一种改进的尖孢镰刀菌的实时荧光定量PCR检测方法   总被引：1，自引：0，他引：1  

黄怀冬  肖姬玲  张屹  阮万辉  魏林  梁志怀 《植物保护》2017,43(4):129-133

为实现荧光定量PCR对土壤病原真菌数量更为高效、灵敏的检测,本研究将液体培养的孢子悬浮液和长年耕作的水稻土制作成孢子浓度为4×10~1~4×10~6 spore/g的带菌土,采用MoBio PowerSoil@DNA Isolation Kit提取模拟带菌土壤总DNA,引入常规PCR预扩增包含qPCR目标序列的1 446bp片段,以预PCR产物为模板进行qPCR,构建荧光定量PCR标准曲线。研究结果表明:以试剂盒提取的带菌土壤总DNA为模板绘制的qPCR标准曲线相关系数r为0.985,检测下限为4×10~3 spore/g土;引入预PCR后,qPCR标准曲线相关系数r为0.974,检测下限为4×10~2 spore/g土,较未引入时提高了10倍,结合不同土壤病原真菌的特异性引物,该检测方法可为土壤病原真菌的有效定量检测提供技术支撑。  相似文献   

基于环介导等温扩增技术检测雪松疫霉根腐病菌   总被引：2，自引：0，他引：2  

纪睿  曾丹丹  廖太林  郑小波  张正光  周锐 《植物检疫》2017,(1):42-47

雪松疫霉根腐病菌(Phytophthora lateralis Tucker et Milbrath)是一种重要的毁灭性植物病原菌,也是我国进境植物检疫性有害生物。目前,我国未有该病害发生的报道,为了防止P.lateralis的传入和扩散,需对其进行快速、准确的检测。本文利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),以Ypt1基因为靶标序列,设计LAMP特异性引物,建立LAMP反应体系,并对灵敏度和特异性进行检测。结果表明:整个检测过程仅需80 min,即可通过肉眼观察直接判定检测结果。在特异性检测中,P.lateralis菌株都能观察到天蓝色的阳性反应,而其他疫霉菌和真菌供试菌株均呈阴性反应。在灵敏度检测中,最低检测限为100 pg/μL。该方法的建立为P.lateralis的检疫鉴定及其所致病害的快速诊断提供了新技术。  相似文献   

油菜茎基溃疡病菌DPO-PCR检测方法的建立     

龙阳  袁俊杰  候翠丽  卢乃会  杨卓瑜  马新华  魏霜 《植物检疫》2017,(4):42-45

根据油菜茎基溃疡病菌ITS基因序列,设计特异性DPO引物,建立检测油菜茎基溃疡病菌的DPO-PCR检测方法,并对其特异性、灵敏性进行评价。结果显示与常规PCR检测方法相比,DPO-PCR反应对退火温度不敏感,具有更强的特异性。利用该方法对10批油菜籽样品进行检测,共检出5批阳性样品,检测结果与常规PCR一致,为油菜茎基溃疡病菌的检测提供了新方法。  相似文献   

一种值得关注的火龙果腐烂病     

王卫芳  何瑞芳  魏楚丹 《植物检疫》2017,(3):39-41

火龙果腐烂病是一种对我国新兴的火龙果果业潜在风险较高的真菌病害。近年来,我国口岸多次从越南进口的火龙果鲜果中截获该病害。本文介绍了其病原真菌仙人掌平脐蠕孢(Bipolaris cactivora(Petrak)Alcorn)地理分布、寄主范围、经济危害性、症状及病原菌形态特征、形态学鉴定和分子检测方法 ;分析了病原菌的进入风险和定殖与扩散风险,提出了加强口岸植物检疫,严防病原菌传入及病害在国内发生扩散和蔓延等风险管理措施。  相似文献   

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

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