Real time PCR. Application in dengue studies

https://doi.org/10.25100/cm.v42i2.778

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Published: Jul 23, 2024
Updated: 2024-07-23
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2024-07-23 (2)
Issue: Vol. 42 No. 2 (2011)
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Authors

Jeanette Prada-Arismendy
Jaime E Castellanos
Abstract
PCR (polymerase chain reaction) is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.
Keywords
  • PCR
  • Real time PCR
  • Molecular biology
  • Dengue virus
  • Housekeeping gene
References

Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H.Specific enzymatic amplification of DNA in vitro: thepolymerase chain reaction. Cold Spring Harb Symp QuantBiol. 1986; 51 Pt 1: 263-73.2.Costa J. Reacción en cadena de la polimerasa (PCR) a tiemporeal. Enferm Infecc Microbiol Clin. 2004; 22: 299-305.3.Arya M, Shergill IS, Williamson M, Gommersall L, Arya N,Patel HRH. Basic principles of real-time quantitative PCR.Expert Rev Mol Diagn. 2005; 5: 209-19.4.Holland PM, Abramson RD, Watson R, Gelfand DH. Detectionof specific polymerase chain reaction product by utilizing the5' ’! 3' exonuclease activity of Thermus aquaticus DNApolymerase. Proc Natl Acad Sci USA. 1991; 88: 7276-80.5.Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneousamplification and detection of specific DNA sequences.Biotechnology. (NY) 1992; 10: 413-7.6.Schneeberger C, Speiser P, Kury F, Zeillinger R. Quantitativedetection of reverse transcriptase-PCR products by means ofa novel and sensitive DNA stain. PCR Methods Appl. 1995; 4:234-8.7.Dhar AK, Roux MM, Klimpel KR. Detection and quantificationof infectious hypodermal and hematopoietic necrosis virusand white spot virus in shrimp using real-time quantitativePCR and SYBR Green chemistry. J Clin Microbiol. 2001; 39:2835-45.8.Skeidsvoll J, Ueland PM. Analysis of double-stranded DNAby capillary electrophoresis with laser-induced fluorescencedetection using the monomeric dye SYBR green I. AnalBiochem. 1995; 231: 359-65.9.Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP.Continuous fluorescence monitoring of rapid cycle DNAamplification. Biotechniques. 1997; 22: 130-1; 134-8.10. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J,Lind K, et al. The real-time polymerase chain reaction. MolAspects Med. 2006; 27: 95-125.11. Cardullo RA, Agrawal S, Flores C, Zamecnik PC, Wolf DE.Detection of nucleic acid hybridization by nonradiativefluorescence resonance energy transfer. Proc Natl Acad SciUSA. 1988; 85: 8790-4.12. Gibson UE, Heid CA, Williams PM. A novel method for realtime quantitative RT-PCR. Genome Res. 1996; 6: 995-1001.13. Heid CA, Stevens J, Livak KJ, Willams PM. Real timequantitative PCR. Genome Res. 1996; 6: 986-94.14. Bär M, Bär D, Lehmann B. Selection and validation ofcandidate housekeeping genes for studies of humankeratinocytes. Review and recommendations. J InvestDermatol. 2009; 129: 535-7.15. Wang X, Seed B. High-throughput primer and probe design.En: Tevfik Dorak M. Real-time PCR. New York: Taylor &Francis Group; 2006. p. 93-106.16. Ririe KM, Rasmussen RP, Wittwer CT. Product differentiationby analysis of DNA melting curves during the polymerasechain reaction. Anal Biochem. 1997; 245: 154-60.17. Mackay IM, Arden, KE, Nitsche A. Real-time PCR in virology.Nucleic Acid Res. 2002; 30: 1292-305.18. Pfaffl MW. A new mathematical model for relativequantification in real-time RT-PCR. Nucleic Acids Res. 2001;29: e45.19. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H. Quantitative real-time RT-PCR data analysis: currentconcepts and the novel «gene expression’s CT difference»formula. J Mol Med. 2006; 84: 901-10.20. Gibson NJ. The use of real-time PCR methods in DNAsequence variation analysis. Clin Chim Acta. 2006; 363: 32-47.21. Valasek MA, Repa JJ. The power of real-time PCR. AdvPhysiol Educ. 2005; 29: 151-9.22. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, VetterEA, et al. Real-Time PCR in clinical microbiology: applicationsfor routine laboratory testing. Clin Microbiol Rev. 2006; 19:165-256.23. Watzinger F, Ebner K, Lion T. Detection and monitoring ofvirus infections by real-time PCR. Mol Aspects Med. 2006;27: 254-98.24. Yeh HY, Yates MV, Chen W, Mulchandani A. Real-timemolecular methods to detect infectious viruses. Sem Cell DevBiol. 2009; 20: 49-54.25. World Health Organization. CDC protocol of realtime RTPCRfor swine influenza A (H1N1). Atlanta: The WHO CollaboratingCentre for influenza at CDC Atlanta; 2009.26. Pantelic L, Sivakumaran H, Urosevic N. Differential inductionof antiviral effects against West Nile virus in primary mousemacrophages derived from flavivirus-susceptible and congenicresistant mice by alpha/beta interferon and poly(I-C). J V i r o l.2005; 9: 1753-64.27. Chomczynski P, Sacchi N. Single-step method of RNA isolationby acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem. 1987; 162: 156-9.28. Castañeda NY, Chaparro-Olaya J, Castellanos JE. Tecnologíade la clonación y expresión génica en un sistema bacteriano ysu aplicación en virología. Rev Facult Med. 2006; 11: 43-53.29. Peirson S, Butler J, Foster R. Experimental validation of noveland conventional approaches to quantitative real-time PCRdata analysis. Nucleic Acids Res. 2003; 31: e73.30. Kenneth J, Schmittgen D. Analysis of relative gene expressiondata using real-time quantitative PCR and the 2- ÄÄCt method.Methods. 2001; 25: 402-8.31. Castellanos JE, Martínez-Gutiérrez M, Hurtado H, Kassis R,Bourhy H, Acosta O,et al. Studying neurotrophin antiviraleffect on rabies-infected dorsal root ganglia cultures. JNeurovirol. 2005; 11: 403-10.

