黄瓜细菌性角斑病菌交叉引物恒温扩增检测技术     

霍亚云  张永江  李为民  朱罗罗  胡林  尤其敏  赵文军 《植物检疫》2017,(5):44-47

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。  相似文献   

基于荧光检测技术的多花黑麦草EST-SSR指纹图谱的构建   总被引：2，自引：0，他引：2  

刘欢  张新全  马啸  张瑞珍  何光武  潘玲  金梦雅 《中国农业科学》2017,50(3):437-450

【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)。  相似文献   

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

LUO Ya-kun  LIANG Lin  WANG Jing  LIU Cun  LIU Qi  LIU Chang  LIN Wen-cheng  CUI Shang-jin 《中国畜牧兽医》2017,44(2):344-349

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.  相似文献   

Research Progress on Resistance Mechanisms and Detecting Technology of Mycobacterium tuberculosis     

LIU Yong-an  LI Xue-rui  LIU Yong-sheng 《中国畜牧兽医》2017,44(6):1884-1889

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.  相似文献   

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

FENG Bin  XIE Zhi-xun  ZHANG Yan-fang  HUANG Jiao-ling  WANG Sheng  FAN Qing  HUANG Li  XIE Li-ji  ZENG Ting-ting  LUO Si-si  DENG Xian-wen  XIE Zhi-qin  LIU Jia-bo  SONG Zhong-bao  LIN Er-ke 《中国畜牧兽医》2017,44(5):1295-1301

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.  相似文献   

Establishment of RT-LAMP for Rapid Detection of Porcine Reproductive and Respiratory Syndrome Virus     

CHEN Xi-wen  WANG Qian  YIN Miao  LI Lian  LUO Wen-tao  YANG Feng  GUO Ai-wei  ZHOU Jie-long  WANG Xiong-qing 《中国畜牧兽医》2017,44(6):1630-1636

  相似文献   

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

LI Lei-ting  LIU Jun-hua  LIU Bao-tao  FU Hong-lei  ZOU Ming 《中国畜牧兽医》2017,44(8):2483-2488

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.  相似文献   

鸡细小病毒半巢式PCR检测方法的建立及初步应用     

奉彬  谢芝勋  张艳芳  黄娇玲  王盛  范晴  黄莉  谢丽基  曾婷婷  罗思思  邓显文  谢志勤  刘加波  宋中宝  林二克 《中国畜牧兽医》2017,44(5):1295-1301

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。  相似文献   

动物源性食品中抗菌药残留的微生物学检测技术研究进展     

吴芹  王玉莲  袁宗辉 《中国畜牧兽医》2017,44(11):3340-3350

抗菌药通过直接用药或用作饲料添加剂投入养殖业中,用于治疗或预防动物疾病。养殖业不当使用抗菌药,不可避免地造成抗菌药在动物源性食品中残留过量。微生物学法是最早用于抗菌药残留检测的方法,较其他检测方法而言,微生物学法操作简单、成本低、高通量,广泛应用于牛奶、畜禽组织等动物源性食品中抗菌药残留快速筛选检测,为后期的化学分析确证法节约大量成本。文章主要综述了微生物学法的发展历程及微生物学法在牛奶、可食性畜禽组织、鸡蛋、水产品、蜂蜜等动物源性食品中抗菌药残留检测的应用情况,为后续研制新微生物学法提供理论依据和实践基础,并对未来的微生物学法的发展做出展望。  相似文献   

基于富集微流控芯片的稻曲病菌孢子光电检测方法   总被引：1，自引：1，他引：0  

杨宁  王盼  张荣标  徐佩锋  孙俊  毛罕平 《农业工程学报》2017,33(20):161-168

针对当前水稻真菌病害发病时间短、传播速度快,缺乏有效早期预警技术的难点,提出一种基于微流控芯片的空气流中水稻真菌病害光电检测方法。该文根据微尺度下孢子富集动力学特征设计了病害孢子高效富集微流控芯片,并结合光电检测系统进行病害孢子的检测。试验根据空气动力学原理以及孢子富集量的大小对微流控芯片通道尺寸进行设置。根据光检测原理和不同浓度孢子在富集区形成的光衰减特性,筛选具有高灵敏度特性的检测光强。在以检测光谱的灵敏度性能为目标并综合考虑线性度的基础上,优选检测波长。以水稻稻曲病菌孢子为检测对象,进行了自动化微流控富集和光电检测试验。试验结果表明:在光源光强1.1×104 cd,波长为650 nm时,利用所述检测方法针对水稻稻曲病菌孢子检测结果(相比与镜检值)误差小于17.5%,根据检测结果建立相关系数为0.992 9的检测模型,具有较好的线性度及可靠性。研究结果为便携式作物病害检测装备的研发提供理论基础。  相似文献