Identification of potyviruses using the polymerase chain reaction with degenerate primers

Langeveld, S.A.; Dore, Jean-Michel; Memelink, Johan; Derks, A.F.L.M.; Vlugt, C.I.M. van der; Asjes, C.J.; Bol, John F.

doi:10.1099/0022-1317-72-7-1531

Cited by 180 publications

(85 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This approach has been shown of practical use to detect most or all isolates of one particular virus or different related viruses within a genus, as shown for potyviruses (2,9), luteoviruses (18), or geminiviruses (20).…”

Section: Discussionmentioning

confidence: 99%

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

et al. 1998

View full text Add to dashboard Cite

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

show abstract

Section: Discussionmentioning

confidence: 99%

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

et al. 1998

View full text Add to dashboard Cite

show abstract

“…The PCR was done in a 20 ml volume solution containing: 12 µL Kapa2G Fast Ready Mix PCR Kit (Thermo Scientific), 4 µL nuclease-free water, 1 µL Poty1 reverse primer 10 µM, 1 µL pCV3t/ AlcarF/U341 forward primer 10 µM, and 1 µL cDNA template. Primer set: Poty1(IDT)/ pGV3t (Sigma) (5'-GGA TTC CGG GTT TTT TTT TTT TTT TTT V-3' (Gibbs and MacKenzie, 1997) and 5'-TGG NCN TGC TAC CAC AAN GG-3' (Chen et al, 2004)); Poty1(IDT)/AlcarF (Sigma) (5'-GGA TTC CGG GTT TTT TTT TTT TTT TTT V-3' (Gibbs and MacKenzie, 1997) and 5'-TGC TGC YTT TGA TAC YTT CGA T-3') (Gambley, 2012)); Poty1 (IDT)/U341 (Sigma) (5'-GGA TTC CGG GTT TTT TTT TTT TTT TTT V-3' (Gibbs and MacKenzie, 1997) and 5'-CCG GAA TTC ATG RTI TGG TGY ATI GAI AAY GG-3' (Langeveld et al, 1991) o C for 6 min. PCR products were separated on electrophoresis 1.5% agarose gels in TBE (Tris-Borate-EDTA) buffer 1x and then stained with ethidium bromide and visualized in a UV transilluminator.…”

Section: Dna Amplification (Pcr)mentioning

confidence: 99%

Elimination of shallot bulb viruses through heat treatment

2017

View full text Add to dashboard Cite

Shallot (Allium cepa L. Aggregatum group) is usually cultivated vegetatively. As a result, viruses tend to accumulate within the host plants and spread to healthy plants every crop cycle, reducing yield and bulb quality. There are a very limited number of studies about the elimination of shallot viruses through heat treatment. The objective of this research was to eliminate shallot viruses through heat treatment to produce virus-free plantlets.The leaves of Biru Lancor with specific visual virus symptoms were detected by Reverse TranscriptionPolymerase Chain Reaction (RT-PCR). Then bulbs of Biru Lancor that were positively infected by viruses were used as materials for heat treatment. The treatments were a control (without treatment), electric treatment at 15 mA for 10 minutes, heat treatment in an incubator at 37°C for 4 weeks, heat treatment in a waterbath at 45°C for 60 minutes, and combination of heat treatment in an incubator at 37°C for 4 weeks and heat treatment in a waterbath at 45°C for 60 minutes. After being subjected to heat treatment, the pseudo stem were cultivated in the MS Medium + 1 mg/L BAP + 1 mg/L IBA.Virus detection by RT-PCR was conducted 28 days after planting using samples of leaves from each plantlet.The results of this research showed that the treatments of electric treatment at 15 mA for 10 minutes and combination of heat treatment in the incubator at 37°C for 4 weeks and heat treatment in the waterbath at 45°C for 60 minutes could suppress the incidence of Shallot latent virus (SLV) until 100%. Heat treatment might have an important role in the degradation of virus particles by boosting Virus-Induced Gene Silencing (VIGS) as plant responses to virus infection.

show abstract

“…Conversely, recent advances in nucleic acid technologies have enabled the development of powerful detection and identification tools. Reverse transcription polymerase chain reaction (RT-PCR) based methods enable fast, accurate detection, quantification and characterization of potyvirus [7]. The present study describes about the detection and identification of the DsMV infecting C. esculenta, through cloning and sequencing of the partial coat protein gene of the virus.…”

mentioning

confidence: 99%

Detection and Identification of Dasheen mosaic virus Infecting Colocasia esculenta in India

et al. 2011

View full text Add to dashboard Cite

Reverse transcription polymerase chain reaction of the infected leaf samples of Colocasia esculenta plants showing severe whitish feathery symptoms were carried out using Potyvirus group specific primers, resulting in an amplicon of 327 bp, encoding the core region of the coat protein gene. Sequencing and BLAST analysis showed that the virus is distinct, closely related to Dasheen mosaic virus (DsMV). Sequence analysis revealed 86 and 96% identity at the nucleotide and amino acid level respectively with the DsMV isolate SY1(accession Number AJ628756). This is the first molecular level characterisation of the DsMV infecting C. esculenta in India.

show abstract

Identification of potyviruses using the polymerase chain reaction with degenerate primers

Cited by 180 publications

References 45 publications

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

Elimination of shallot bulb viruses through heat treatment

Detection and Identification of Dasheen mosaic virus Infecting Colocasia esculenta in India

Contact Info

Product

Resources

About