Japanese Journal of Phytopathology
Online ISSN : 1882-0484
Print ISSN : 0031-9473
ISSN-L : 0031-9473
Detection of Hop Latent Viroid (HLVd) Using Reverse Transcription and Polymerase Chain Reaction(RT-PCR)
Tatsuji HATAYAKatsuyuki HIKAGENarushi SUDATatsuya NAGATAShifang LIYutaka ITOGATokio TANIKOSHIEishiro SHIKATA
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1992 Volume 58 Issue 5 Pages 677-684

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Abstract

A viroid-like RNA was detected in low molecular weight RNAs extracted from virus-free hops by two-dimensional polyacrylamide gel electrophoresis (PAGE). It migrated considerably faster than hop stunt viroid in 5% PAGE containing 8M urea under a denaturing condition. A cDNA fragment was amplified from the low molecular weight RNAs containing the viroid-like RNA by reverse transcription and polymerase chain reaction (RT-PCR) using a pair of hop latent viroid (HLVd) specific primers. The amplified cDNA product of expected size was detected by agarose gel electrophoresis. The full-length cDNA of the viroid-like RNA in RT-PCR products was cloned and sequenced. The established nucleotide sequence of the inserted cDNA was completely identical to that previously reported for HLVd. The total nucleic acids were extracted from hops cultivated in Japan, and the presence of HLVd were examined by dot blot hybridization and RT-PCR. In a hop sample, HLVd was not detected by dot blot hybridization, but detected by RT-PCR. The result indicates that RT-PCR is potentially more sensitive than dot blot hybridization using a radioactive probe. HLVd was detected in all mother cultivars tested. However, the two virus-free clones obtained by meristem culture showed negative reaction in both dot blot hybridization and RT-PCR detection methods. It is also shown that HLVd probably can be eliminated by meristem culture as indicated in several plant viruses and viroids

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