Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays

Szemes, Marianna; Bonants, P.J.M.; Weerdt, M. de; Banér, Johan; Landegren, Ulf; Schoen, C.D.

doi:10.1093/nar/gni069

Cited by 124 publications

(94 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4). This strategy has been used for fungi and nematodes (Szemes et al 2005;van Doorn et al 2007). In another strategy, Engel et al (2010) used a random primed PCR amplification before hybridization in the development of a microarray designed to detect 44 viruses of the grapevine.…”

Section: Crop-oriented Diagnosis: Multiplexing and Probe Arraysmentioning

confidence: 99%

New Developments in Pathogen Detection and Identification

Nolasco¹

2012

Acta Hortic.

View full text Add to dashboard Cite

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.

show abstract

Section: Crop-oriented Diagnosis: Multiplexing and Probe Arraysmentioning

confidence: 99%

New Developments in Pathogen Detection and Identification

Nolasco¹

2012

Acta Hortic.

View full text Add to dashboard Cite

show abstract

“…The molecular ring is then amplified by universal primers and labelled with a fluorophore. The amplicon, that must include the Zip Code, is then hybridized to an UA [52].…”

Section: Ligation Detection Reaction-universal Arrays (Ldr-ua)mentioning

confidence: 99%

Potentials and limitations of molecular diagnostic methods in food safety

Lauri

Mariani

2008

Genes Nutr

View full text Add to dashboard Cite

Molecular methods allow the detection of pathogen nucleic acids (DNA and RNA) and, therefore, the detection of contamination in food is carried out with high selectivity and rapidity. In the last 2 decades molecular methods have accompanied traditional diagnostic methods in routine pathogen detection, and might replace them in the upcoming future. In this review the implementation in diagnostics of four of the most used molecular techniques (PCR, NASBA, microarray, LDR) are described and compared, highlighting advantages and limitations of each of them. Drawbacks of molecular methods with regard to traditional ones and the difficulties encountered in pathogen detection from food or clinical specimen are also discussed. Moreover, criteria for the choice of the target sequence for a secure detection and classification of pathogens and possible developments in molecular diagnostics are also proposed.

show abstract

“…6 Padlock probes are linear oligonucleotides that can detect target sequences that are complementary at both ends. 7,8 In the presence of the target, the padlock probe circularizes via DNA ligation and acts as a template for RCA, which generates a long single-stranded DNA molecule; a probe conjugated with a fluorescent label can be visualized as a 1-μm-sized dot under a fluorescence microscope. This technique has been applied to the detection of specific point mutations in the mitochondrial genome of individual cells, 4 replication of porcine circovirus type 2 in cultured cells and infected tissues, 9 mRNA expression within a cell in combination with a reverse transcription reaction, 5,10 mRNA sequencing, 11 and protein localization in combination with an antigen-antibody reaction, also known as proximity ligation assay.…”

Section: Introductionmentioning

confidence: 99%