Visualized And Simultaneous Detection Of Plant Pathogens Using Protein Macroarray

Protein chip technology has become a useful and crucial tool for large scale and high throughput bioanalysis. In narrow sense, the definition of protein chip and protein array can be changed with each other. According the spotting size, protein chip can be divided into two types: microarray and macroarray. In the nature conditions, animals and plants may be infected by multiple pathogens in the growth cycle. Immunology and molecular biology methods are currently used to evaluate the infected information of plant sample. Based on double antibody sandwich principle, a protein macroarray system was developed to detect multiple pathogens of solanaceous plant. On nitrocellulose membrane, capture antibodies are spotted and they can specific bind the target antigens. After washing, the macroarray was incubated with detection antibodies labeled with alkaline phosphatase. Then, the macroarrays were incubated with specific BCIP/NBT alkaline phosphatase substrate. The result can be examined by naked eye or scanned with a common scanner. On the sensitivity test experiment, protein macroarray showed a higher or equal detectability than enzyme linked immunosorbent assay (ELISA). Moreover,24 seed samples were used to test the practicability of protein macroarray, results showed a high consistent (240/240) with ELISA assay. Further more, a protein macroarray to Acidovorax avenae subsp. citrulli (Aac) was developed. This macroarray can detect Aac as low as 2.3×105 cfu/mL with outstanding specificity and repeatability. Protein macroarray not only has the single indexed detectability, but also has the multiplexed detectability. The plant sample used in the assay is not necessary to special handling and can avoid nucleic acid extraction, reverse transcription and molecular labeling. The operation of macroarray was very conveniently, detection result can be examined by naked eye or scanned with a common scanner to semiquantitative or quantitative analysis. This work provided a rapid and simultaneous detection to multiple plant pathogens and has a prospective prospect.