黄瓜细菌性角斑病菌交叉引物恒温扩增检测技术

黄瓜细菌性角斑病是我国黄瓜生产上的重要病害之一,其病原菌为Pseudomonas syringae pv.lachrymans。根据该病原菌甘油醛-3-磷酸脱氢基因保守序列设计引物和探针,建立了交叉引物恒温扩增和核酸试纸条检测技术。菌体DNA检测灵敏度可达0.55 ng,纯菌直接检测灵敏度基本可达到单个细菌。所测试的5株黄瓜细菌性角斑病菌和染病黄瓜叶片均为阳性,其他13株对照菌株均为阴性。该方法灵敏度高,且操作简单,对设备要求低等,适合基层实验室应用。     

基于最大类间方差法的黄瓜病害叶片分割

以黄瓜病害叶片为研究对象,采用直方图阈值分割法去除背景,分别采用最大类间方差法(Otsu)和边缘检测法来分割黄瓜叶片中的病害部位,对比这2种方法的分割效果。最后,对已有的最大类间方差法进行了改进,对病害叶片图像的红色分量进行了病斑分割。结果表明,边缘检测法分割出来的病斑部位轮廓具有不完整性,而最大类间方差法的分割效果较好。采用最大类间方差法对黄瓜病害叶片分割取得了较理想的效果,为后续病害识别奠定了基础。     

耕层残膜回收机的研究

地膜覆盖技术已成为农业生产中不可或缺的技术,极大地促进了农业发展和进步。但是农用的塑料薄膜大都是不可分解的,长时间留在土壤里,不但会对作物造成严重的危害,影响作物正常的生长和产量,还会影响机械作业的质量。要在收获庄稼后,及时地回收残膜,避免造成"白色污染"和塑料资源浪费。为此介绍了残膜回收机在国内外的发展现状以及典型的残膜回收机的结构与工作原理,并提出存在的问题和未来的发展建议。     

基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建

【目的】构建基于EST-SSR荧光标记的多花黑麦草(Lolium multiflorumLam.)DNA指纹鉴定体系,为多花黑麦草品种鉴定提供高通量技术手段,为不同品种的合理应用提供参考依据,有效保护农民利益和育种权益。【方法】利用表型性状差异大的3个品种(特高Tetragold、长江2号Changjiang No.2和阿伯德Aubade),通过聚丙烯酰胺凝胶电泳从200对EST-SSR引物中筛选出扩增条带清晰、多态性好、扩增稳定的30对引物。在筛选出的每对引物5′端添加荧光标记FAM后,采用毛细管法通过DNA分析仪检测200个单株不同等位变异的扩增片段,从30对EST-SSR引物中筛选出25对扩增稳定的荧光引物,建立基于高通量荧光SSR标记的多花黑麦草品种鉴定体系。【结果】通过25对EST-SSR引物构建的DNA指纹图谱来进行10个多花黑麦草材料的品种鉴定。25对EST-SSR引物共检测到127个等位基因,等位变异扩增片段长度范围为51—249 bp,每对引物可检测到有效等位基因数为2—11个,特异等位基因最多可检测到11个(N101),平均每对引物4.00个;多态性位点的比率范围为33.33%—100.00%。平均PIC值为0.702,Shannon指数最大为3.322(N101),平均为1.929,基因多样性指数变幅为0.159—0.500,平均0.318,可鉴别的材料数为0—10个;其中14对特征引物在10个品种(系)上可检测出25个特异等位基因。综合来看,引物N101在200对引物中鉴别效率最高,可直接将10个多花黑麦草品种(系)区分开,在长江2号、赣选1号和川农2号上同时检测出特异等位基因。但由于多花黑麦草在品种间与品种内变异均较高,因此为鉴定更多材料,从25对引物中选择了6对扩增和检测效果较好的引物(N54、N101、N146、N151、N154、N156),6对引物可检测到的稳定等位基因数均不小于19个,在达伯瑞和邦德上最多可检测到22个等位基因。通过6对高效引物构建了10个多花黑麦草品种(系)的DNA指纹图谱,包括标准模式图、图谱代码和图谱QR编码。首次利用EST-SSR荧光标记毛细管电泳检测,为10个多花黑麦草材料分别构建了唯一的指纹代码和QR编码。【结论】利用6对高效引物构建了多花黑麦草SSR高通量鉴定体系,其中荧光引物N101多态性最高,可直接鉴别10个多花黑麦草品种(系)。     

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples.     

