Quantitative PCR for detection of Citrus tristeza virus in Colombia

Luis Miguel Solano-Luna, Edisson Chavarro-Mesa, Jorge Evelio Ángel-Diaz


Real-time quantitative PCR (qRT-PCR) was applied using SYBR Green for the specific detection of Citrus tristeza virus (CTV) in Colombian. Genomic RNA (gRNA) was amplified using primers designed from conserved sequences in the open reading frames (ORF’s) 1b and 2. We obtained a 186 bp product with neither dimers nor non-specificity. The analysis of the melting curve showed a peak between 81 °C and 83 °C, with a correlation coefficient of 0.998 and an efficiency of 99.1 %. The amplification of the 186 bp fragment resulted in the standard curve, which allowed the quantitative analysis of the samples with a detection range between 1x108 and 1x103 genomic RNA copies, with low variation coefficients. CTV accumulation was higher in foliar and fruit tissue than in the bark, and the differences observed among several citrus species susceptible to infection were minimal. The highest concentration of virus was found in the upper third of the analyzed plants, followed by the lower third and finally the middle third. The qRT-PCR is a specific and sensitive method, with a practical interest for the detection of viral diseases in citrus plants and other crops of commercial interest.


absolute quantification; citrus diseases; Closterovirus; detection of plant pathogens; real-Time PCR SYBR Green


(1) Caicedo A., Muñoz O., Triviño A., Gómez JE. Bases para la certificación de plántulas de cítricos libres de enfermedades en Colombia. Novedades Técnicas. 2006; 7: 9-16.

(2) Morales J., Acosta O., Tamayo P., Peñaranda J. Characterization of Citrus tristeza virus isolates from Colombia. Rev. Protección Veg. 2013; 28(1): 45-53.

(3) Ruiz-Ruiz S., Moreno P., Guerri J., Ambros S. Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific Real-Time Reverse Transcription-polymerase chain reaction method using TaqMan LNA probe. Phytopathology. 2009; 99: 307-315. DOI: http://doi.org/10.1094/PHYTO-99-3-0307.

(4) Dawson WO., Bar-Joseph M., Garnsey SM., Moreno P. Citrus tristeza virus: Making an Ally from an Enemy. Annu. Rev. Phytopathol. 2015; 53: 137-55. DOI: http://doi.org/10.1146/annurev-phyto-080614-120012.

(5) Oliveros GO., Martínez SN., Torres R., Acosta O. CPm gene diversity in field isolates of Citrus tristeza virus from Colombia. Archives of Virology. 2009; 154(12): 1933-1937. DOI: http://doi.org/10.1007/s00705-009-0530-6.

(6) Costa A., Nunes W., Zanutto C., Müller G. Stability of Citrus tristeza virus protective isolates in field conditions. Pesquisa Agropecuaria Bras. 2010; 45(7): 693-700. DOI: http://doi.org/10.1590/S0100-204X2010000700009.

(7) Ruiz-Ruiz S., Moreno P., Guerri J., Ambros S. A Real-Time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues. Journal of virogical methods. 2007; 145: 96-105. DOI: http://doi.org/10.1016/j.jviromet.2007.05.011.

(8) Harper SJ. Citrus tristeza virus: evolution of complex and varied genotypic groups. Front. Microbiol. 2013; 4: 93. DOI: http://doi.org/10.3389/fmicb.2013.00093.

(9) Febres VJ., Ashoulin L., Mawassi M., Frank A., Bar-Joseph M., Manjunath KL., Lee RF., Niblett CL. The p27 protein is present at one end of Citrus tristeza virus particles. Phytopathology. 1996; 86: 1331-1335.

(10) Saponari M., Manjunath K., Yokomi R. Quantitative detection of Citrus tristeza virus in citrus and aphids by Real-Time reverse transcription-PCR (TaqMan). Journal of Virogical Methods. 2008; 147: 43-53. DOI: http://doi.org/10.1016/j.jviromet.2007.07.026.

