scholarly journals An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1370-1374 ◽  
Author(s):  
Mohamad Chikh-Ali ◽  
Stewart M. Gray ◽  
Alexander V. Karasev
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Rna Extraction ◽  
Leaf Tissue ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potato Leaf ◽  
Extraction Step ◽  
First Time ◽  
Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

scholarly journals Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 641-644 ◽  
Author(s):  
Manphool S. Fageria ◽  
Mathuresh Singh ◽  
Upeksha Nanayakkara ◽  
Yvan Pelletier ◽  
Xianzhou Nie ◽  
...  
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Current Season ◽  
Seed Market ◽  
Polymerase Chain ◽  
Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

Prevalence and strain composition of potato virus Y circulating in potato fields in China’s north-central province Shanxi

Plant Disease ◽  
2021 ◽  
Author(s):  
Pengcheng Ding ◽  
Dexin Chen ◽  
Haixu Feng ◽  
Jiao Li ◽  
Hui Cao ◽  
...  
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Potato Production ◽  
Central China ◽  
Shanxi Province ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Single Strain ◽  
North Central ◽  
Production Areas

Potato is an important crop in Shanxi province located in north-central China. During 2019-2020, 319 potato leaf samples were collected from eight locations distributed in three major potato production areas in Shanxi. Bio-chip detection kit revealed the presence of several potato viruses, and among them potato virus Y (PVY) was the most common one, reaching the incidence of 87.8% of all symptomatic samples. The immuno-captured multiplex reverse transcription (RT)-PCR was used to identify strains for all 280 PVY-positive samples, unveiling 242 samples infected with a single strain of PVY (86.4%) and 38 (13.6%) with a mixed infection. Of samples with a single-strain infection, PVY -SYR-II accounted for 102 (42.1%), followed by PVYN-Wi (33, 13.6%) , PVY -SYR-I (28, 11.6%), 261-4 (22, 9.1%), PVYNTNa (20, 8.3%), PVYNTNb (19, 7.9%), and PVY -SYR-III (18, 7.4%). Seven isolates representing different recombinants were selected for whole genome sequencing. Phylogenetic and recombination analyses confirmed the RT-PCR based strain typing for all seven strains of PVY found in Shanxi. SXKL-12 is the first SYR-III strain from potato reported from China. However, unlike that in other known SYR-III isolates, the region positioned from 1,764 to1,902 nt in SXKL-12 shared the highest sequence identity of 82.2% with an uncharacterized PVY isolate, JL-23, from China. Interestingly, the PVYN-Wi isolate SXZY-40 also possessed a more divergent sequence for the region positioned from 6,156 to 6,276 nt than other N-Wi isolates known to date, sharing the highest identity of 86.6% with an uncharacterized Chinese PVY isolate, JL-11. Pathogenicity analysis of dominant strains PVY -SYR-II and PVYN-Wi in six local popular potato cultivars revealed that Kexin 13, Helan 15 and Jizhangshu 12 were susceptible to these two strains with mild mottling or mosaic symptoms expression, while three cultivars, Jinshu 16, Qingshu 9, Xisen 6 were found fully resistant.

Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

2005 ◽  
Vol 124 (1-2) ◽  
pp. 65-71 ◽  
Author(s):  
Boris Pastorino ◽  
Maël Bessaud ◽  
Marc Grandadam ◽  
Severine Murri ◽  
Hugues J. Tolou ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Detection And Quantification

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽  
2005 ◽  
Vol 89 (6) ◽  
pp. 605-610 ◽  
Author(s):  
Xianzhou Nie
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

scholarly journals First Report of Potato virus Y Strain N-Wilga Infecting Chrysanthemum in China

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1589-1589 ◽  
Author(s):  
X. L. Liu ◽  
Q. Wei ◽  
B. Hong ◽  
X. T. Zhao
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Chrysanthemum Morifolium ◽  
Rt Pcr ◽  
Pcr Assay ◽  
First Report ◽  
Chlorotic Mottle ◽  
Leaf Yellowing ◽  
Chrysanthemum Morifolium Ramat

