Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction.
We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) to detect six citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV) and Citrus viroid OS (CVd-OS) and Apple stem grooving virus (ASGV, synonym: Citrus tatter leaf virus (CTLV)) from citrus plants. The multiplex RT-PCR was also designed to distinguish CVd-I-LSS (a distinct variant of CBLVd) from CBLVd. By the multiplex RT-PCR, one to eight fragments specific to the pathogens were simultaneously amplified from one sample and identified by their specific molecular sizes in 6% polyacrylamide gel electrophoresis. The results of the multiplex RT-PCR were consistent with those of other diagnoses, such as uniplex RT-PCR, to detect each of the pathogens. The multiplex RT-PCR provides a simple and rapid method for detecting various viroids and ASGV in citrus plants, which will help diagnose many citrus plants at a time.
Ito
Takao
T
Department of Citrus Research, National Institute of Fruit Tree Science, Kuchinotsu, Nagasaki 859-2501, Japan. itotaka@affrc.go.jp
Ieki
Hiroyuki
H
Ozaki
Katsumi
K
D002957:Citrus / Q000821:virology*; D004591:Electrophoresis, Polyacrylamide Gel; D010942:Plant Viruses / Q000302:isolation & purification*; D012328:RNA Viruses / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014772:Viroids / Q000302:isolation & purification