J Bacteriol Virol.  2015 Mar;45(1):54-61. 10.4167/jbv.2015.45.1.54.

Development and Verification of Nested PCR Assay for Detection of Tobacco rattle virus in Plant Quarantine

• Lee S
• Lee JY
• Shin YG
• Lee SH
• Ahn TY

• KMID: 2168705
• DOI: http://doi.org/10.4167/jbv.2015.45.1.54

Abstract

Tobacco rattle virus (TRV) is a plant pathogen belonging to the Group IV positive-sense single-stranded RNA viruses. TRV causes disease in various plants (e.g., potato, tomato and tobacco), for which it was classified as a controlled quarantine virus in Korea. This study aimed to develop specific primer sets for the rapid detection of TRV. Two RT-PCR primer sets were developed for specific detection of TRV. Furthermore, nested primer sets were also developed, which is required for high sensitivity detection in plant quarantine. The RT-PCR and nested PCR products had the following sizes: set 5 (1,096-->540 bp), and set 7 (878-->756 bp), respectively. In addition, a modified positive-control plasmid was also developed for use as a positive control in TRV quarantine. The diagnostic system for TRV detection was verified using samples from Korean quarantine sites for the last five years (2009-2014). A total of 83 cases were detected among various import crops. This system for detection of TRV will continuously contribute to plant quarantine in the future.

Keyword

Tobacco rattle virus; Nested PCR; Quarantine

MeSH Terms

Korea
Lycopersicon esculentum
Plants*
Plasmids
Polymerase Chain Reaction*
Quarantine*
RNA Viruses
Solanum tuberosum
Tobacco*

Figure

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  Figure 1. Map of primer design for detection of TRV

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  Figure 2. Results of selection of TRV RT-PCR primer sets. Panel (A), First selection of specific RT-PCR primer sets for the detection of TRV. M, 100 bp step DNA Ladder maker (Genepia, Korea); dot, selected PCR primer set; Lane number, primer set for detection of TRV. Panel (B), Second selection of specific RT-PCR primer sets for the detection of TRV. Lane M, 100 bp step DNA Ladder maker (Genepia); lane AM, Alfalfa mosaic virus; lane Ar, Arabis mosaic virus; lane CM, Cucumber mosaic virus; lane Pe, Pepper mottle virus; lane PY, Potato virus Y; lane RM, Ribgrass mosaic virus; lane TG, Tobacco mild-green mosaic virus; lane TM, Tobacco mosaic virus; lane To, Tomato mosaic virus; lane TS, Tomato spotted wilt virus.

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  Figure 3. RT-PCR sensitivity test on the selective primer sets for TRV.

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  Figure 4. Result of selected RT-PCR and nested-PCR products for the detection of TRV. Lane M, 100 bp step DNA Ladder; lane 1, final selected primer set #5 (1,096 bp); lane 2, nested-PCR product from primer set #5 (543 bp); lane 3, final selected primer set #7 (880 bp); lane 4, nested-PCR product from primer set #7 (760 bp).

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  Figure 5. BamH I sequence insertion of the TRV genetically modified-positive control plasmids.

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