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Identification of Ranunculus Mild Mosaic Virus on Ranunculus asiaticus Collected from Border Intercepted Ranunculus and the Development and Application of the Virus Detection Reagents
 RanMMV)。
RanMMV)。

Author:Chin-Chih Chen*, Jia-Yi Liao, Ming-Ling Liao, Yu-Chi Liu, and Fen-Lang Chiang

Abstract:

<i>Ranunculus asiaticus</i> cut flowers with mosaic symptoms imported from The Netherlands were intercepted by quarantine at the border customs. The potential causal agents were identified and the detection tools were developed in this study. Indirect enzyme-linked immunosorbent assay (indirect ELISA) was performed with various antibodies against different ornamental viruses preserved in the laboratory. All the tested samples were positively reacted with the commercial potyvirus monoclonal antibody (produced by Agdia Inc., Elkhart, IN, USA) and could be positively detected with the potyvirus-degenerated primers (HRP-5/Oligo-dT(14) to amplify a 1.3-kbp amplicon in reverse transcription-polymerase chain reactions (RT-PCRs). Sequencing data showed the nucleic acid sequences of the amplified amplicons were similar to that of ranunculus mild mosaic virus (RanMMV). Both of the nucleotide and deduced amino acid sequences of the cloned cDNA shared more than 98.6% identities with that of RanMMV coat protein gene (GenBank Accession No. LC387972) indicating the intercepted virus is a RanMMV isolate. The further designed RanMMV primer pair (RanMM-u/ RanMM-d) can effectively detect the RanMMV on imported mosaic cut flowers and bulb tissues of<i> R. asiaticus</i>, and generate a predicted 1.04 kbp amplicon. The cDNA of the RanMMV coat protein open reading frame (CP ORF), containing 825 nucleotides, was constructed in the expression vector of pET28a(+) by artificial gene synthesis, and then the fusion protein (approximately Mt. 30.5 kDa) was expressed in the Escherichia coli BL21 host. The resulting RanMMV coat protein was used as an antigen to prepare a polyclonal antibody against RanMMV. The produced polyclonal antibody against RanMMV can successfully detect RanMMV on the imported R. asiaticus in immunoassays. RanMMV has not been found in Taiwan and this is the first interception case of the virus at the border. The detection reagents of RanMMV have been successfully developed in this study which might be helpful in improving the efficiency of border quarantine and inspection on imported Ranunculus materials. 

Key words: Ranunculus asiaticus, Potyvirus, Polyclonal antibody, RT-PCR detection.

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