Abstract
Many methods can be used for arriving in a correct virus disease diagnose, and the serological techniques are the most used methods for plant virus identification. The indirect enzyme-linked immunosorbent assay (Indirect-ELISA) or plate-trapped antigen ELISA (PTA-ELISA) has been useful for detection of viruses in a wide range of situations, especially to test a large number of samples in a relatively short period of time. Immune-biological Companies have developed practical kits for direct ELISA or double antibody sandwich (DAS-ELISA), but neither Company has developed kits for PTA-ELISA. As a single universal antibody-conjugate is used for detection of a wide range of plant viruses, the PTA-ELISA technique is more economical, practical and suitable for virus detection. Considering also the great problem of including infectious plant viruses in DAS-ELISA kits, a simple kit for PTA-ELISA was developed for plant virus identification. Extracts from infected plant tissues were added into the ELISA plate wells, which were sealed with plastics and maintained in the refrigerator and Laboratory conditions for different periods of time. The plates were tested by the regular PTA-ELISA and after 20 months of incubation, the plate showed excellent results when used for detection of six virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus in infected plant tissues. The ELISA plate trapped virus together with its specific antiserum could constitute a simple kit, which will permit the exchange of antisera among virologists without transferring infectious viruses from one laboratory to another to be used as control.
Highlights
The diseases caused by virus are generally difficult to control and several of them cause serious problems for the crop productions
enzymelinked immunosorbent assay (ELISA) plates trapped antigens were prepared with six virus species from the family Secoviridae, Comovirus genus (Squash mosaic virus, SQMV and Cowpea severe mosaic virus, CPSMV), family Bromoviridae, Cucumovirus genus (Cucumber mosaic virus, CMV), family Potyviridae, Potyvirus genus (Cowpea aphidborne mosaic virus, CABMV and Zucchini yellow mosaic virus, ZYMV) and Sobemovirus (Papaya lethal yellowing virus, PLYV) in infected plant tissues (NASCIMENTO et al, 2010; SILVEIRA et al, 2005)
The antigen trapped plates were tested at 30-days intervals by the regular plate-trapped antigen (PTA)-ELISA method, following the steps 100 μL of the polyclonal virus antisera produced in rabbits previously absorbed with extracts from healthy plants that were produced in the Plant Virus Laboratory at University of Ceará (UFC), and diluted to 2,000 were added into the wells
Summary
The diseases caused by virus are generally difficult to control and several of them cause serious problems for the crop productions. An adequate diagnosis procedure with a correct virus identification and determination of its distribution in the field are essential for establishing efficient control measures. For this reason, biological, serological and molecular methods have been developed for identification and detection of plant virus infections in the field. The correct diagnose of a plant virus diseases can be accomplished by one or a combination of methods involving the morphological, physical, biological, cytological, serological and molecular properties of the viruses, but serology is one of the most specific and easy method to obtain a rapid and precise identification of plant viruses. The great value of the serological methods for virus identification is based on the specific reaction between the antigens and its specific antibody
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