A Two-tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/subtypes

Overview

Journal Biomed Res Int

Publisher Wiley

Specialties Biomedical Engineering
Biotechnology
General Medicine

Date 2013 Aug 29

PMID 23984344

Citations 12

Authors

Jin Li

Shunxiang Qi

Chen Zhang

Xiumei Hu

Hongwei Shen

Mengjie Yang

Ji Wang

Miao Wang

Wenbo Xu

Xuejun Ma

Affiliations

Soon will be listed here.

Abstract

There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20-200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.

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References

Rand K, Rampersaud H, Houck H . Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP. J Clin Microbiol. 2011; 49(7):2449-53. PMC: 3147849. DOI: 10.1128/JCM.02582-10. View

Fauci A, Touchette N, Folkers G . Emerging infectious diseases: a 10-year perspective from the National Institute of Allergy and Infectious Diseases. Emerg Infect Dis. 2005; 11(4):519-25. PMC: 3320336. DOI: 10.3201/eid1104.041167. View

Pabbaraju K, Tokaryk K, Wong S, Fox J . Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections. J Clin Microbiol. 2008; 46(9):3056-62. PMC: 2546709. DOI: 10.1128/JCM.00878-08. View

Nolte F, Marshall D, Rasberry C, Schievelbein S, Banks G, Storch G . MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses. J Clin Microbiol. 2007; 45(9):2779-86. PMC: 2045250. DOI: 10.1128/JCM.00669-07. View

Hu X, Zhang Y, Zhou X, Xu B, Yang M, Wang M . Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay. J Clin Microbiol. 2011; 50(2):288-93. PMC: 3264198. DOI: 10.1128/JCM.05828-11. View

Bibby D, McElarney I, Breuer J, Clark D . Comparative evaluation of the Seegene Seeplex RV15 and real-time PCR for respiratory virus detection. J Med Virol. 2011; 83(8):1469-75. PMC: 7166927. DOI: 10.1002/jmv.22125. View

Gadsby N, Hardie A, Claas E, Templeton K . Comparison of the Luminex Respiratory Virus Panel fast assay with in-house real-time PCR for respiratory viral infection diagnosis. J Clin Microbiol. 2010; 48(6):2213-6. PMC: 2884497. DOI: 10.1128/JCM.02446-09. View

Xu Z, Cheng W, Li B, Li J, Lan B, Duan Z . Development of a real-time PCR assay for detecting and quantifying human bocavirus 2. J Clin Microbiol. 2011; 49(4):1537-41. PMC: 3122821. DOI: 10.1128/JCM.00196-10. View

Henrickson K . Advances in the laboratory diagnosis of viral respiratory disease. Pediatr Infect Dis J. 2004; 23(1 Suppl):S6-10. DOI: 10.1097/01.inf.0000108187.63151.ea. View

10.

Brittain-Long R, Westin J, Olofsson S, Lindh M, Andersson L . Prospective evaluation of a novel multiplex real-time PCR assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate. J Clin Virol. 2010; 47(3):263-7. PMC: 7108433. DOI: 10.1016/j.jcv.2009.12.010. View

11.

Merante F, Yaghoubian S, Janeczko R . Principles of the xTAG respiratory viral panel assay (RVP Assay). J Clin Virol. 2008; 40 Suppl 1:S31-5. PMC: 7128903. DOI: 10.1016/S1386-6532(07)70007-0. View

12.

Barenfanger J, Drake C, Leon N, Mueller T, Troutt T . Clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study. J Clin Microbiol. 2000; 38(8):2824-8. PMC: 87120. DOI: 10.1128/JCM.38.8.2824-2828.2000. View

13.

Li H, McCormac M, Estes R, Sefers S, Dare R, Chappell J . Simultaneous detection and high-throughput identification of a panel of RNA viruses causing respiratory tract infections. J Clin Microbiol. 2007; 45(7):2105-9. PMC: 1932978. DOI: 10.1128/JCM.00210-07. View

14.

Qin M, Wang D, Huang F, Nie K, Qu M, Wang M . Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer. J Virol Methods. 2010; 168(1-2):255-8. DOI: 10.1016/j.jviromet.2010.04.031. View

15.

Tabone T, Mather D, Hayden M . Temperature switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs. BMC Genomics. 2009; 10:580. PMC: 2795770. DOI: 10.1186/1471-2164-10-580. View

16.

Krunic N, Yager T, Himsworth D, Merante F, Yaghoubian S, Janeczko R . xTAG RVP assay: analytical and clinical performance. J Clin Virol. 2008; 40 Suppl 1:S39-46. PMC: 7129559. DOI: 10.1016/S1386-6532(07)70009-4. View

17.

Calvo C, Garcia-Garcia M, Blanco C, Vazquez M, Frias M, Perez-Brena P . Multiple simultaneous viral infections in infants with acute respiratory tract infections in Spain. J Clin Virol. 2008; 42(3):268-72. PMC: 7108242. DOI: 10.1016/j.jcv.2008.03.012. View

18.

Li J, Mao N, Zhang C, Yang M, Wang M, Xu W . The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes. BMC Infect Dis. 2012; 12:189. PMC: 3462154. DOI: 10.1186/1471-2334-12-189. View

19.

Bruijnesteijn van Coppenraet L, Swanink C, van Zwet A, Nijhuis R, Schirm J, Wallinga J . Comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems. J Virol Methods. 2010; 169(1):188-92. PMC: 7119677. DOI: 10.1016/j.jviromet.2010.07.032. View

20.

Cilla G, Onate E, Perez-Yarza E, Montes M, Vicente D, Perez-Trallero E . Viruses in community-acquired pneumonia in children aged less than 3 years old: High rate of viral coinfection. J Med Virol. 2008; 80(10):1843-9. PMC: 7166914. DOI: 10.1002/jmv.21271. View