López María M, Bertolini Edson, Olmos Antonio, Caruso Paola, Gorris María Teresa, Llop Pablo, Penyalver Ramón, Cambra Mariano
Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial. 46113, Moncada, Valencia, Spain.
Int Microbiol. 2003 Dec;6(4):233-43. doi: 10.1007/s10123-003-0143-y. Epub 2003 Sep 11.
Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.
检测植物材料、载体或天然宿主中的有害病毒和细菌对于确保安全和可持续农业至关重要。在过去几年中，现有技术已取得显著进展，以实现对病原体的快速可靠检测，从样品中提取目标物对于优化检测非常重要。对于病毒，通过将植物材料或昆虫载体印迹或挤压到膜上来简化样品制备。为提高细菌检测技术的灵敏度，建议在液体或固体培养基中进行预富集步骤。当需要分析大量样品时，血清学和分子技术目前是最合适的。许多植物病原体都有特异性单克隆和/或重组抗体，这有助于血清学检测的特异性。分子检测可通过使用柱或机器人自动从病原体中纯化核酸来优化。PCR的新变体，如在单个封闭管中的简单或多重巢式PCR、协同PCR以及对扩增子的实时监测或定量PCR，可在单次检测中对一种或几种病原体进行高灵敏度检测。核酸分析的最新进展是微阵列技术，但它需要通用的DNA/RNA提取和预扩增方法以提高检测灵敏度。许多植物病原体基因组测序所带来的研究进展，特别是在蛋白质组学时代，为这些微生物灵敏且特异的检测技术的未来发展提供了新的信息来源。
