Oligonucleotide microarray with a minimal number of probes for the detection and identification of thirteen genera of plant viruses

Abstract

A major challenge facing agriculture at present is the development of techniques that can screen field samples and other plant materials simultaneously for the presence of many viruses. Microarray techniques show promise in this regard, as their high throughput nature can potentially detect a range of viruses using a single test. In this paper we present an array that can detect a wide spectrum of 169 plant virus species from 13 different genera. The array was constructed using an automated probe design protocol which generated a minimal number of probes to detect viruses at the genus level. The designed arrays showed a high specificity and sensitivity when tested with a set of standard virus samples. Field samples collected from a severe disease outbreak of Panax notoginseng farms in Yunnan, China, in 2001 were screened, where a potyvirus infection was identified associated with the disease.

Fig. 1

Probe design workflow of the microarray-based plant virus detection.

Fig. 2

Sensitivity test of PVX with RNA dilutions of (A) 10⁰, (B) 10¹ and (C) 10² fold using microarray, and (D) RT-PCR. The array image is a computer generated pseudo-color image. The construction of the microarray is described in Supplementary File 1. The target viruses of each probe are described in the data table of GEO platform GPL8484. Probes targeting Potexvirus were highlighted with red rectangles. Probes not included in the red rectangles were probes targeting the other virus genera. Some probes targeting Potexvirus were not positive, as can be seen in the lower right rectangle of each image, because they may target the other viruses, other than PVX, in the genus Potexvirus.

Fig. 3

Sequence alignment of TRSV and schematic view of genome structure. (A) Probe sequence alignment with TRSV RNA 1 and RNA 2. (B) Genome structure of the TRSV and location of the probes specific to TRSV.

Fig. 4

Detection of TRSV using the microarray. The three probes targeting TRSV were nepo_5_848, nepo_5_836 and nepo_5_901, and all hybridized positively to the virus. The array image is a computer generated pseudo-color image. Probes targeting Nepovirus were highlighted with red rectangles. Probes not included in the red rectangles were probes targeting the other virus genera. Some spots targeting the other virus genera were visible on the image, but they were all below the detection threshold, as explained in the Section 2. Some probes targeting Nepovirus were not positive, as can be seen in the lower right rectangle of each image, because they may target the other viruses, other than TRSV, in the genus Nepovirus.

Fig. 5

Sequence alignment of the unknown Potyvirus and schematic view of genome structure. (A) Probe sequence alignment with PVY and the unknown Potyvirus detected from P. notoginseng samples. (B) Genome structure of the unknown Potyvirus and location of the probes. The genome structure and coordinates were simulated from PVY.