Research Progress on Resistance Mechanisms and Detecting Technology of Mycobacterium tuberculosis

In recent years, the constantly emerging of drug resistant Mycobacterium tuberculosis brings unprecedented pressures to treat tuberculosis. In recent studies, it has found that not only bovine-type Mycobacterium tuberculosis can infect people, but human-type Mycobacterium tuberculosis can also infect cattle, which is also brings threats and challenges to the development of the cattle industry. And how to establish quick, handy, sensitive, highly specific and inexpensive resistance testing methods is the key to control the spread of the disease.In this review, it introduced not only the mechanism of action of isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide and so on, but also the traditional detection methods of drug resistance of mycobacteria (luciferase system, microscopic observation of drug susceptibility, proportion method, Bactec MGIT 960 System)and molecular detection methods (reverse hybriditation-based line probe assay (LiPA), Gene Xpert automatic detection system, PCR-single-strand conformation polymorphism analysis(PCR-SSCP),PCR-restriction fragment length polymorphism(PCR-RFLP), DNA sequencing, gene chip, RNA/RNA mismatch assay, which may be helpful to the further and clinical treatment and research for tuberculosis.     

Development and Preliminary Application of a Semi-nested PCR Assay for Detection of Chicken Parvovirus

In order to set up and optimize a semi-nested PCR for rapid detection of chicken parvovirus (ChPV), three specific primers were designed according to conserved sequences of NS 1 gene of ChPV. The specificity and sensitivity of ChPV semi-nested PCR were tested, and the assay was applied to detect 48 clinical samples. The specificity and sensitivity tests showed that this semi-nested PCR was only sensitive to ChPV for amplifying specific band of 186 bp and it could detect 5.62 fg/μL of ChPV DNA, without any sensitivity to other viruses, such as Newcastle disease virus, H9 subtype avian influenza virus, Marek's disease virus, infectious laryngotracheitis virus and infectious bronchitis virus. 48 chicken samples were detected and the positive rate was 16.67% (8/48). The results of our study demonstrated that the optimized semi-nested PCR could be a method that was suitable for clinical detection of ChPV.     

Residue Analysis of Five Kinds of Penicillins in Eggs by High Performance Liquid Chromatography-Ultraviolet Detection

This study was aimed to develop a method for simultaneous determination of five kinds of penicillins in eggs by high performance liquid chromatography-ultraviolet detection (HPLC-UVD). The eggs samples were extracted by acetonitrile,degreased by n-hexane,concentrated by rotary evaporation,derivatization,and then was separated by HPLC. The results showed that the calibration curves of five kinds of penicillins were linear correlated in the concentration ranges of 10 to 1 000 μg/kg with the correlation coefficients >0.999,which the limits of detection and quantification were 1.5 μg/kg (S/N=3) and 5.0 μg/kg (S/N=10),respectively. The average recoveries were 62.04% to 93.13%,the relative standard deviations were 3.91% to 13.69%.And the intra-day and inter-day RSDs were 3.98% to 14.71% and 4.26% to 12.71%,respectively. The developed method was suitable for detecting five kinds of penicillins residues in eggs with high repeatability and sensitivity.     

鸡细小病毒半巢式PCR检测方法的建立及初步应用

为建立一种快速、特异、灵敏的检测鸡细小病毒（chicken parvovirus,ChPV）的方法,根据ChPV的保守基因NS1设计了3条特异性引物,建立并优化了能快速检测ChPV的半巢式PCR方法,对其进行特异性和敏感性试验,并用所建立的方法对48份临床样品进行了检测。特异性和敏感性试验结果显示,建立的半巢式PCR只对ChPV敏感,扩增产物为186 bp的特异性条带;其最低能检测到5.62 fg/μL的ChPV DNA;而对鸡新城疫病毒、H9亚型禽流感病毒、马立克氏病病毒、鸡传染性喉气管炎病毒、鸡传染性支气管炎病毒不敏感。临床检测结果显示,同时对48份临床样品进行检测,检出率为16.67%（8/48）,提示广西区内鸡群存在ChPV感染。本研究建立的ChPV半巢式PCR方法适用于ChPV的临床检测。