Baldi Paolo, La Porta Nicola
IASMA Research and Innovation Centre, Fondazione Edmund MachTrento, Italy.
MOUNTFOR Project Centre, European Forest InstituteTrento, Italy.
Front Plant Sci. 2017 Jun 8;8:944. doi: 10.3389/fpls.2017.00944. eCollection 2017.
In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. , a Gram-negative bacterium belonging to the family , is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of infection have been reported in other European Countries. At least five different subspecies of have been reported and classified: , and . A sixth subspecies () has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field.
在与植物致病细菌的永无休止的斗争中,一个主要目标是对感染微生物进行早期鉴定和分类。属于 科的革兰氏阴性细菌 也不例外,因为这种病原体具有广泛的传播媒介和寄主植物,其中许多可能长时间携带病原体而不表现出任何症状。直到最近几年,由 引起的大多数疾病都在北美洲和南美洲被报道,但最近这种苛求性病原体引起的橄榄快速衰退综合征在普利亚(意大利东南部)广泛传播,并且在其他欧洲国家也报道了几例 感染病例。至少已报道并分类了五种不同的亚种: 、 和 。最近还提出了第六个亚种( )。因此,开发快速可靠的方法以便在感染的最初阶段检测到病原体至关重要,从而防止这种危险细菌的进一步传播。为此,诸如酶联免疫吸附测定(ELISA)和免疫荧光等经典免疫学方法并不总是足够灵敏。然而,已经开发出利用特异性引物扩增基因组DNA靶区域的基于聚合酶链反应(PCR)的方法,并且这些方法正成为检测和鉴定许多细菌种类的有力工具。本综述的目的是说明最常用的PCR方法在 研究中的应用,范围从经典PCR到几种基于PCR的检测方法:随机扩增多态性DNA(RAPD)、定量实时PCR(qRT-PCR)、巢式PCR(N-PCR)、免疫捕获PCR(IC-PCR)、短串联重复序列(SSRs,也称为可变数目串联重复序列VNTR)、单核苷酸多态性(SNP)和多位点序列分型(MLST)。还提到了特定靶标的扩增和序列分析。描述并讨论了过去几年在该病原体基于DNA的分类方面取得的快速进展,并列出了为不同方法设计的特异性引物,以便为该领域的所有研究人员提供一个简洁有用的工具。