ELISA (Enzyme-Linked Immunosorbent Assay)
Immunoassays are chemical tests utilizing antibodies to detect pathogens such as viruses, bacteria, and fungi, and are routinely used in medical and agricultural disease diagnostics. Antibodies can specifically recognize the unique molecular structure (antigen) of pathogens and bind to them. The formation of antibody-antigen complexes are then detected via an indicator reaction. Immunoassays are highly sensitive and specific, including immunoblots, immunofluoresence, ELISA, and lateral flow assays (strip tests).
We use a double antibody sandwich ELISA for plant disease diagnosis. To detect a specific pathogen, a test well on a microtiter plate is coated with a particular (capture) antibody that specifically binds particles of the pathogen we test for in your sample. The non-target pathogens are then washed out. The secondary antibody conjugated to an enzyme is added to the test well. If the target pathogen is present and bound by the first antibody, the conjugate will be bound and unbound conjugate is washed out. After adding a colorless substrate that can be broken down into a colored one by the enzyme conjugate, color will be developed in the test wells if enzyme conjugate attached to a pathogen is present.
PCR (polymerase chain reaction)
Each species of pathogens carries a unique DNA or RNA signature that differentiates it from other organisms. We use PCR to detect pathogens by specifically amplifying target nucleic acid sequences from select microbes present in samples collected from complex biological environments using specific primers. During the PCR process, a set of primers specifically bind on the target nucleic acid sequence and the DNA fragment flanked by two primers is copied by an enzyme, resulting in doubled amount in each cycle. PCR is normally performed for 30 – 40 cycles producing more than 1 billion times of the target DNA fragment. Therefore PCR is the most sensitive of the existing rapid methods to detect plant pathogens.