Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses

Abstract

Maoka, T., Sugiyama, S., Maruta, Y., and Hataya, T. 2010. Application of cDNA macroarray for simultaneous detection of 12 potato viruses. Plant Dis. 94:1248-1254. A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors.Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 10 2 to 4 × 10 6 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 10 4 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

Aphelenchoides besseyi Christie (Nematoda: Aphelenchoididae), agente causal del amachamiento del frijol común

“…Se consideraron estadísticamente positivas las muestras cuyo valor de absorbancia fue superior al valor del promedio para el control negativo más tres veces el valor de la desviación estándar (De Breuil et al, 2008;Maoka et al, 2010).…”

Standardized RT-PCR Conditions for Detection and Identification of Eleven Viruses of Potato and Potato spindle tuber viroid

“…Even more recently the use of micro-or macroarrays for potato virus detection and identification has been reported (Boonham et al 2003;Agindotan and Perry 2008;Maoka et al 2010). Still in the developmental stage is a "universal plant virus microarray" that may be able to detect most plants viruses in a single test (Bagewadi et al 2010).…”

Breeding of Pest and Disease Resistant Potato Cultivars in Japan by Using Classical and Molecular Approaches

“…Twelve viruses have been identified as causal agents of viral diseases affecting potatoes in Japan (Maoka et al 2010). Among the 12 viruses, PVY is the most frequently found in Japanese potato production areas.…”