(11) Bertolini E., Moreno A., Capote N., Olmos A., Vidal E., Pérez J., Cambra M. Quantitative detection of Citrus tristeza virus in plant tissues and single aphid by Real-Time RT-PCR. Plant Pathology. 2008; 120: 177-188. DOI: .

(12) Gómez G. Estado actual de la citricultura en cifras, p. 12-20. En: Gómez B. G, Caicedo A. A, Gil V. L, (eds.). Tecnología para el cultivo de cítricos en la región Caribe colombiana, Produmedios, Colombia, 2008.

(13) Rodríguez P., Romero de Pérez G., Guzmán M. Detección del virus de la tristeza de los cítricos por serología, microscopía e hibridación in situ. Revista Colombiana de Biotecnología. 2009; XI(1): 94-106.

(14) Satyanarayana T., Bar-Joseph M., Mawassi M., Albiach-Martí MR., Ayllón MA., Gowda S., Hilf ME., Moreno P., Garnsey SM., Dawson WO. Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees. Virology. 2001; 280: 87-96. DOI: http://doi.org/10.1006/viro.2000.0759.

(15) Ananthakrishnan G., Venkataprasanna T., Roy A., Brlansky R. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR. Journal of Virological Methods. 2010; 64(1-2): 75-82. DOI: http://doi.org/10.1016/j.jviromet.2009.12.001.

(16) Folimonova SY., Robertson CJ., Shilts T., Folimonov AS., Hilf ME. Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus. J. Virol. 2010; 84: 1314-25. DOI: http://doi.org/10.1128/JVI.02075-09.

(17) Heid CA., Stevens J., Livak KJ., Williams PM. Real time quantitative PCR. Genome Res. 1996; 6: 986-994. DOI: http://doi.org/10.1101/gr.6.10.986.

(18) Freeman WM., Walker SJ., Vrana KE. Quantitative RT-PCR: pitfalls and potential. Biotechniques. 1999; 26: 112-125.

(19) Bustin SA. Quantification or mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 2002; 29: 23-29. DOI: http://doi.org/10.1677/jme.0.0290023.

(20) Mackay IM., Arden KE., Nitsche A. Real time PCR in virology. Nucleic Acids Res. 2002; 30: 1292-1305. DOI: za href="http://doi.org/10.1093/nar/30.6.1292">http://doi.org/10.1093/nar/30.6.1292.

(21) Whelan JA., Rusell NB., Whelan MA. A method for the absolute quantification of cDNA using real-time PCR. Journal of Immunological Methods. 2003; 278: 261-269. DOI: http://doi.org/10.1016/S0022-1759(03)00223-0.

(22) Tan BH., Lim EA., Liaw JC., Seah SG., Yap EP. Diagnostic value of real-time capillary thermal cycler in virus detection. Expert Rev. Mol. Diagn. 2004; 4: 219-230. DOI: http://doi.org/10.1586/14737159.4.2.219.

(23) Roy A., Ananthakrishnan G., Hartung JS., Brlansky RH. Development Citrus tristeza virus and application of a multiplex reverse transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates. Phytopathology. 2010; 100: 1077-1088. DOI: http://doi.org/10.1094/PHYTO-04-10-0102.

(24) Mirmajlessi S., Loit E. General principles of real-time PCR: a technology for quantitative detection of phytopathogens. Journal of medical and bioengineering. 2016; 5(1), February.

(25) Rutledge RG., Coté C. Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Research. 2003; 31: 93. DOI: http://doi.org/10.1093/nar/gng093.

(26) Casal J., Mateu E. Tipos de muestreo. Rev. Epidem. Med. Prev. 2003; 1: 3-7.

(27) Lee C., Kim J., Shin SG., Hwang S. Absolute and relative qPCR quantification of plasmid copy number in Escherichia coli. Journal of Biotechnology. 2006; 123: 273-280. DOI: http://doi.org/10.1016/j.jbiotec.2005.11.014.

