Dataset Information

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea.

ABSTRACT: Potato mop-top virus (PMTV) is classified as a plant quarantine virus in Korea. It is tested at import inspection sites. In this study, two sets of reverse transcription-hemi-nested polymerase chain reaction primers were developed to diagnose PMTV. In addition, modified- positive control plasmid was developed for the identification of false positive results in plant quarantine. The assay is expected to be useful for improving the detection and diagnosis of PMTV.

SUBMITTER: Lee S

PROVIDER: S-EPMC5446823 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Publications

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea.

Lee S S Lee J Y JY Kim H H Kim J H JH Rho J Y JY

Indian journal of microbiology 20160927 2

Potato mop-top virus (PMTV) is classified as a plant quarantine virus in Korea. It is tested at import inspection sites. In this study, two sets of reverse transcription-hemi-nested polymerase chain reaction primers were developed to diagnose PMTV. In addition, modified- positive control plasmid was developed for the identification of false positive results in plant quarantine. The assay is expected to be useful for improving the detection and diagnosis of PMTV. ...[more]

PMID: 28611505

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Project description:The evolutionary divergence of Potato mop-top virus (PMTV), a tri-partite, single-stranded RNA virus, is exceptionally low, based on the analysis of sequences obtained from isolates from Europe, Asia and North America. In general, RNA viruses exist as dynamic populations of closely related and recombinant genomes that are subjected to continuous genetic variation. The reason behind the low genetic variation of PMTV remains unclear. The question remains as to whether the low variability is a shared property of all PMTV isolates or is a result of the limited number of isolates characterized so far. We hypothesized that higher divergence of the virus might exist in the Andean regions of South America, the centre of potato domestication. Here, we report high variability of PMTV isolates collected from 12 fields in three locations in the Andean region of Peru. To evaluate PMTV genetic variation in Peru, we generated full-length cDNA clones, which allowed reliable comparative molecular and pathobiological characterization of individual isolates. We found significant divergence of the CP-RT and 8K sequences. The 8K cistron, which encodes a viral suppressor of RNA silencing, was found to be under diversifying selection. Phylogenetic analysis determined that, based on the CP-RT sequence, all PMTV isolates could be categorized into three separate lineages (clades). Moreover, we found evidence for recombination between two clades. Using infectious cDNA clones of the representatives of these two clades, as well as reassortants for the RNA-CP genomic component, we determined the pathobiological differences between the lineages, which we coined as S (for severe) and M (for mild) types. Interestingly, all isolates characterized previously (from Europe, Asia and North America) fall into the S-type clade, whereas most of the Peruvian isolates belong to the M-type. Taken together, our results support the notion of the single introduction of PMTV from the centre of potato origin to Europe, and subsequent spread of the S-type into Asia and USA. This is also supported by the suggested novel classification of isolates based on genetic constellations.

Project description:BackgroundHexaploid wheat (Triticum aestivum L.) possesses a large genome that contains 1.6 x 1010 bp of DNA. Isolation of a large number of gene sequences from complex gene families with a high level of gene sequence identity from genomic DNA is therefore difficult and time-consuming. Bacterial artificial chromosome (BAC) libraries can be useful for such work. Here we report on an efficient approach for rapid isolation and sequencing of the low molecular weight glutenin subunit gene family from the 'Glenlea' wheat BAC library via primer-template mismatch PCR using universal primers, primer walking using hemi-nested touchdown (TD) PCR, and followed by direct sequencing of PCR products.ResultsFor the primer-template mismatch PCR, the universal primers were designed based on conserved gene coding regions of consensus sequences. The effects of the universal primer-template mismatches on the efficiency of standard PCR amplification were investigated after assembly of sequences from different primers amplifying the same BAC clones. Single or multiple mismatches were observed at 5' terminal, internal and the penultimate position, respectively. These mismatches included the transition mispairs G:T, T:G, A:C and the transversion mispairs A:A, A:G, G:G, G:A. Two or more primer-template mismatches reduced PCR product yield approximately from 2-fold to 10-fold compared to PCR product yield without the primer-template mismatch. For the hemi-nested TD PCR, primers were designed based on the known sequences obtained and/or published. The hemi-nested TD PCR increased both specificity and yield by high and low annealing temperatures in two consecutive amplifications. Comparison of two methods for purifying PCR products prior to sequencing showed that purification using MultiScreen384-PCR filter plates had an advantage over ethanol purification because greater numbers of sequencing reactions could be performed from comparable volumes of PCR reactions.ConclusionThis approach was fast, easy and cost-effective for isolation and sequencing of genes from complex gene families. It may be suitable for (i) isolation of other complex gene families and/or gene homologues from BAC libraries, (ii) for characterization of multi-copy repetitive elements pending availability of BAC libraries, and (iii) for filling in gaps in shotgun BAC sequencing.

Project description:A mechanistic understanding of robust self-assembly and repair capabilities of complex systems would have enormous implications for basic evolutionary developmental biology as well as for transformative applications in regenerative biomedicine and the engineering of highly fault-tolerant cybernetic systems. Molecular biologists are working to identify the pathways underlying the remarkable regenerative abilities of model species that perfectly regenerate limbs, brains, and other complex body parts. However, a profound disconnect remains between the deluge of high-resolution genetic and protein data on pathways required for regeneration, and the desired spatial, algorithmic models that show how self-monitoring and growth control arise from the synthesis of cellular activities. This barrier to progress in the understanding of morphogenetic controls may be breached by powerful techniques from the computational sciences-using non-traditional modeling approaches to reverse-engineer systems such as planaria: flatworms with a complex bodyplan and nervous system that are able to regenerate any body part after traumatic injury. Currently, the involvement of experts from outside of molecular genetics is hampered by the specialist literature of molecular developmental biology: impactful collaborations across such different fields require that review literature be available that presents the key functional capabilities of important biological model systems while abstracting away from the often irrelevant and confusing details of specific genes and proteins. To facilitate modeling efforts by computer scientists, physicists, engineers, and mathematicians, we present a different kind of review of planarian regeneration. Focusing on the main patterning properties of this system, we review what is known about the signal exchanges that occur during regenerative repair in planaria and the cellular mechanisms that are thought to underlie them. By establishing an engineering-like style for reviews of the molecular developmental biology of biomedically important model systems, significant fresh insights and quantitative computational models will be developed by new collaborations between biology and the information sciences.

Dataset Information

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea.

Publications

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of <i>Potato Mop</i>-<i>Top Virus</i> at Quarantine Inspection Sites in Korea.

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