Optimization of a Real-Time RT-PCR Assay and its Comparison with ELISA, Conventional RT-PCR and the Grow-out Test for Large Scale Diagnosis of Potato virus Y in Dormant Potato Tubers

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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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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Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2021.369 ◽

2021 ◽

pp. 1-18

Author(s):

Jonathan B. Gubbay ◽

Heather Rilkoff ◽

Heather L. Kristjanson ◽

Jessica D. Forbes ◽

Michelle Murti ◽

...

Keyword(s):

Positive Predictive Value ◽

Real Time ◽

Reverse Transcription ◽

Predictive Value ◽

Large Scale ◽

Rdrp Gene ◽

Nucleic Acid Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Test Probability

Abstract Objectives Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false positive test. This study assessed positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Methods A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the four groups with higher pre-test probability (individual group range 50·0% to 85·0%). Conclusions Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

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

Plant Disease ◽

10.1094/pdis-02-14-0154-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1589-1589 ◽

Cited By ~ 3

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.

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

Plant Disease ◽

10.1094/pd-90-0935 ◽

2006 ◽

Vol 90 (7) ◽

pp. 935-940 ◽

Cited By ~ 121

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.

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Improvement of Potato virus Y (PVY) detection and quantitation using PVYN- and PVYO-specific real-time RT-PCR assays

Journal of Virological Methods ◽

10.1016/j.jviromet.2006.01.019 ◽

2006 ◽

Vol 134 (1-2) ◽

pp. 261-266 ◽

Cited By ~ 30

Author(s):

Valérie Balme-Sinibaldi ◽

Michel Tribodet ◽

Flora Croizat ◽

Pierre Lefeuvre ◽

Camille Kerlan ◽

...

Keyword(s):

Real Time ◽

Potato Virus ◽

Potato Virus Y ◽

Rt Pcr ◽

Pcr Assays

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Cytochrome c oxidase mRNA as an internal control for detection of Potato virus Y and Potato leafroll virus from single aphids by a co-amplification RT-PCR assay

Journal of Virological Methods ◽

10.1016/j.jviromet.2006.08.007 ◽

2006 ◽

Vol 138 (1-2) ◽

pp. 152-159 ◽

Cited By ~ 7

Author(s):

Changzheng He ◽

Teresa A. Molen ◽

Xingyao Xiong ◽

Gilles Boiteau ◽

Xianzhou Nie

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Potato Virus ◽

Internal Control ◽

Potato Virus Y ◽

Potato Leafroll Virus ◽

Rt Pcr ◽

Pcr Assay ◽

Leafroll Virus

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RT-PCR and Real-Time RT-PCR Methods for the Detection of Potato Virus Y in Potato Leaves and Tubers

Methods in Molecular Biology - Plant Virology Protocols ◽

10.1007/978-1-4939-1743-3_2 ◽

2014 ◽

pp. 13-26 ◽

Cited By ~ 4

Author(s):

Tyler D. B. MacKenzie ◽

Xianzhou Nie ◽

Mathuresh Singh

Keyword(s):

Real Time ◽

Potato Virus ◽

Potato Virus Y ◽

Rt Pcr

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Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721994810 ◽

2021 ◽

pp. 104063872199481

Author(s):

Yixin Xiao ◽

Fan Yang ◽

Fumin Liu ◽

Hangping Yao ◽

Nanping Wu ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Minor Groove Binder ◽

Rt Pcr ◽

Influenza A Viruses ◽

Pcr Assay ◽

Infectious Dose ◽

The Matrix ◽

Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

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