基于锁式探针技术的菜豆晕疫病菌检测技术研究

根据菜豆晕疫病菌的一段特异促旋酶亚组B(gyr B)基因序列,设计锁式探针和扩增引物,优化体系反应条件,建立了基于锁式探针菜豆晕疫病菌滚环扩增特异性检测体系。试验结果表明该检测体系能够从供试菌株中特异性检测出菜豆晕疫病菌。该体系检测DNA的阈值为600 fg/μL,与传统PCR相当;检测菌悬液检测阈值1.3×10~3 cfu/m L,比传统PCR高10倍,在模拟样品检测中也显示了更适合于样品的检测。     

番茄细菌性叶斑病菌超分支滚环扩增快速检测技术

根据番茄细菌性叶斑病菌(Pseudomonas syringae pv. tomato,Pst)的一段特异蛋白基因序列,按照锁式探针的设计原理设计探针和扩增引物,优化系列反应条件,建立了特异性的Pst超分支滚环扩增技术。试验结果表明：该检测技术能够从供试的10种不同的病原菌中特异性地检测出番茄细菌性叶斑病菌,DNA检测的最低浓度为500 fg/μL,检测灵敏度高于常规PCR。     

重组酶聚合酶扩增技术在植物病原快速检测中的应用

植物病原的快速检测对于植物病害防控具有重要意义。重组酶聚合酶扩增(recombinase polymerase amplification,RPA)是近年建立的等温核酸扩增技术,具有灵敏度高、特异性强、适用于现场快速检测等特点,已在多个领域广泛应用。本文对重组酶聚合酶扩增技术的原理、特性、检测方法及其在植物病原体检测领域的应用进展加以综述。     

用滚环扩增技术检测双生病毒

 本文报道了利用滚环扩增技术(rolling circle amplification,RCA)放大双生病毒的靶核酸信号,进而利用限制性内切酶片段长度多态性(RFLP)检测不同类型双生病毒的新技术体系。研究结果表明,利用RCA-RFLP对于已知的单组份和双组份以及带有卫星的菜豆金色花叶病毒属病毒都能够有效检测,对采自浙江杭州地区的田间样品进行RCA-RFLP检测的结果与利用简并引物PA和PB进行PCR扩增的结果相吻合。相比传统的PCR检测方法,用RCA-RFLP体系检测双生病毒灵敏性更高,操作更简便。     

环介导等温扩增技术在植物病原物检测中的应用

环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种新型的由环介导的等温核酸扩增分子技术,不仅特异性强、操作简便、成本低,还能快速、高效地检测病原物,为植物病害的防控提供更精准的防治适期,从而可以减少农药的滥用。本文主要针对LAMP技术的原理、发展、优缺点、在真菌、细菌、病毒等多种植物病原物检测及在抗药性检测中的应用进行总结,并结合国内外研究进展对其应用前景进行了分析。     

基于PCR技术的植物病原真菌检测技术研究进展

植物病原真菌是可以在植物上引起病害的一类重要病原物,对该类病原的快速检测是对其进行植物检疫、监测预报及病害防治必不可少的基础工作.近年来,以PCR为基础的分子检测技术的日益发展使得植物病原真菌的检测更加快速、灵敏和可靠.本文对近年来基于PCR技术的植物病原真菌检测方法进行了介绍与评述,这些方法包括ITS-PCR、巢式PCR、多重PCR、PCR ELISA和实时荧光定量PCR技术.     

植物病原真菌检测技术研究进展

传统的植物病害诊断方法往往根据病组织的症状、病原菌的特征以及研究人员的经验进行。但某些病原引起的症状相似,例如胡萝卜的细菌枯萎病与交链孢黑斑病、病毒与难养菌、不同病原物复合侵染造成的症状。有些病原菌不能进行纯培养,因而,单靠传统的分离培养、生物学测定等?..     

免疫金银染色技术及其在植物病原细菌研究中的应用

免疫金银染色技术及其在植物病原细菌研究中的应用冯家望莫晓凤（拱北动植物检疫局５１９０２０）自从１９７１年Ｆａｕｌｋ和Ｔａｙｌｏｒ〔１〕首次报道用胶体金标记抗体研究细胞表面抗原以来，免疫金银染色（ｌｍｍｕｎｏ－ｇｏｌｄｓｔａｉｎｉｎｇ）技术已被成功地应...     

基于PCR技术的植物病原菌分子定量检测技术研究进展

植物病原菌的菌源量是病害发生和流行的重要因子之一,对其精准的定量测定或检测可大大提高植物病害预测的准确性,本文对实时荧光定量PCR (qPCR)与数字PCR在植物病原菌定量检测、以及基于RNA水平的real-time PCR和基于核酸染料(EMA/PMA)与qPCR相结合的技术在植物病原菌活体定量检测中的应用进行了综述,并展望其在植物病害流行和预测中的应用前景。     

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.     

Biocontrol of soil-borne plant pathogens: Concepts and their application

A few soil-borne plant pathogens have been controlled successfully by commercial formulations of biocontrol agents, but many attempts to develop biocontrol inoculants, although promising under experimental conditions, have met with difficulties in practice. The reasons for this are discussed in this review, which outlines some of the major findings on the behaviour of microbial inoculants in soil. It is emphasized that biocontrol also occurs naturally in current agricultural practice and can be exploited purposely, but it is vulnerable to disruption by agrochemicals or mismanagement. The future of biocontrol of soilborne plant pathogens probably lies in integrated (biorational) control systems that combine the use of commercial inoculants, where appropriate, with management practices that maintain and enhance the natural biocontrol systems.     

Exploiting generic platform technologies for the detection and identification of plant pathogens

The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.