Development and Evaluation of a Four-tube Real Time Multiplex PCR Assay Covering Fourteen Respiratory Viruses, and Comparison to Its Corresponding Single Target Counterparts

Overview

Journal J Clin Virol

Specialty Microbiology

Date 2011 May 17

PMID 21571585

Citations 48

Authors

Rogier R Jansen

Janke Schinkel

Sylvie Koekkoek

Dasja Pajkrt

Marcel Beld

Menno D de Jong

Richard Molenkamp

Affiliations

Soon will be listed here.

Abstract

Background: Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, like culture and antigen detection. However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR's is limited for most published respiratory multiplex real time PCR assays.

Objectives: Development and extensive analysis of an internally controlled multiplex real time rt-PCR for detection of respiratory viruses.

Study Design: The assay was validated in comparison to single-target PCR's using plasmid targets and prospectively collected nasopharyngeal aspirates.

Results: Using plasmid targets the multiplex format was found to be as least as sensitive and specific as the single-target PCR and no competition was observed when different targets were present at different amounts in one tube. Clinical validation showed high concordance for all viruses tested except for samples with low levels of enterovirus.

Conclusion: This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus.

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References

Molenkamp R, van der Ham A, Schinkel J, Beld M . Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics. J Virol Methods. 2007; 141(2):205-11. DOI: 10.1016/j.jviromet.2006.12.007. View

Lu X, Holloway B, Dare R, Kuypers J, Yagi S, Williams J . Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses. J Clin Microbiol. 2007; 46(2):533-9. PMC: 2238069. DOI: 10.1128/JCM.01739-07. View

Mahony J, Blackhouse G, Babwah J, Smieja M, Buracond S, Chong S . Cost analysis of multiplex PCR testing for diagnosing respiratory virus infections. J Clin Microbiol. 2009; 47(9):2812-7. PMC: 2738055. DOI: 10.1128/JCM.00556-09. View

Brittain-Long R, Nord S, Olofsson S, Westin J, Anderson L, Lindh M . Multiplex real-time PCR for detection of respiratory tract infections. J Clin Virol. 2007; 41(1):53-6. PMC: 7172039. DOI: 10.1016/j.jcv.2007.10.029. View

Tapparel C, Cordey S, van Belle S, Turin L, Lee W, Regamey N . New molecular detection tools adapted to emerging rhinoviruses and enteroviruses. J Clin Microbiol. 2009; 47(6):1742-9. PMC: 2691104. DOI: 10.1128/JCM.02339-08. View

Puppe W, Weigl J, Aron G, Grondahl B, Schmitt H, Niesters H . Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract pathogens. J Clin Virol. 2004; 30(2):165-74. DOI: 10.1016/j.jcv.2003.10.003. View

Wang W, Ren P, Sheng J, Mardy S, Yan H, Zhang J . Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays. J Virol Methods. 2009; 162(1-2):40-5. PMC: 7119720. DOI: 10.1016/j.jviromet.2009.07.004. View

Beld M, Minnaar R, Weel J, Sol C, Damen M, van der Avoort H . Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control. J Clin Microbiol. 2004; 42(7):3059-64. PMC: 446274. DOI: 10.1128/JCM.42.7.3059-3064.2004. View

van Elden L, van Kraaij M, Nijhuis M, Hendriksen K, Dekker A, Rozenberg-Arska M . Polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia. Clin Infect Dis. 2001; 34(2):177-83. PMC: 7109912. DOI: 10.1086/338238. View

10.

Freymuth F, Vabret A, Cuvillon-Nimal D, Simon S, Dina J, Legrand L . Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness. J Med Virol. 2006; 78(11):1498-504. PMC: 7159369. DOI: 10.1002/jmv.20725. View

11.

Tapparel C, Junier T, Gerlach D, Van-Belle S, Turin L, Cordey S . New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses. Emerg Infect Dis. 2009; 15(5):719-26. PMC: 2687021. DOI: 10.3201/eid1505.081286. View

12.

van de Pol A, van Loon A, Wolfs T, Jansen N, Nijhuis M, Breteler E . Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients with respiratory symptoms. J Clin Microbiol. 2007; 45(7):2260-2. PMC: 1932984. DOI: 10.1128/JCM.00848-07. View

13.

Scheltinga S, Templeton K, Beersma M, Claas E . Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR. J Clin Virol. 2005; 33(4):306-11. PMC: 7185544. DOI: 10.1016/j.jcv.2004.08.021. View

14.

Gunson R, Collins T, Carman W . Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. J Clin Virol. 2005; 33(4):341-4. PMC: 7108440. DOI: 10.1016/j.jcv.2004.11.025. View