Reverse-Transcription Polymerase Chain Reaction Detection of Citrus Tristeza Virus in Aphids

A rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection of citrus tristeza virus (CTV) in three aphid species. Seven CTV isolates from a worldwide isolate collection were used for aphid acquisition feeding by three aphid species. These included the most efficient CTV vector, the brown citrus aphid, Toxoptera citricida; the melon aphid, Aphis gossypii; and the green peach aphid, Myzus persicae, a non-vector for CTV. A short procedure for nucleic acid extraction from single or groups of aphids was developed. Nucleic acid extracts from 1, 3, 5, and 10 aphids with acquisition-access periods of 24 and 48 h were reverse transcribed and amplified using primers for the coat protein gene of the Florida B3 (T-36) isolate of CTV. PCR-amplified fragments of approximately 670 bp were obtained from all the isolates tested and the amplified product from the aphids fed on citrus infected with isolate B3 was confirmed as the CTV coat protein gene by digesting with various restriction enzymes. This technique will be useful in investigations of CTV-vector-plant interactions and CTV epidemiology.