ISHS Acta Horticulturae 386: XVI International Symposium on Fruit Tree Virus diseases
SENSITIVE DETECTION OF APPLE CHLOROTIC LEAF SPOT VIRUS FROM INFECTED APPLE OR PEACH TISSUE USING RT-PCR, IC-RT-PCR, OR MULTIPLEX IC-RT-PCR |
| Authors: | L. Nemchinov, A. Hadidi, J.A. Foster, T. Candresse, T. Verderevskaya |
| Abstract:
DNA primers specific for apple chlorotic leaf spot virus (ACLSV) were constructed based on the nucleotide sequence of a 358 nt segment of the viral coat protein gene. Primers were utilized for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and amplification by immunocapture (IC)-RT-PCR of a 358 bp cDNA fragment from extracts of ACLSV-infected apple, peach, or tobacco tissue from Moldova, France, or the U.S. This fragment represents the overlapping between the 50K cell to cell movement protein and coat protein gene of ACLSV (German et al., 1990). Multiplex IC-RT-PCR assay was also developed for simultaneous detection of ACLSV and plum pox virus (PPV) from infected tissue. In this assay, two cDNA fragments were amplified - the 358 bp ACLSV cDNA fragment and a 220 bp PPV cDNA fragment. Southern blot hybridization of the amplified products to a digoxigenin (DIG)-labeled cRNA probe of the cloned 358 bp ACLSV cDNA was used to confirm the detection of ACLSV from infected tissue using PCR amplification. DIG-labeled cRNA probe of cloned PPV cDNA of the 3' non-coding region was also used to confirm the detection of PPV from infected tissue. No DNA was detected in amplified extracts from uninfected tissue. | |
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