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a commercially important ornamental grown worldwide, and is also extensively used as an edible and medicinal plant. In the present work, viruses and viroids infecting chrysanthemum were investigated in China in 2012 and 2013. Typical viral symptoms were observed in field-grown chrysanthemum with leaf yellowing and mottled leaves in Wenjiang District, Sichuan Province, China. The incidence of these symptoms in the field was 12.3%. Chrysanthemum virus B (CVB), Tomato aspermy virus (TAV), Cucumber mosaic virus (CMV), Tobacoo mosaic virus (TMV), Chrysanthemum stunt viroid (CSVd), and Chrysanthemum chlorotic mottle viroid (CChMVd), which had previously been reported to infect chrysanthemum in China (2,3), were not detected by RT-PCR assay. Since these symptomatic chrysanthemum plants grew next to a tobacco field, viruses affecting tobacco were suspected as possible cause. Sixteen symptomatic leaves and 12 non-symptomatic leaves were collected and tested for Potato virus Y (PVY) presence using commercial PVY-specific DAS-ELISA kits (Catalog no. PSA20001, Agdia) Six samples were found positive for PVY. RT-PCR tests using specific primers for CP gene (CP-F 5′-ACTGTGATGAATGGGCTTATG-3′; CP-R 5′-GGCATATATGGTTCCTTTTTG-3′) (4) amplified a single, expected 218-bp DNA fragment from chrysanthemum extracts from all six samples positive for PVY in ELISA. These six PCR fragments were sequenced and found 100% identical to each other. The sequence (GenBank Accession No. KJ174515) shared 99% identity with corresponding sequences of several PVY isolates (NC_001616, EF026076, HM590407, and JQ924288). The same six positive samples were subjected to a multiplex RT-PCR assay (1) to identify the PVY strain type, and all six PVY samples from Sichuan were found to belong to the PVYN-Wi strain. To our knowledge, this is the first report of the PVYN-Wi strain infecting chrysanthemum in Sichuan, China. References: (1) M. Chikh Ali et al. Plant Dis. 10:1370, 2013. (2) E. A. Nassar et al. Int. J. Virol. 8:14, 2012. (3) H. Yamamoto et al. J. Gen. Plant Pathol. 71:156, 2005. (4) J. Q. Zhang et al. J. Phytopathol. 161:92, 2013.

scholarly journals A Multiplex PCR Assay to Characterize Potato virus Y Isolates and Identify Strain Mixtures

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 935-940 ◽  
Author(s):  
James H. Lorenzen ◽  
Lisa M. Piche ◽  
Neil C. Gudmestad ◽  
Teresa Meacham ◽  
Pat Shiel
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Infection Type ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Strain ◽  
Multiplex Pcr Assay ◽  
Strain Type ◽  
Pcr Assays

Potato virus Y (PVY) has become a serious problem for the seed potato industry, with increased incidence and rejection of seed lots submitted for certification. New PVY strains and strain variants have emerged in recent decades in Europe and North America, including the PVYN strain that causes veinal necrosis in tobacco, and strain variants that represent one or three recombination events between the common strain (PVYO) and PVYN. Several reverse transcription-polymerase chain reaction (RT-PCR) assays have been described that characterize PVY isolates as to strain type, but they are limited in their ability to detect some combinations of mixed strain infections. We report here the development of a single multiplex RT-PCR assay that can assign PVY strain type and detect mixed infections with respect to the major strain types. Validation of this assay was achieved using 119 archived PVY isolates, which had been previously characterized by serology and bioassay and/or previously published RT-PCR assays. Results for single-strain isolates were comparable to previous results in most cases. Interestingly, 16 mixed infections were distinguished that had previously gone undetected. The new multiplex RT-PCR assay will be useful for researchers and seed production specialists interested in determining PVY infection type using a single assay.

scholarly journals First Identification of an Unusual Recombinant Potato virus Y Strain in Potato in Mexico

Plant Disease ◽  
2010 ◽  
Vol 94 (10) ◽  
pp. 1262-1262 ◽  
Author(s):  
L. Robles-Hernández ◽  
A. C. González-Franco ◽  
J. Hernández-Huerta ◽  
T. M. Meacham ◽  
O. V. Nikolaeva ◽  
...  
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Sandwich Elisa ◽  
Detection Methods ◽  
Molecular Characteristics ◽  
Rt Pcr ◽  
Central State ◽  
Strain Specificity ◽  
Potato Leaf ◽  
Systemic Vein

