Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVY NTN and the mild PVY N , were monitored. Microarray and quantitative real-time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate. Additionally, symptom severity and development was observed and the amount of virus isolate accumulated in systemically infected leaves was evaluated, where a significantly higher amount of PVY NTN was detected. Microarray analysis revealed 572, 1288 and 1706 differentially expressed genes at 0AE5, 12 and 48 h post-inoculation, respectively in cv. Igor, with a similar pattern observed in cv. Nadine. Microarray and quantitative real-time PCR results implied an earlier accumulation of sugars and lower photosynthesis in leaves inoculated with the aggressive isolate than in leaves inoculated with the mild isolate. The PVY NTN isolate did not activate early differential expression of the Fe-superoxide dismutase and pectin methylesterase inhibitor (PMEI) genes, indicating a delay in plant response relative to that following PVY N inoculation. Differences in the expression of the b-glucanase-I gene were also observed in early plant responses to inoculation with each virus isolate.

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Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log 10 dynamic range, a reproducible (R 2 > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%–98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.

Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative realtime RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log 10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.

Since their conception in the late 1990s, microarray techniques have become a tool of choice for monitoring pangenomic gene expression. Although there are a large number of variations on the basic methodology the general approach remains standard and involves the comparison of a "test" RNA with a "control" RNA; in this case "healthy" and "virus-infected" plants. The protocol itself can be broken down into five main parts: RNA extraction, cDNA synthesis, hybridization, array scanning, and data analysis. The method presented is optimized for use with arrays based on glass slides spotted with cDNA, in this case 15,264 cDNAs from Solanum tuberosum. The labeling technique presented involves two steps: hybridization of cDNA produced using oligo-dT linker primers to the array and hybridization with a DNA dendrimer reagent comprising sequence complementary to the linker sequence bound to a fluorescent dye. We also present the use of the R environment for data analysis, generating statistical support for differential gene expression observed.