RT-PCR detection and identification of three species of cucumoviruses with a genus-specific single pair of primers

Reverse transcription and polymerase chain reaction (RT-PCR) was used for detection and identification of three cucumoviruses (cucumber mosaic virus, CMV; peanut stunt virus, PSV; tomato aspermy virus, TAV) in various plants sources with a single pair of primers, designed as CPTALL-3 and CPTALL-5. The pair of cucumovirus genus-specific primers that flank the coat protein gene were designed and used to amplify a DNA fragment of approximately ranging from 938 to 966 bp. The RT-PCR with the set of primers specifically amplified the target size of DNA fragment in all the tested cucumoviruses (CMV S-TA, S-IB and S-II, PSV and TAV). No DNA product of any length was produced when brome mosaic virus or tobacco mosaic virus RNA was used as templates. The cucumoviruses examined were differentiated by PCR-restriction fragment length polymorphism with different enzymes. This indicates that the designed primers are only specific for the cucumoviruses and useful for reliable information of identification of members of the Cucumovirus genus, (C) 1999 Elsevier Science B.V. All rights reserved.