Development of a diagnostic system to detect potato virus T using RT-PCR and nested PCR

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.

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Specific Detection of Chikungunya Virus Using a RT-PCR/Nested PCR Combination

Journal of Veterinary Medicine Series B ◽

10.1046/j.1439-0450.2002.00535.x ◽

2002 ◽

Vol 49 (1) ◽

pp. 49-54 ◽

Cited By ~ 42

Author(s):

M. PFEFFER ◽

B. LINSSEN ◽

M. D. PARKER ◽

R. M. KINNEY

Keyword(s):

Nested Pcr ◽

Chikungunya Virus ◽

Rt Pcr ◽

Specific Detection

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Development of RT-PCR and Nested PCR for Detecting Four Quarantine Plant Viruses Belonging to Nepovirus

Research in Plant Disease ◽

10.5423/rpd.2013.19.3.220 ◽

2013 ◽

Vol 19 (3) ◽

pp. 220-225 ◽

Cited By ~ 16

Author(s):

Siwon Lee ◽

Eun-Ha Kang ◽

Yong-Gil Shin ◽

Su-Heon Lee

Keyword(s):

Nested Pcr ◽

Plant Viruses ◽

Rt Pcr

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Detection and genome characterization of Potato virus Y isolates infecting potato (Solanum tuberosum L.) in La Union (Antioquia, Colombia)

Agronomía Colombiana ◽

10.15446/agron.colomb.v34n3.59014 ◽

2016 ◽

Vol 34 (3) ◽

pp. 317-328 ◽

Cited By ~ 4

Author(s):

Pablo Gutiérrez S. ◽

Mauricio Marín M. ◽

Daniel Muñoz E.

Keyword(s):

Solanum Tuberosum ◽

Potato Virus ◽

Potato Virus Y ◽

Seed Tuber ◽

Rt Pcr ◽

Potato Plants ◽

Nextgeneration Sequencing ◽

Prevalent Strain ◽

Detailed Molecular Analysis

Potato virus Y (PVY) is one of the most severe viruses affecting the production of potato (Solanum tuberosum) in the world. This study presents a detailed molecular analysis using nextgeneration sequencing (NGS), IC-RT-qPCR and RT-PCR on the PVY isolates infecting seed-tubers and foliage of potato plants cv. Diacol-Capiro in La Union (Antioquia, Colombia). Analysis of incidence by IC-RT-qPCR in 15 random leaf samples of three cultivation plots and fifteen sprouting tuber eye-buds reveal infection levels between 13.4 and 80%; a higher incidence of 86.7% was observed in seed-tuber samples with threshold cycle (Ct) values as low as 24.3. Genome assembly from a bulk of foliage samples resulted in a consensus PVY genome (PVY_LaUnionF) of 9,702 nt and 399 polymorphic sites within the polyprotein ORF; while the assembled genome from sprouts of tubers has 9,704 nt (PVY_LaUnionT) and contained only six polymorphic nucleotide sites. Phylogenetic analysis demonstrates that the PVY isolates from leaf samples are in the recombinant PVYNTN group (sequence identity >99%); while those from tuber sprouts are in the PVYN/NTN group with identities above 95%. Sanger sequencing of viral capsid suggests the presence of a third variant related to PVYO, a prevalent strain reported in potato fields worldwide.

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

Plant Disease ◽

10.1094/pdis-09-21-1950-re ◽

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.

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Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control

Plant Disease ◽

10.1094/pd-90-0185 ◽

2006 ◽

Vol 90 (2) ◽

pp. 185-189 ◽

Cited By ~ 34

Author(s):

ZhiYou Du ◽

JiShuang Chen ◽

Chuji Hiruki

Keyword(s):

Potato Virus ◽

Internal Control ◽

18S Rrna ◽

Degradation Kinetics ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Potato Viruses ◽

Das Elisa

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

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The Recombinant Potato virus Y (PVY) Strain, PVYNTN, Identified in Potato Fields in Victoria, Southeastern Australia

Plant Disease ◽

10.1094/pdis-05-20-0961-sc ◽

2020 ◽

Vol 104 (12) ◽

pp. 3110-3114

Author(s):

Mariana Rodriguez-Rodriguez ◽

Mohamad Chikh-Ali ◽

Steven B. Johnson ◽

Stewart M. Gray ◽

Nellie Malseed ◽

...

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Strain Typing ◽

Rt Pcr ◽

Growing Seasons ◽

Fta Cards ◽

Southeastern Australia ◽

Whole Genomes ◽

Pcr Assays

Potato virus Y (PVY) is one of the main viruses affecting potato in Australia. However, molecular characterization of PVY isolates circulating in potato in different states of Australia has not yet been thoroughly conducted. Only nonrecombinant isolates of three biological PVY strains collected from potato were reported previously from Western Australia and one from Queensland. Here, PVY isolates collected from seed potato originating in Victoria, Australia, and printed on FTA cards, were subjected to strain typing by RT-PCR, with three isolates subjected to whole genome sequencing. All the 59 PVY isolates detected during two growing seasons were identified to be recombinants based on two RT-PCR assays. No nonrecombinant PVY isolates were identified. All the RT-PCR typed isolates belonged to the PVYNTN strain. Sequence analysis of the whole genomes of three isolates suggested a single introduction of the PVYNTN strain to Australia but provided no clues as to where this introduction originated. Given the association of the PVYNTN strain with potato tuber damage, growers in Australia should implement appropriate strategies to manage PVYNTN in potato.

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Development of a Diagnostic System for Novel Influenza A(H7N9) Virus Using a Real-Time RT-PCR Assay in Japan

Japanese Journal of Infectious Diseases ◽

10.7883/yoken.jjid.2014.136 ◽

2015 ◽

Vol 68 (2) ◽

pp. 113-118 ◽

Cited By ~ 7

Author(s):

Ikuyo Takayama ◽

Hitoshi Takahashi ◽

Mina Nakauchi ◽

Shiho Nagata ◽

Masato Tashiro ◽

...

Keyword(s):

Real Time ◽

Influenza A ◽

Diagnostic System ◽

H7n9 Virus ◽

Rt Pcr ◽

Pcr Assay ◽

Novel Influenza A

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Potato virus Y (PVY) detection in a single aphid by one-step RT-PCR with boiling technique

Entomological Research ◽

10.1111/1748-5967.12170 ◽

2016 ◽

Vol 46 (4) ◽

pp. 278-285 ◽

Cited By ~ 4

Author(s):

Juil Kim ◽

Deok Jea Cha ◽

Min Kwon ◽

Rameswor Maharjan

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Rt Pcr ◽

One Step

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

Plant Disease ◽

10.1094/pdis-02-13-0161-sr ◽

2013 ◽

Vol 97 (10) ◽

pp. 1370-1374 ◽

Cited By ~ 31

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.

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