Standardized RT-PCR Conditions for Detection and Identification of Eleven Viruses of Potato and Potato spindle tuber viroid

Crosslin, J. M.; Hamlin, Launa L.

doi:10.1007/s12230-011-9198-z

Cited by 34 publications

(14 citation statements)

References 38 publications

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“…In addition, uniplex RT-PCR was also performed for detection of PMTV, PVM and PotLV using primers reported previously (Nie et al, 2008;Crosslin & Hamlin, 2011). Upon confirmation of PVY's presence in some of the samples, three sets of RT-PCR assays including the P1 gene-based RT-PCR , the RJ-based RT-PCR and the multiplex RT-PCR described by Lorenzen et al (2006) were used to analyze the PVY genotype/strain type in the samples.…”

Section: Virus Sample Collectionmentioning

confidence: 99%

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

Nie

Singh

2013

Canadian Journal of Plant Pathology

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Post-harvest screening of potato 'Kennebec' revealed a Potato virus Y (PVY) incidence of 15.8%, a rate that is unusually high for a cultivar possessing a high level of field resistance to the virus. Randomly selected tubers were planted in a field plot and the resulting plants were monitored. Approximately 16% of plants developed symptoms ranging from mild mosaic symptoms to severe necrosis/rugosity/stunting. ELISA and RT-PCR analysis revealed that infections with Potato virus S (PVS), Potato virus X (PVX) and PVY, mostly in mixed-infections, occurred commonly in 14 sampled plants. Two strains, namely the common strain (PVY • ) and the recombinant tuber necrotic strain (PVY NTN ) were identified in the PVY-positive plants. In general, mild mosaic was associated with infections with PVX and PVS; intermediated mosaic was associated with PVS and PVY NTN infections; whereas severe leaf deformation/necrosis/drop symptoms were associated with PVY NTN and PVX co-infections, or with PVY • and either PVS or PVX co-infections. Virus-free plantlets of potato 'Kennebec' were mechanically inoculated with PVX, PVY • , and PVY NTN alone or with PVX+PVY • or PVX+PVY NTN combinations in the greenhouse. Single infections with PVY • or mixed-infections with PVX+PVY • or PVX+PVY NTN incited severe mosaic symptoms and systemic necrosis soon after inoculation; whereas single infections with PVX and PVY NTN induced mild to intermediate mosaic symptoms only. The most severe symptoms occurred in the mixed-inoculation with PVX+PVY NTN , demonstrating dramatic synergism between these strains. Similarly, profound PVX and PVY NTN synergism was also found in tobacco and Physalis floridana plants, suggesting that the strain of PVY plays an important role in the level of synergistic reactions between PVX and PVY on host plants. Résumé:Le criblage consécutif à la récolte de la pomme de terre 'Kennebec' a révélé une incidence de 15,8 % du virus Y de la pomme de terre, soit un taux anormalement élevé pour un cultivar possédant un fort degré de résistance au virus en champ. Des tubercules choisis aléatoirement ont été plantés dans une parcelle de terrain et les plants qui en ont résulté ont été suivis de près. Environ 16 % des plants ont développé des symptômes variant des symptômes bénins de la mosaïque à ceux plus graves de la nécrose en passant par la rugosité ou le rabougrissement. Des tests ELISA et des analyses par RT-PCR ont révélé que des infections causées par les virus S (VSPT), X (VXPT) et Y (VYPT) de la pomme de terre, principalement sous forme d'infections mixtes, sont apparues couramment chez 14 plants échantillonnés. Deux souches, à savoir la souche ordinaire (VYPT • ) et la souche recombinante causant la nécrose des tubercules (VYPT NTN ), ont été identifiées chez les plants réceptifs au VYPT. En général, la mosaïque bénigne était associée aux infections causées par le VXPT et le VSPT; la mosaïque moyennement virulente, aux infections causées par le VSPT et le VYPT NTN , tandis que la déformation grave du feuillage, la nécrose et le...

