New Developments in Pathogen Detection and Identification

Abstract

A b s tractBy the turn of the century most of the methods for amplifying the pathogen detection signal had already been developed. However only a few were successfully incorporated into routine diagnosis schemes. At present, real time PCR became the predominant choice with signal transduction based on fluorescence generated by intercalating dyes or fluorescent resonant energy transfer. Real-time PCR is no more exclusively restricted to niches for which no alternative was feasible; its use is widening and is substituting or complementing other techniques like ELISA. On the low technology side and judging from the success in medical fields, Loop Mediated Isothermal Amplification (LAMP) appears as an interesting alternative. Convergence into a single platform led to the concept of crop-oriented diagnosis, e.g, a unique device or system that could be used in the detection of the relevant pathogens of a crop. However PCR by itself is not enough robust to support the degree of multiplexing needed and fluorescence detection systems are limited to a reduced number of dyes that can be used simultaneously. Initial attempts to develop alternative systems relied in the use of microarrays but suffer from limited sensitivity. Low Density Arrays, in which individual PCR reactions are spatially separated and done in parallel appear an interesting option, already available for grapevine. All these assays only provide an answer regarding the presence of certain pathogens for which there is an a priori suspicion. Recently introduced high massively parallel sequencing coupled to metagenomic analysis appears to be a major breakthrough in diagnosis, enabling the non-targeted diagnosis of bacteria, virus, fungi and novel agents in a single assay. These have been implemented among others for citrus and grapevine.

Grapevine leafroll-associated virus 3

“…The LAMP method relies on the isothermal amplification of a target sequence by a strand displacing DNA polymerase and four primers with six target areas. This method has been applied for the detection of viruses including GLRaV-3 by adding reverse transcriptase to the LAMP protocol (RT-LAMP) (Nolasco, 2010; Pietersen and Walsh, 2012). …”

Grapevine leafroll-associated virus 1

“…The LAMP method relies on the isothermal amplification of a target sequence by a strand displacing DNA polymerase and four primers with six target areas. This method has been applied for the detection of viruses including GLRaV-3 by adding reverse transcriptase to the LAMP protocol (RT-LAMP) (Nolasco, 2010;Pietersen and Walsh, 2012).…”