Primer Modification Improves Rapid and Sensitive
            <i>In Vitro</i>
            and Field-Deployable Assays for Detection of High Plains Virus Variants

Arif, Mohammad; Aguilar-Moreno, Guadalupe Stefanny; Wayadande, Astri; Fletcher, Jacqueline; Ochoa-Corona, F. M.

doi:10.1128/aem.02340-13

Cited by 35 publications

(34 citation statements)

References 25 publications

(41 reference statements)

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“…Rapid diagnosis can be difficult in cases where a virus is not well recognized as infecting a particular plant host or geographical region and is further complicated where the virus differs from known strains. Currently, most methods in use for virus detection are species-specific (Arif et al, 2014) (e.g. PCR or ELISA).…”

Section: Introductionmentioning

confidence: 99%

Detection, discrimination and discovery of a new Tobacco streak virus strain

Dutta

Ali

Melcher

2015

Journal of Virological Methods

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Soybean plants that exhibited symptoms of virus infection were sampled from different counties of Oklahoma. These plants were tested serologically for 15 viruses known to infect soybean plants. Fifty-seven samples that exhibited typical virus-like symptoms did not test positive for any of the 15 viruses used in a dot-immunobinding assay (DIBA). Four samples were pooled and used for next generation sequencing using the 454-Roche protocol. Sequence and phylogenetic analysis of the sequences obtained revealed infection with a distinct strain of Tobacco streak virus (TSV). TSV was one of the 15 viruses initially tested for using DIBA and had tested negative. TSV belongs to the genus Ilarvirus and has been reported as a causal agent of bud blight in soybean crops in Brazil and the United States. Out of 10 reported primer pairs for TSV reverse transcription-polymerase chain reaction (RT-PCR), only two had the potential, based on sequence similarity, to amplify part of the genome of the distinct strain of TSV found in Oklahoma and only one was actually able to amplify the region. In this study, a new primer pair, specific to all known TSV and capable of amplifying the Oklahoma strain (TSV-OK), was designed from a highly conserved region of coat protein (CP) sequences and end-point PCR and quantitative RT-PCR detection methods were developed and their sensitivity assayed. This is the first report of specific primers designed from this highly conserved region in the CP of TSV for detection of TSV. Twenty-three of the 57 DIBA soybean samples that initially tested negative were retested with the new specific end-point PCR method and found positive for TSV infection.

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Section: Introductionmentioning

confidence: 99%

Detection, discrimination and discovery of a new Tobacco streak virus strain

Dutta

Ali

Melcher

2015

Journal of Virological Methods

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“…Besides the selection of unique and conserved targets, primer and probe design plays a very important role in developing a robust and specific multiplex TaqMan qPCR assay [17,29]. Assay specificity can also be enhanced using dual-quenched probes [16,30]; that is, an internal quencher, TAO or ZEN, was added to each probe to reduce the distance from reporter to quencher, leading to low background signal and greater precision ( Table 2).…”

Section: Discussionmentioning

confidence: 99%

VIDHOP, viral host prediction with Deep Learning

Mock

Viehweger

Barth

et al. 2019

Preprint

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Zoonosis, the natural transmission of infections from animal to human, is a far-reaching global problem. The recent outbreaks of Zika virus and Ebola virus are examples of viral zoonosis, which occur more frequently due to globalization. In case of a virus outbreak, it is helpful to know which host organism was the original carrier of the virus. Once the reservoir or intermediate host is known, it can be isolated to prevent further spreading of the viral infection. Recent approaches aim to predict a viral host based on the viral genome, often in combination with the potential host genome and using arbitrary selected features. This methods have a clear limitation in either the amount of different hosts they can predict or the accuracy of the prediction. Here, we present a fast and accurate deep learning approach for viral host prediction, which is based on the viral genome sequence only. To assure a high prediction accuracy we developed an effective selection approach for the training data, to avoid biases due to a highly unbalanced number of known sequences per virus-host combinations. We tested our deep neural network on three different virus species (influenza A virus, rabies lyssavirus, rotavirus A) and reached for each virus species a AUC between 0.94 and 0.98, outperforming previous approaches and allowing highly accurate predictions while only using fractions of the viral genome sequences. We show that deep neural networks are suitable to predict the host of a virus, even with a limited amount of sequences and highly unbalanced available data. The deep neural networks trained for this approach build the core of the virus host predicting tool VIDHOP (VIrus Deep learning HOst Prediction). doi: bioRxiv preprint shown to perform well with character sequences 28 , such as DNA or RNA sequences, potentially allowing for a furthermore increase in the prediction quality. Author Contributions

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“…Primers with a 5′-flap have been used to improve the efficiency of the PCR reaction [56–59] and to enhance the quality of sequencing [60]. Such primers have also been reported to increase the fluorescent signal of real-time PCR [56, 57].…”

Section: Discussionmentioning

confidence: 99%

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

et al. 2017

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Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

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Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Cited by 35 publications

References 25 publications

Detection, discrimination and discovery of a new Tobacco streak virus strain

Detection, discrimination and discovery of a new Tobacco streak virus strain

VIDHOP, viral host prediction with Deep Learning

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

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