Potato virus Y (PVY) has been reported in potato crops in Mexico (3), with tobacco necrotic variants found in the central State of Mexico (4). Nevertheless, many individual states are currently declared PVY free and distribution of individual strains of PVY in potato in different states of Mexico and in different solanaceous crops had not yet been studied. A limited field PVY survey was conducted on potato in the State of Chihuahua in August 2009. More than 900 random potato leaf samples were collected from cvs. Snowden, Atlantic, FL1867, Felsina, Fianna, Gigant, and Alpha. Seven were found to be PVY-positive and had been collected from cvs. Fianna, Snowden, and FL1867. The PVY status of the collected samples was initially determined with the PVY-specific Immunostrips (Bioreba, Reinach, Switzerland) and by double-antibody sandwich-ELISA using the polyclonal PVY detection kit (Agdia, Elkhart, IN). To determine the strain specificity of these PVY isolates following ELISA tests, the infected original samples were inoculated onto tobacco plants at the four-leaf stage and symptom appearance and development were observed for 8 weeks side-by-side with control isolates PB-Oz (PVYO), N4 (PVYNTN), and Mont (PVYN) (1), followed by the standard PVY strain typing by reverse transcription (RT)-PCR (2). Only one of the PVY-positive samples, originally from symptomless potato cv. Fianna, induced systemic PVY infection in tobacco by producing stunting, mosaic, and vein clearing. No systemic vein necrosis, characteristic of isolates Mont and N4, was observed in Nicotiana tabacum cvs. Burley, Xanthi, or Samsun after inoculation with this isolate during all 8 weeks of observation. This isolate, PVY-M3, was typed as a PVY recombinant by RT-PCR, with two recombinant junctions characteristic of European PVYNTN strains (2). It was further analyzed by triple-antibody sandwich-ELISA using four PVYO and PVYN strain-specific monoclonal antibodies. Monoclonals 1F5 (Agdia) and SASA-N (Scottish Agriculture Science Agency [SASA], Edinburgh) reacted to this isolate and identified PVY-M3 serologically as PVYN serotype, characteristic of other PVYNTN recombinants. Monoclonals MAb2 (Agdia) and SASA-O (SASA), specific to PVYO and PVYC strains, did not react to PVY-M3. Taken together, the combination of biological, serological, and molecular characteristics define this recombinant isolate from Mexico as belonging to the same PVY strain group represented by the isolate PVY-L26 (1). To our knowledge, this is the first report of such an unusual PVYNTN recombinant strain from Mexico. Presence of this isolate, with no vein necrotic symptoms induced on tobacco and with PVYNTN genome, will necessitate development of new detection methods for the seed potato industry in Mexico. References: (1) X. Hu et al. Virus Res. 143:68, 2009. (2) J. L. Lorenzen et al. Plant Dis. 90:935, 2006. (3) L. P. Moreno et al. Rev. Mex. Fitopatol. 22:187, 2004. (4) V. R. Ramirez-Rodriguez et al. Virol. J. 6:48, 2009.

scholarly journals Ocorrência de vírus em batata em sete estados do Brasil

2009 ◽  
Vol 27 (4) ◽  
pp. 490-497 ◽  
Author(s):  
Antônio Carlos de Ávila ◽  
Paulo Eduardo de Melo ◽  
Lindolfo R Leite ◽  
Alice K Inoue-Nagata
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Leaf Roll ◽  
Potato Virus X ◽  
Potato Leaf Roll Virus ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Potato Virus S ◽  
Das Elisa

As viroses causam rápida degenerescência dos tubérculos-sementes de batata. Em condições tropicais, em que a presença de afídeos vetores é constante e a estrutura das populações de vírus é dinâmica, a pressão das doenças é enorme. Conhecer essa dinâmica é uma ferramenta importante para a sustentabilidade da produção de batata. Realizou-se um levantamento abrangente da ocorrência de viroses em batata no Brasil, além de estudar-se a distribuição das estirpes de Potato virus Y (PVY) associadas ao mosaico da batata. Em 2005 e 2006 foram visitadas lavouras em sete estados brasileiros, coletando-se folíolos com sintomas de viroses (1.256 amostras) e amostras aleatórias (360 amostras). Foi feita também uma estimativa visual da incidência de mosaico e enrolamento-das-folhas em vários dos campos visitados. Das 1.256 amostras suspeitas, 840 apresentaram reação positiva em teste sorológico para PVY (66,9%), 128 para Potato leaf roll virus (PLRV) (10,2%), 79 para Potato virus S (PVS) (6,3%) e nenhuma para Potato virus X (PVX). Os resultados dos testes de detecção por DAS-ELISA, biológico e RT-PCR mostraram a presença quase absoluta do subgrupo necrótico de PVY, em sua maioria PVY NTN. A análise de uma sub-amostragem em todos os municípios visitados confirmou que essa variante está hoje presente nos sete estados visitados. Amostras de PVY NTN foram obtidas das cultivares Asterix, Atlantic, Agata, Achat, Baronesa, Baraka, Bintje, Caesar, Cupido, Marijke, Monalisa, Panda e Vivaldi, que apresentaram diferentes níveis de suscetibilidade. As amostras aleatórias revelaram um quadro muito similar ao encontrado com as amostras sintomáticas. PLRV foi identificado em MG, BA, PR e SC, em várias lavouras de forma muito freqüente. PVS foi identificado nesses mesmos estados e também em SP. PVX foi detectado em apenas uma amostra tomada ao acaso em Serra do Salitre (MG). O contraste entre a avaliação visual dos sintomas e os resultados do teste de detecção por ELISA revelou a possibilidade de infecção latente por PVY em níveis relevantes na cultivar Asterix.

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