Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).
A quantitative and multiplex real-time RT-PCR assay was developed to detect Citrus tristeza virus (CTV) along with plant mRNA, which serves as an internal control to ascertain RNA extraction quality. The real-time technique was validated against 39 CTV strains from around the world as well as with the aphid vector, Aphis gossypii, given a 48 h acquisition access period on a CTV source plant. The assay was effective for quantitation of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 1 fg was consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than ELISA or traditional RT-PCR. The real-time RT-PCR assay developed is a valuable new tool for detection and titer quantitation of CTV.
Saponari
Maria
M
USDA, Agricultural Research Service, 9611 S. Riverbend Avenue, Parlier, CA 93648, USA.
Manjunath
Keremane
K
Yokomi
Raymond K
RK
D000818:Animals; D001042:Aphids / Q000821:virology*; D002957:Citrus / Q000821:virology*; D017790:Closterovirus / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D010935:Plant Diseases; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D012367:RNA, Viral; D012313:RNA