Colombia Médica Vol. 42 Nº 2, 2011 (Abril-Junio)32. Gomes-Ruiz AC, Nascimento RT, de Paula SO, Lopes daFonseca BA. SYBR green and TaqMan Real-Time PCRassays are equivalent for the diagnosis of dengue virus type 3infections. J Med Virol. 2006; 78: 760-3.33. Halstead SB. Pathogenesis of dengue: Challenges to molecularbiology. Science. 1988; 239: 476-81.34. Wang WK, Sung TL, Tsai YC, Kao CL, Chang SM, King CC.Detection of dengue virus replication in peripheral bloodmononuclear cells from dengue virus type 2-infected patientsby a reverse transcription-real-time PCR assay. J ClinMicrobiol. 2002; 40: 4472-8.35. Mashimo T, Lucas M, Simon-Chazottes D, Frenkiel MP,Montagutelli X, Ceccaldi PE, et al. A nonsense mutation in thegene encoding 2’-5’-oligoadenylate synthetase/L1 isoform isassociated with West Nile virus susceptibility in laboratorymice. Proc Natl Acad Sci USA. 2002; 99: 11311-16.36. Stürzenbaum SR, Kille P. Control genes in quantitativemolecular biological techniques: the variability of invariance.Comp Biochem Physiol Part B Biochem Mol Biol. 2001; 130:281-9.37. Urosevic N, van Maanen M, Mansfield JP, Mackenzie JS,Shellam GR. Molecular characterization of virus-specificRNA produced in the brains of Flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitisvirus. J Gen Virol. 1997; 78: 23-9.38. Prada J, Castellanos JE, Rincón V. El genotipo de oligo-adenilato sintetasa 1b no está relacionado con susceptibilidada la infección por virus dengue en macrófagos de diferentesespecies de roedores. Acta Biol Colomb. 2006; 11: 162-3.39. Prada J, Rincón V, Castellanos JE. Relación entre el genotipode la enzima oligoadenilato sintetasa (Oas1b) y la susceptibi-lidad a la infección por dengue en ratones. Infectio. 2006; 10:139.40. Houghton-Triviño N, Salgado DM, Rodríguez JA, Bosch I,Castellanos JE. Levels of soluble ST2 in serum associatedwith severity of dengue due to tumour necrosis factor alphastimulation. J Gen Virol. 2010; 91: 697-706.41. Houghton-Triviño N, Salgado D, Rodríguez J, Bosch I, Cas-tellanos JE. Los niveles del inmunoregulador STST2s ycitoquinas pro-inflamatorias, pero no la carga viral, secorrelacionan con severidad en pacientes con infección agudapor Dengue. Infectio. 2008; 12: S51.42. Martínez-Gutiérrez M, Trujillo A, Castellanos JE, Vélez ID,Mañes S, Gallego JC. La lovastatina afecta el proceso deensamblaje del virus dengue más que la replicación en dossistemas de cultivo celular. Biomedica. 2009; 29: 192-3

How to Cite
Prada-Arismendy, J., & Castellanos, J. E. (2024). Real time PCR. Application in dengue studies. Colombia Medica, 42(2), 243–258. https://doi.org/10.25100/cm.v42i2.778 (Original work published June 14, 2011)
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