Assessment of Arthropod Pathogen Microarray sensitivity

In order to estimate the sensitivity of the arthropod pathogen microarray (APM) two positive control samples were prepared in the presence and absence of pathogen-free honey bee RNA. A full-length (9,264 nucleotide) Drosophila C virus (DCV) clone was in vitro transcribed, serially diluted into honey bee RNA, reverse-transcribed, amplified, dye-labeled and hybridized to the APM as described above. Detection of at least 3 of the 8 unique DCV oligonucleotides and their reverse complements resulted in an estimated DCV detection level of 1.9×10⁵ genome copies (1 pg DCV genomic RNA) in an A. mellifera RNA (1 µg) background. Similarly, detection of a BQCV genome segment (452 nt), corresponding to one array oligo and its reverse complement, diluted into either pathogen-free honey bee RNA (0.5 µg) or water indicated detection limits of 1.2×10⁵ genome segment copies (30 fg BQCV RNA segment) and 1.2×10⁴ genome segment copies (3 fg BQCV RNA segment) respectively.