In-house standardization and validation of a multiplex RT-PCR assay for the detection of 13 respiratory viruses

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Hernán Vargas
Ángela Diaz
Yamile Celis
Liliana Díaz
Sandra Gómez
Jenny Sánchez
Carlos Golijow
Patricia Arce


Background.  Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, such as culture and antigen detection. Objective. Standardization and validation of a multiplex real-time PCR assay for the detection of 13 respiratory viruses. Methods. The assay was validated using RNA control targets and comparing results to single-target PCR’s. Results. Using RNA controls the multiplex format was found to be as sensitive and specific as the single-target PCRs, and no competition was observed between targets.  The efficiencies for most of the reactions were approximately 90%, but a lower efficiency was found for Parainfluenza 2 with a rate of amplification in each cycle of 86.63%. On the other hand, a higher efficiency was observed in respiratory syncytial virus A and respiratory syncytial virus B ((93.07% each).  Conclusion: This multiplex RT-PCR format shows an adequate efficiency, demonstrating an excellent sensitivity, specificity and repeatability for all the studied respiratory viruses. 

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1. Salez N., Vabret A., Leruez M., Andreoletti L., Carrat F. et al. (2015). Evaluation of four Commercial Multiplex Molecular Test for the Diagnosis of acute respiratory Infections. Plos One 10 (Vol 6); 1-17.
2. Sanghavi S, Bullota A, Husain S, Rinaldo C. (2012). Clinical evaluation of multiplex real time PCR panels for rapid detection of respiratory viral infections. Journal of medical Virology. (Vol 84); 162-169.
3. Ferkol T. Schraufnagel, D. (2014). The Global Burden of respiratory disease. Ann Am Thorac Soc (Vol 11); 404–406.
4. Watanabe A, Carraro E, Camarago C, Puerari D, Guatura S. et al. (2013). Human adenovirus detection among immunocompetent and immunocompromised patients presenting acute respiratory infection. Revista da Sociedad Brasileira de Medicine Tropical 46 (2); 161-165.
5. Pavia A. (2011). Viral Infections of the lower respiratory tract; Old viruses, new viruses, and the role of diagnosis. Clin Infect Dis. (Vol 4); 284- 289.
6. Krause J, Panning M, Hengel H, Henneke P. (2014). The role of multiplex PCR in respiratory tract infections in children. Dtsch Arztebl Int; (111); 639-45.
7. Weissenbacher MC, MM Ávila. Los virus como causa de IRA alta y baja en niños: características generales y diagnóstico, in Infecciones respiratorias en niños 1999, OPS - OMS: http://; 89 - 105.
8. Liolios L, Jenney A, Spelman D, Kotsimbos T, Catton M. et al. (2001). Comparison of a Multiplex Reverse TranscriptionPCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens. J Clin Microbiol, 39(8); 2779-2783.
9. Syrmis, MW, Whiley DM, Thomas M, Mackay I, Williamson J. et al. (2004). A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples. J. Mol. Diagn. 6(2); 125–131.
10. Bellau-Pujol S, Vabret A, Legrand L, Dina J, Gouarin S. et al. (2005). Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses. J Virol Methods.
126(1–2); 53-63.
11. M”hadheb M, Harrabi M, Souii A, Jrad N, Gharbi J. (2015). Multiplex RT-PCR and Indirect Immnofluorescence Assay for Detection and Subtyping of human Influenza Virus in Tunisia. Curr Microbiol. (70); 324-329.
12. Fuller JA, Kariuki M, Bigogo G, Aura B, Maurice OO. et al. (2013). Association of the CT Values of Real-Time PCR of Viral Upper Respiratory Tract Infection With Clinical Severity, Kenya. Journal of Medical Virology. (Vol 85); 924-932.
13. Sidoti F, Bergalllo M, Costa C, Cavallo R. (2013). Alternative Molecular Test for Virological Diagnosis. Mol Biotechnol. (53); 352-362.
14. Pretorius M. Madhi S, Cohen C, Naidoo, Groome M. et al. (2012). Respiratory Viral Coinfections Identified by a 10-Plex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay in Patients Hospitalized With Severe Acute Respiratory illness-South Africa, 2009-2010. Journal of Infectious Diseases (206); S159-S165.
15. Martins RB, Carney S, Goldemberg D, Bonine L, Cruz L. et al. (2014). Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection. Mem. Inst. Oswaldo Cruz. Rio de Jainero. Vol (109) (6); 716-721.
16. Heim A, Ebnet C, Harste G, Pring P. (2003). Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J Med Virol. 70(2); 228-239.
17. Gunson R, Collins T, Carman W. (2005). Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 33(4); 341-344.
18. Lu X, Chittaganpitch M, Olsen S, Mackay I, Sloots T. et al. (2006). Real-Time PCR Assays for Detection of Bocavirus in Human Specimens. J Clin Microbiol. 44(9); 3231-3235.
19. Boivin G, Cote S, Déry P, Serres G, Bergeron M. (2004). Multiplex Real-Time PCR Assay for Detection of Influenza and Human Respiratory Syncytial Viruses. Journal of clinical Microbiology. (Vol 42); 45-51.
20. Rheem I, Park J, Kim T, Wan J. (2012) Evaluation of a Multiplex Real-time PCR Assay for the Detection of Respiratory Viruses in Clinical Specimens. Ann Lab Med; 32;399-406.
21. Dorak, T. (2006). Data analysis and reporting, in Real-Time PCR.Taylor & Francis; 39-61.
22. Fraga D., M.T., Fenster S. 2008. Real-time PCR, in Current protocols: essential laboratory techniques, W.E.A. Gallagher S. R. John Wiley & Sons, Inc., Hoboken, NJ; 10.3.1–10.3.34.
23. Millan J, Yunda L. An Open-Access Web-based medical image atlas for collaborative Medical image sharing, processing, Web Semantic searching and analysis with uses in medical training,
research and second opinion of cases. Nova. 2014;12(22):14350



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