Multiplex RT-PCR reaction for simultaneous detection of Tomato torrado virus and Pepino mosaic virus co-infecting Solanum lycopersicum
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Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute Władysława Węgorka 20, 60-318 Poznań, Poland
Przemysław Wieczorek
Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute Władysława Węgorka 20, 60-318 Poznań, Poland
Submission date: 2013-02-27
Acceptance date: 2013-07-30
Journal of Plant Protection Research 2013;53(3):289–294
The tomato (Solanum lycopersicum L.) is cultivated all over the world and is a vegetable of significant economic importance. However, an increased production of the vegetable is directly connected with an elevated occurrence of pathogens limiting the production efficiency of the vegetable. Both, Tomato torrado virus and Pepino mosaic virus have been found to be serious disease factors. When not controlled, these viruses can significantly decrease tomato cultivation. In this article, we report a multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol for simultaneous detection of both, Tomato torrado virus (ToTV) and Pepino mosaic virus (PepMV) in virus infected plants. The assay was designed to specifically amplify the conserved regions of genomic ribonucleic acid (RNA) of both viruses. Moreover, the glycerandehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control of amplification to exclude false-negative assay results. High-resolution melt analysis of generated RT-PCR products was additionally performed to increase sensitivity and double-check the specificity of the reaction without the need of subsequent complementary deoxyribonucleic acid (cDNA) sequencing.
The authors have declared that no conflict of interests exist.
Agindotan B.O., Shiel P.J., Berger P.H. 2007. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR. J. Virol. Methods 142 (1–2):1–9.
Amari K., Gonzalez-Ibeas D., Gómez P., Sempere R.N., Sanchez-Pina M.A., Aranda M.A., Diaz-Pendon J.A., Navas-Castillo J., Moriones E., Blanca J., Hernandez-Gallardo M.D., Anastasio G. 2008. Tomato torrado virus is transmitted by Bemisia tabaci and infects pepper and eggplant in addition to tomato. Plant Dis. 92 (7): 1139.
Alfaro-Fernández A., Del Carmen Córdoba-Sellés M., Juárez M., Herrera-Vásquez J.A., Sánchez-Navarro J.A., Del Carmen Cebrián M., Font M.I., Jorda C. 2010a. Occurrence and geographical distribution of the ‘Torrado’ Disease in Spain. J. Phytopathol. 158 (7–8): 457–469.
Alfaro-Fernández A., Cebrián M.C., Herrera-Vásquez J.A., Córdoba-Sellés M.C., Sánchez-Navarro J.A., Jordá C. 2010b. Molecular variability of Spanish and Hungarian isolates of Tomato torrado virus. Plant Pathol. 59: 785–793.
Bru D., Martin-Laurent F., Philippot L. 2008. Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Apel. Environ. Microbio. 74 (5): 1660–1663.
Budziszewska M., Obrępalska-Stęplowska A., Wieczorek P., Pospieszny H. 2008. The nucleotide sequence of a Polish isolate of Tomato torrado virus. Virus Genes. 37 (3): 400–406.
Cheng J., Jiang Y., Rao P., Wu H., Dong Q., Wu Z., Ding X., Guo J. 2013. Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting- curve analysis with EvaGreen. Arch. Virol. 158 (2): 379–386.
Chen S.N., Gu H., Wang X.M., Chen J.S., Zhu W.M. 2011. Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting tomato using 18S rRNA as an internal control. Acta Biochim. Biophys. Sinica. 43: 465–471.
Gambley C.F., Thomas J.E. , Persley D.M., Hall B.H. 2010. First Report of Tomato torrado virus on Tomato from Australia. Plant Dis. 94 (4): 486.
Gómez P., Sempere R.N., Amari K., Gómez-Aix C., Aranda M.A. 2010. Epidemics of Tomato torrado virus, Pepino mosaic virus and Tomato chlorosis virus in tomato crops: do mixed infections contribute to torrado disease epidemiology? Annu. Appl. Biol. 156 (3): 401–410.
Gurtler V., Grando D., Mayall B.C., Wang J., Ghaly-Derias S. 2012. A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis. J. Microbiol. Methods 90 (3): 167–81.
Hanssen I.M., Paeleman A., Vandewoestijne E., Van Bergen L., Bragard C., Lievens B., Vanachter A.C.R.C., Thomma B.P.H.J. 2009. Pepino mosaic virus isolates and differential symptomatology in tomato. Plant Pathol. 58 (3): 450–460.
Hanssen I.M., Thomma B.P.H.J. 2010. Pepino mosaic virus: a successful pathogen that rapidly evolved from emerging to endemic in tomato crops. Mol. Plant Pathol. 11 (2): 179–189.
Hasiów-Jaroszewska B., Pospieszny H., Borodynko N. 2009. New necrotic isolates of Pepino mosaic virus representing the Ch2 genotype. J. Phytopathol. 157 (7–8): 494–496.
Herrera-Vasquez J.A., Alfaro-Fernández A., Cordoba-Selles M.C., Cebrian M.C., Font M.I., Jorda C. 2009. First report of Tomato torrado virus infecting tomato in single and mixed infections with Cucumber mosaic virus in Panama. Plant Dis. 93 (2): 198.
Mao F., Leung W.Y., Xin X. 2007. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 7 (1): 76–117.
Menzel W., Jelkmann W., Maiss E. 2002. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J. Virol. Methods 99: 81–92.
Owczarzy R., Vallone P.M., Gallo F.J., Paner T.M, Lane M.J., Benight A.S. 1997. Predicting sequence-dependent melting stability of short duplex DNA oligomers. Biopolymers 44: 217–239.
Pospieszny H., Borodynko N., Obrępalska-Stęplowska A., Hasiów B. 2007. The first report of Tomato torrado virus in Poland. Plant Dis. 91 (10), p. 1364.
Pospieszny H., Budziszewska M., Hasiów-Jaroszewska B., Obrępalska-Stęplowska A., Borodynko N. 2010. Biological and molecular characterization of Polish isolates of Tomato torrado virus. J. Phytopathol. 158 (1): 56–62.
Quito-Avila D.F., Martin R.R. 2012. Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections. J. Virol. Methods. 179 (1): 38–44.
Stadhouders R., Pas S.D., Anber J., Voermans J., Mes T.H., Schutten M. 2010. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5’ nuclease assay. J. Mol. Diagn. 12 (1): 109–117.
Tajiri-Utagawa E., Hara M., Takahashi K., Watanabe M., Wakita T. 2009. Development of a rapid high-throughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses. J. Clin. Microbiol. 47: 435–40.
Verbeek M., Dullemans A.M., van den Heuvel J.F.J.M., Maris P.C., van der Vlugt R.A.A. 2007. Identification and characterization of Tomato torrado virus, a new plant picorna-like virus from tomato. Arch. Virol. 152: 881–890.
Wieczorek P., Budziszewska M., Obrępalska-Stęplowska A. 2013. Analysis of expression of GAPDH gene in Solanum lycopersicum L. infected with Tomato torrado virus (ToTV). [Analiza ekspresji genu GAPDH w Solanum lycopersicum L. podczas infekcji wirusem nekrozy pomidora – Toto]. Prog. Plant Prot./Post. Ochr. Roślin 53 (2): 227–231.
Yin Z., Chrzanowska M., Michalak K., Zagórska H, Zimnoch-Guzowska E. 2012. Recombinants of PVY strains predominate among isolates from potato crop in Poland. J. Plant Prot. Res. 52 (2): 214–219.