The Use of Nucleic Acid Probes for the Detection of Prunus Necrotic Ringspot Virus and Prune Dwarf Virus

Scott, S. W.; Bowman-Vance, V.; Bachman, E. J.

doi:10.17660/actahortic.1992.309.8

Cited by 10 publications

(10 citation statements)

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“…PNRSV, an economically important virus of Prunus spp., has been detected, by biological assay, serological methods (Halk et al ., 1984; Mink & Aichele, 1984; Scott et al ., 1989), molecular hybridization (Scott et al ., 1992; Crosslin et al ., 1992) including nonradioactive riboprobes (Heuss‐LaRosa et al ., 1995) and RT‐PCR (Rowhani et al ., 1995; Spiegel et al ., 1996). However, no attempts appear to have been made to use the same material and compare the three methods simultaneously.…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts

et al. 1998

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Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL ¹1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL ¹1 and 4 ng mL ¹1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits. Materials and Methods Plant and virus sourcePetals from a field-grown PNRSV-infected nectarine (Prunus persica) tree were homogenized and served as inoculum for cucumber (Cucumis sativus cv. National Pickling) plants. After several passages, virus was Plant Pathology (1998) 47, 780-786 780 ᮊ 1998 BSPP

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Section: Discussionmentioning

confidence: 99%

Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts

et al. 1998

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“…The reliability of PNRSV detection by ELISA is limited to the periods of active growth and becomes unreliable when stem elongation ceases (Scott et al, 1992). The reliability of PNRSV detection by ELISA is limited to the periods of active growth and becomes unreliable when stem elongation ceases (Scott et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

Improved detection of prunus necrotic ringspot virus by the polymerase chain reaction

et al. 1996

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The reverse transcription-polymerase chain reaction (RT-PCR) technique was used for detection of prunus necrotic ringspot virus in dormant peach trees which tested negative by ELISA. Total RNA extracted from bark tissue, using a lithium chloride based method, were used for reverse transcription and subsequent amplification of viral sequences. The PCR product, about 300 base pairs in size, was analyzed by gel electrophoresis and visualized by ethidium bromide staining. In some cases, PCR products were not clearly visible in the stained gel and became distinct only following hybridisation with a 32p-labelled virus specific probe. The RT-PCR assay described in this paper is sensitive enough for detection of PNRSV in dormant woody bark tissue and could be incorporated into testing protocols during post-entry quarantines for rapid initial screening of imported budwood and in virus elimination programs.

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“…Therefore, great efforts have been made during recent years to improve the sensitivity and speed of molecular diagnostic methods. Both molecular hybridization-based methods (5,17,19,23,27) and PCR-based methods (8,19,20,21,23,29) detect these viruses even in low concentration limits. As a next step towards development of faster and more time-saving methods, attempts have been made to simultaneously detect at least two viruses.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

Saade¹,

Aparicio²,

Sánchez‐Navarro³

et al. 2000

Phytopathology®

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The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

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The Use of Nucleic Acid Probes for the Detection of Prunus Necrotic Ringspot Virus and Prune Dwarf Virus

Cited by 10 publications

References 0 publications

Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts

Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts

Improved detection of prunus necrotic ringspot virus by the polymerase chain reaction

Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

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