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Section: Virus Sample Collectionmentioning

confidence: 99%

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

Nie

Singh

2013

Canadian Journal of Plant Pathology

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“…The offspring are next inspected visually for virus symptoms and/or tested by the enzyme-linked immunosorbent assay (ELISA) (Zahn et al 2010). Alternatively, tubers can be tested directly by RT PCR (Crosslin and Hamlin 2011) or real-time RT-PCR ). However, the cost and complexity of immunological and molecular methods restricts their use to dedicated, specialized laboratories .…”

Section: Introductionmentioning

confidence: 99%

A One-Step, Real-Time Reverse Transcription Loopmediated Isothermal Amplification Assay to Detect Potato Virus Y

et al. 2015

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Potato viruses such as Potato virus Y (PVY) cause diseases that affect potato quality and thus damage potato production worldwide. Current tests for viral infection use double-antibody sandwich enzyme linked immunosorbent assays (DAS ELISA) or reverse transcription polymerase chain reaction (RT-PCR)/real-time RT-PCR. Despite many advantages, these assays have a number of drawbacks that affect cost and time of diagnosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) allows fast detection of target RNA. Here, we developed a closed-tube real-time RT LAMP assay for fluorescent detection of PVY. Specific RT-LAMP primers were designed to target the conserved region of the sequence encoding the PVY coat protein. The assay was specific and facilitated sensitive PVY detection in a single tube at 65°C. The time-topositive values depended on the PVY concentration in tested samples. The effectiveness of RT LAMP in testing field-grown plants compared favorably with DAS ELISA and RT-PCR; under the tested conditions, RT-LAMP was about 1000-fold more sensitive than DAS ELISA and lateral flow assay (LFA) and about 10-fold more sensitive than RT PCR. Thus, this fluorescent RT-LAMP assay has great potential for routine detection of PVY. 1000 veces más sensible que DAS ELISA y que el ensayo de flujo lateral, y como 10 veces más sensible que RT-PCR. La efectividad de RT-LAMP al probarla en plantas en el campo se comparó favorablemente con DAS ELISA y RT-PCR. En consecuencia, presenta un gran potencial para detección de rutina del PVY.

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“…The reaction was performed in 25 ul containing 2.5 ul 20 mm solution of dNTP, 1.25 ul of 10 x RT buffer, 1.25 ul of 10 x PCR buffer, 0.2 ul of 5U M-MuLV reverse transcriptase, 0.2 ul of 5U Taq DNA-polymerase, 0.5 ul of 50 uM sense and antisense primers (for PVM or PVS), 2 ul of total RNA sample. For PVM detection the following oligonucleotide primers were used: forward 5'-gccacatcygaggacatgat-3', reverse 5'-gtgagctcsggaccattcat-3'; for PVS detection: forward 5'-gaggctatgctggagcagag-3', reverse 5'-aatctcagcgccaagcatcc-3' [12]. Reaction of amplification was performed on "Mastercycler Gradient" (Eppendorf, Germany) under following conditions: 1 cycle: 37˚C-60 min; 1 cycle: 94˚C-5 min; 42 cycles: 9˚C-1 min, 60˚C-1 min, 72˚C-1 min; 1 cycle: 72˚C-5 min; 4˚C-storage.…”

Section: Methodsmentioning

confidence: 99%

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i> against Phytopathogenic Rna-Viruses

Шарипова¹,

Rockstroh²,

Balaban³

et al. 2015

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Standardized RT-PCR Conditions for Detection and Identification of Eleven Viruses of Potato and Potato spindle tuber viroid

Abstract: Standardized RT-PCR procedures were devel-

Cited by 34 publications

References 38 publications

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

A One-Step, Real-Time Reverse Transcription Loopmediated Isothermal Amplification Assay to Detect Potato Virus Y

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i> against Phytopathogenic Rna-Viruses

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Standardized RT-PCR Conditions for Detection and Identification of Eleven Viruses of Potato and Potato spindle tuber viroid

Abstract: Standardized RT-PCR procedures were devel-

Cited by 34 publications

References 38 publications

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVYNTNand PVY° strains

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVYNTNand PVY° strains

A One-Step, Real-Time Reverse Transcription Loopmediated Isothermal Amplification Assay to Detect Potato Virus Y

Antiviral Effect of Ribonuclease from &lt;i&gt;Bacillus pumilus&lt;/i&gt; against Phytopathogenic Rna-Viruses

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Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

Response of potato, tobacco andPhysalis floridanaplants to mixed infection with PVX, PVY^NTNand PVY° strains

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i> against Phytopathogenic Rna-Viruses