(28) Cambra M., Gorris MT., Olmos A., Martinez MC., Roman MP., Bertolini E., Lopez A., Carbonell EA. European diagnostic protocols (DIAG-PRO) for Citrus Tristeza Virus in adult trees. In: Duran-villa N, Milne R. G., Da Graca J. V., (Eds.). En: Procedings of the 15th conference of the International Organization of citrus virologists. IOCV, Riverside, CA, 2002.

(29) Narváez G., Skander BS., Ayllon MA., Rubio L., Guerri J., Moreno P. A new procedure to differentiate Citrus tristeza virus isolates by hybridisation with digoxigenin-labelled cDNA probes. J. Virol. Methods. 2000; 85: 83-92. DOI: http://doi.org/10.1016/S0166-0934(99)00158-5.

(30) Nolasco G., de Blas C., Torres V., Ponz F. A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. J. Virol. Methods. 1993; 45: 201-218. DOI: http://doi.org/10.1016/0166-0934(93)90104-Y.

(31) Olmos A., Cambra M., Esteban O., Gorris MT., Terrada E. New device and method for capture, reverse transcription and nested PCR in a single closed-tube. Nucleic Acids Res. 1999; 27: 1564-1565. DOI: http://doi.org/10.1093/nar/27.6.1564.

(32) Permar TA., Garnsey SM., Gumpf DJ., Lee RF. A monoclonal antibody which discriminates strains of Citrus tristeza virus. Phytopathology. 1990; 80: 224-228. DOI: http://doi.org/10.1094/Phyto-80-224.

(33) Roistacher CN. Graft-transmissible diseases of citrus. En: Handbook for Detection and Diagnosis. FAO, Rome, Italy, 1991

(34) Vela C., Cambra M., Cortés E., Moreno P., Miguet JG., Pérez de San Román C., Sanz A. Production and characterization of monoclonal antibodies specific for Citrus tristeza virus and their use for diagnosis. J. Gen. Virol. 1986; 67: 91-96. DOI: http://doi.org/10.1099/0022-1317-67-1-91.

(35) Papin JF., Vahrson W., Dittmer DP. SYBR green-based real-time quantitative PCR assay for detection of West Nile virus circumvents false-negative results due to strain variability. J. Clin. Microbiol. 2004; 42: 1511-1518. DOI: < a href="http://doi.org/10.1128/JCM.42.4.1511-1518.2004">http://doi.org/10.1128/JCM.42.4.1511-1518.2004.

(36) Mendoza A., Salazar A., Alvarado O., Cruz MA., Barrera HA. Caracterización molecular de razas severas y débiles del virus de la tristeza de los cítricos. Ciencias UANL. 2005; 8: 250-258.

(37) Loconsole G., Saponari M., Savino V. Development of Real-Time PCR based assays for simultaneous and improved detection of citrus virus. Plant Pathology. 2010; 128: 251-259. DOI: http://doi.org/10.1007/s10658-010-9653-6.

(38) Sambrook J, Fritschi EF., Maniatis T. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York, 1989.

DOI: https://doi.org/10.19053/01228420.v15.n1.2018.7789

Article Metrics

Abstract Views

Metrics Loading ...


  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.


Revista Ciencia y Agricultura - ISSN: 0122-8420

Indexed by: REDIBDialnetCAB Abstracts, Gale Cengage-Informe Académico.


Licencia de Creative Commons

Los artículos de la Revista Ciencia y Agricultura son difundidos bajo Creative Commons Attribution 4.0 International

Sede Central Tunja–Boyacá–Colombia
Avenida Central del Norte 39-115
PBX: (57+8) 7405626
portalweb@uptc.edu.co Comentarios de este sitio
Horario de atención y servicio telefónico
8:00 a.m. a 12:00 m y 2:00 p.m a 6:00 p.m.

Atención al Ciudadano
Línea Gratuita: 01 8000 942024
Tel: (57+8) 7428263
Notificaciones Judiciales
Notificaciones de aviso

Institución de Educación Superior sujeta a inspección y vigilancia por el Ministerio de Educación Nacional
Sistema OJS - Metabiblioteca |