multiplex PCR is to amplify several segments of target DNA simultaneously and thereby to conserve template DNA, save time, and minimize expense. The success of the experiment is dependent on primer design. However, this can be a dreary task as there are many constrains such as melting temperatures, primer length, GC content and complementarity that need to be optimized to obtain a good PCR product. In our investigations, we found few primer design tools for multiplex PCR and there was no suitable tool for our partners who want to use a multiplex PCR genotypic assay. The tool draws on a genetic algorithm where stochastic approaches based on the concept of biological evolution, biological genetics and genetic operations on chromosomes are used to find an optimal solution for multiplex PCR. The presented experimental results indicate that the proposed algorithm is able to find a set of primer pairs that not only obey the design properties but also work in the same tube.     

具有单一热源的对流双温PCR微流控装置的研究

为了克服传统连续流动聚合酶链式反应(PCR)微系统经常需要多个加热源来获取PCR所需要的温度,且集成或者非集成的泵系统也需要用来驱动PCR溶液连续流动,从而实现PCR扩增的缺点,研究了一种具有单一热源的对流双温PCR微系统.通过复合肋片技术在沟槽铜片上形成对流双温PCR所需要的温度,由流体密度差形成的自然对流来驱动闭环...     

Polymerase chain reaction compatibility of adhesive transfer tape based microfluidic platforms

Laser patterned adhesive transfer tapes are a rapid, versatile, and low cost option to fabricate microfluidic platforms. In this work, we examined the compatibility with polymerase chain reaction (PCR) of different types of adhesive tape materials patterned with a CO₂ laser cutter. Acrylic, polyimide, and silicone-based tapes were considered. We performed a systematic study on off-the-shelf adhesive tapes with respect to fluid handling, PCR inhibition, reagent loss, and on-chip PCR reaction. A novel microfluidic PCR approach was implemented that combines the advantages of previously reported systems. It uses a thermal gradient from a single heating element and the thermocycling was carried out by passing the reaction mixture back and forth in a microfluidic channel strategically placed along the thermal gradient. Only the silicone-based tapes were compatible with on-chip PCR. The overall fabrication process takes less than 30 min, uses only off-the-shelf finished or semi-finished materials, and is amenable to large-scale reel-to-reel processing.