Development of a droplet digital polymerase chain reaction (ddPCR) assay for the detection of Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seeds

Vargas-Hernández, Brenda Yazmín; Pool-Ramírez, José Abrahán; Núñez-Muñoz, Leandro Alberto; Calderón-Pérez, Berenice; Torre-Almaráz, Rodolfo De La; Hinojosa-Moya, Jesús; Xoconostle‐Cázares, Beatriz; Ruíz-Medrano, Roberto

doi:10.1016/j.jviromet.2022.114466

Cited by 12 publications

(8 citation statements)

References 17 publications

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“…These advantages make ddPCR a valuable addition to the virologist's toolbox (Kojabad et al, 2021). In general, the ddPCR technique is characterized by its high detection sensitivity (10 copies) in samples (Vargas‐Hernández et al, 2022). In this study, the sensitivity assay showed that the limit of detection developed ddPCR method for GSIV was 3.7 ± 0.4 copies/reaction.…”

Section: Discussionmentioning

confidence: 99%

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

Meng

Jiang

Xie

et al. 2023

Journal of Fish Diseases

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Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1°C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV‐2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real‐time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real‐time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load.

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Section: Discussionmentioning

confidence: 99%

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

Meng

Jiang

Xie

et al. 2023

Journal of Fish Diseases

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“…Although bioassays play a key role in several fields of plant virology, such as host range determination [57][58][59], symptomatology [60,61], virus replication, plant-pathogen interactions [62,63], and determination of function(s) of viral proteins or genes and host genes (gene knockdown via virus induced gene silencing) [64,65], the biological assay has always been paled into insignificance in virus quantification, because the sensitivity and reproducibility of this method might be misevaluated. Undoubtedly, molecular methods such as q(RT)-PCR or digital (RT)-PCR could detect very low concentrations or copy numbers of virus genomes [66,67], which is beyond the ability of a bioassay. The establishment of a successful bioassay or infectiosity test requires a minimum number of virus particles which is equal to the infective dose or the lowest values of boundary or the lowest concentration of virus particles that could be detected by this method.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host

Zarghani

Monavari

Ehlers

et al. 2022

Plants

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Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

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“…Several nucleic acid amplification‐based protocols, including RT‐quantitative PCR (RT‐qPCR) and droplet digital PCR (ddPCR), have been developed to detect ToBRFV in leaf and seed samples (Chanda et al, 2021b ; Fidan et al, 2021 ; Luigi et al, 2022 ; Menzel & Winter, 2021 ; Panno et al, 2019b ; Vargas‐Hernández et al, 2022 ). Yan et al ( 2021b ) reported a quadruplex RT‐PCR protocol that allows for the simultaneous detection of four tomato‐infecting viruses, ToBRFV, TMV, ToMV, and TSWV, from the same sample in one reaction.…”

Section: Detection and Diagnosismentioning

confidence: 99%

“…NGS approaches have been used successfully to detect ToBRFV and have been instrumental in determining full genomic sequences of ToBRFV, and useful for understanding evolutionary relationships and the spread of ToBRFV worldwide. However, NGS methods are not the preferred choice for ToBRFV detection due to the costs (monetary, expertise, and infrastructure) and processing time required compared to amplificationbased approaches.Several nucleic acid amplification-based protocols, including RT-quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR), have been developed to detect ToBRFV in leaf and seed samples(Chanda et al, 2021b;Fidan et al, 2021;Luigi et al, 2022;Menzel & Winter, 2021;Panno et al, 2019b;Vargas-Hernández et al, 2022) Yan et al (2021b). reported a quadruplex RT-PCR protocol that allows for the simultaneous detection of four tomato-infecting viruses, ToBRFV, TMV, ToMV, and TSWV, from the same sample in one reaction.…”

mentioning

confidence: 99%

Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide

Zhang

Griffiths

Marchand

et al. 2022

Molecular Plant Pathology

101

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Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm‐1 , Tm‐2 , and Tm‐2 2 in tomato and L 1 and L 2 alleles in pepper. Currently, no commercial ToBRFV‐resistant tomato cultivars are available. Integrated pest management‐based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long‐term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment‐friendly strategy for pathogen control. Taxonomy Tomato brown rugose fruit virus belongs to the genus Tobamovirus , in the family Virgaviridae . The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus . Genome and virion The ToBRFV genome is a single‐stranded, positive‐sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod‐shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. Disease symptoms Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose do...

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Development of a droplet digital polymerase chain reaction (ddPCR) assay for the detection of Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seeds

Cited by 12 publications

References 17 publications

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host

Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide

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