Plant virus diagnosis
Plant virus diagnosis
If you're diagnosing many samples with the same virus, ELISA is the method of choice. My concern however is diagnosing a few samples where virus (and sometimes host) identity is obscure and also samples with multiple viruses. The first step is to determine if the sample has a virus infection, the second is to determine the general nature of the virus ( classification) and the final step is to identify the specific virus. Often knowledge of classification combined with host identity (and perhaps symptoms) suggests virus identity. DNA based methods are not suited to the first two steps.
Minipurification - This is a partial virus purification followed by gel electrophoretic separation and detection of capsid proteins. It's sensitive, reasonably universal and can be completed in a day. pH and temperature sensitivity of viruses are easily checked as is sensitivity to common proteases. It's also useful for monitoring cultures for contamination.
Protein phenol extraction, followed by SDS gel electrophoresis. This is quicker and simpler than minipurification, but not as sensitive. It will miss low concentration viruses or viruses whose capsids overlap with host proteins. It can however pick up inclusion body proteins and capsid proteins of insoluble viruses which makes it a useful complement to minipurification. It can also sample small tissue areas, making it especially useful for viruses with localized symptoms.
dsRNA analysis works extremely well for some virus groups, but poorly for others. It's less universal than minipurification. It requires larger tissue volumes.
Inoculation to Chenopodium quinoa works well for many viruses. I often inoculate minipurified samples to C. quinoa. If the plant becomes infected, virus can be detected by phenol extraction of single lesions, or by minipurification of larger tissue volumes. Symptoms on C. quinoa can be helpful, but many viruses give similar symptoms. One must be careful to recognize and avoid contamination.
I don't have an electron microscope readily available, but EM is a convenient standard technique. Minipurified samples are especially convenient for EM. Combining EM with serology is convenient for specific diagnosis.
What about symptoms?
Diagnosing viruses in the field isn't like bird watching. Virus symptoms depend on host genetics and physiology such that virus A under appropriate conditions exhibits symptoms of virus B. Furthermore it's clear that small (single nucleotide) genetic differences can influence symptoms. Symptoms are extremely useful, but need to be interpreted with caution.