Comparison of Methods for Virus Detection in <i>Allium</i> spp.

Dovas, Chrysostomos Ι.; Hatziloukas, Efstathios; Salomon, R.; Barg, E.; Shiboleth, Yoel Moshe; Katis, N. I.

doi:10.1046/j.1439-0434.2001.00705.x

Cited by 47 publications

(36 citation statements)

References 19 publications

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“…However, virus diagnosis in in vitro planting material requires a more reliable and sensitive method. Reverse transcriptase-polymerase chain reaction (RT-PCR) can detect minute amounts of target RNA and is suitable for detecting viruses in very small tissue samples with extremely low virus titer, as found in meristem-tip culture (Dovas et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine

et al. 2003

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The plum pox virus (PPV) and prunus necrotic ringspot virus (PNRSV) cause serious disease problems in stone-fruit trees. In this work, the possibility of obtaining plant material free from these viruses through thermotherapy and meristem-tip culture from infected nectarine shoots (Prunus persica var. nectarina Max, cv. 'Arm King') was studied. In addition, the detection of these viruses in in vitro cultures and young acclimatized plantlets with double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was studied. Meristem-tip explants (0.8-1.3 mm) derived from sprouted buds of winter wood and spring shoots from field grown plants had a 2-5% regeneration response. However, application of thermotherapy to potted nectarine trees (3 weeks at a maximum temperature of 35 degrees C) facilitated excision of longer meristem tips (1.3-2.0 mm) that resulted in a significantly higher regeneration response (38%) in woody plant medium (WPM) without plant growth regulators. Such explants formed multiple shoots with the addition of 8 microM benzylaminopurine and 0.8 microM indoleacetic acid. When they were tested for the presence of PPV and PNRSV, 86% and 81% were found to be virus-free as detected by DAS-ELISA and multiplex RT-PCR, respectively. Individual shoots excised from virus-free cultures readily rooted in vitro (half-strength WPM plus 2 microM indolebutyric acid) and grew to plantlets. The combination of an efficient protocol for virus elimination and the establishment of highly sensitive diagnostics resulted in the production of nectarine plants free from PPV and PNRSV.

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Section: Introductionmentioning

confidence: 99%

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine

et al. 2003

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“…A simple and economical sample preparation method is therefore required. A RT-PCR technique using crude nucleic acid extracts from infected plants has been reported previously (Borja and Ponz 1992;Dovas et al 2001), and the optimum extraction buffer for sample preparation has been investigated (Dovas and Katis 2002;Singh et al 2002). In reports of this method, sensitivity was low (Nishiguchi et al 1995;Wetzel et al 1992).…”

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Use of crude sap for one-step RT-PCR-based assays of Bean yellow mosaic virus and the utility of this protocol for various plant?virus combinations

Uga

2005

J Gen Plant Pathol

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One-step reverse transcription-polymerase chain reaction (one-step RT-PCR) was successfully adapted to detect Bean yellow mosaic virus in the crude sap of infected dwarf gentian plants (Gentiana scabra). The virus was detected directly from crude sap at dilutions up to 10 6 -fold (W/ V), which is 100 times more sensitive than indirect enzymelinked immunosorbent assay or bioassay after mechanical inoculation, and equally as sensitive as immunocapture RT-PCR. This one-step RT-PCR method using crude sap was also useful for detecting 16 virus species from ten genera with isometric, filamentous, and rod-shaped particles from 32 plant species in 15 families.

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“…Samples were tested for the presence of allexiviruses by reverse transcription (RT)-PCR. Total RNA was extracted from 100 mg of leaves using an RNeasy Plant Minikit (Qiagen) according to the manufacturer's protocol and RT-PCR was performed using primers designed in conserved regions at the 3′-end of open reading frame 6 and the 3′ end of the non coding region of allexivirus sequences (Dovas et al 2001). Expected amplicons of approximately 200 bp were obtained from all the samples tested.…”

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confidence: 99%