Archives of Virology

, Volume 164, Issue 7, pp 1843–1850 | Cite as

Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control

  • Juju Qi
  • Xinna Li
  • Yi Zhang
  • Xinxin Shen
  • Guowei Song
  • Jing Pan
  • Tao Fan
  • Ruihuan Wang
  • Lixin LiEmail author
  • Xuejun MaEmail author
Brief Report


Human respiratory syncytial virus (RSV) is a common viral pathogen that causes lower respiratory tract infections in infants and children globally. In this study, we developed a duplex reverse transcription recombinase-aided amplification (duplex-rtRAA) assay containing an internal control in a single closed tube for the detection of human RSV. The internal control in the amplification effectively eliminated false-negative results and ensured the accuracy of the duplex-rtRAA system. We first developed and evaluated a universal singleplex-rtRAA assay for RSV. The sensitivity of this assay for RSV was determined as 4.4 copies per reaction, and the specificity was 100%. Next, a duplex-rtRAA assay with an internal control was established. The sensitivity of the duplex-rtRAA assay approached 5.0 copies per reaction, and no cross-reaction with other common respiratory viruses was observed. The two detection methods (singleplex-rtRAA and duplex-rtRAA) developed in this study were used to test 278 clinical specimens, and the results showed absolute consistency with RSV RT-qPCR analysis, demonstrating 100% diagnostic sensitivity and specificity. These data indicate that the duplex-rtRAA has great potential for the rapid detection of RSV with a high sensitivity.



We acknowledge the Children’s Hospital of Hebei Province, China, for providing clinical specimens.


This work was supported by grants from China Mega-Project for Infectious Disease (2018ZX10711001, 2017ZX10104001 and 2017ZX10302301-004-002). A Chinese patent is pending.

Compliance with ethical standards

Conflict of interest

All the authors approved the final manuscript and they have no conflict of interest to declare.

Ethical approval

All aspects of the study were performed in accordance with national ethics regulations and approved by the Institutional Review Boards of National Institute for Viral Disease Control and Prevention, Center for Disease Control and Prevention of China.

Supplementary material

705_2019_4230_MOESM1_ESM.doc (48 kb)
Supplementary material 1 (DOC 47 kb)
705_2019_4230_MOESM2_ESM.doc (46 kb)
Supplementary material 2 (DOC 46 kb)


  1. 1.
    Ye S, Wang T (2018) Laboratory epidemiology of respiratory viruses in a large children’s hospital. Medicine 97:e11385CrossRefGoogle Scholar
  2. 2.
    Englund JA, Sullivan CJ, Jordan MC, Dehner LP, Vercellotti GM, Balfour HH (1988) Respiratory syncytial virus infection in immunocompromised adults. Ann Intern Med 109:203CrossRefGoogle Scholar
  3. 3.
    Falsey AR, Treanor JJ, Betts RF, Walsh EE (1992) Viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings. J Am Geriatr Soc 40:115–119CrossRefGoogle Scholar
  4. 4.
    Chen J, Hu P, Zhou T, Zheng T, Zhou L, Jiang C, Pei X (2018) Epidemiology and clinical characteristics of acute respiratory tract infections among hospitalized infants and young children in Chengdu, West China, 2009–2014. BMC Pediatr 18:216CrossRefGoogle Scholar
  5. 5.
    Mufson MA, Örvell C, Rafnar B, Norrby E (1985) Two distinct subtypes of human respiratory syncytial (RS) virus. Virus Res 3:2111CrossRefGoogle Scholar
  6. 6.
    Mazur NI, Higgins D, Nunes MC, Melero JA, Langedijk AC, Horsley N, Buchholz UJ, Openshaw PJ, Mclellan JS, Englund JA (2018) The respiratory syncytial virus vaccine landscape: lessons from the graveyard and promising candidates. Lancet Infect Dis. Google Scholar
  7. 7.
    Barenfanger J, Drake C, Leon N, Mueller T, Troutt T (2000) Clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study. J Clin Microbiol 38:2824Google Scholar
  8. 8.
    Rogers BB, Shankar P, Jerris RC, Kotzbauer D, Anderson EJ, Watson JR, O’Brien LA, Uwindatwa F, Mcnamara K, Bost JE (2015) Impact of a rapid respiratory panel test on patient outcomes. Arch Pathol Lab Med 139:636CrossRefGoogle Scholar
  9. 9.
    Jonathan N (2006) Diagnostic utility of BINAX NOW RSV—an evaluation of the diagnostic performance of BINAX NOW RSV in comparison with cell culture and direct immunofluorescence. Ann Clin Microbiol Antimicrob 5:13CrossRefGoogle Scholar
  10. 10.
    Reis AD, Fink MC, Machado CM, Paz Jde PJP Jr, Oliveira RR, Tateno AF, Machado AF, Cardoso MR, Pannuti CS (2008) Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants. Revista Do Instituto De Medicina Tropical De São Paulo 50:37CrossRefGoogle Scholar
  11. 11.
    Borek AP, Clemens SH, Gaskins VK, Aird DZ, Valsamakis A (2006) Respiratory syncytial virus detection by Remel Xpect, Binax now RSV, direct immunofluorescent staining, and tissue culture. J Clin Microbiol 44:1105–1107CrossRefGoogle Scholar
  12. 12.
    Falsey AR, Formica MA, Walsh EE (2002) Diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-PCR to viral culture and serology in adults with respiratory illness. J Clin Microbiol 40:817–820CrossRefGoogle Scholar
  13. 13.
    Bonroy C, Vankeerberghen A, Boel A, Beenhouwer HD (2010) Use of a multiplex real-time PCR to study the incidence of human metapneumovirus and human respiratory syncytial virus infections during two winter seasons in a Belgian paediatric hospital. Clin Microbiol Infect 13:504–509CrossRefGoogle Scholar
  14. 14.
    Mentel R, Wegner U, Bruns R, Gürtler L (2003) Real-time PCR to improve the diagnosis of respiratory syncytial virus infection. J Med Microbiol 52:893–896CrossRefGoogle Scholar
  15. 15.
    Yan L, Zhou J, Zheng Y, Gamson AS, Roembke BT, Nakayama S, Sintim HO (2014) Isothermal amplified detection of DNA and RNA. Mol Biosyst 10:970–1003CrossRefGoogle Scholar
  16. 16.
    Compton J (1991) Nucleic acid sequence-based amplification. Nature 350:91–92CrossRefGoogle Scholar
  17. 17.
    Deiman B, Schrover C, Moore C, Westmoreland D, Wiel PVD (2007) Rapid and highly sensitive qualitative real-time assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology. J Virol Methods 146:29–35CrossRefGoogle Scholar
  18. 18.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63CrossRefGoogle Scholar
  19. 19.
    Hoos J, Peters RM, Tabatabai J, Grulichhenn J, Schnitzler P, Pfeil J (2017) Reverse-transcription loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs. J Virol Methods 242:53–57CrossRefGoogle Scholar
  20. 20.
    Vincent M, Yan X, Kong H (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep 5:795CrossRefGoogle Scholar
  21. 21.
    Piepenburg O, Williams C, Stemple D (2006) Na: DNA detection using recombination proteins—art. no. e204. PLoS Biol 4:1115–1121CrossRefGoogle Scholar
  22. 22.
    Bei L, Cheng HR, Yan QF, Huang ZJ, Shen GF, Zhang ZF, Yinv LI, Deng ZX, Lin M (2010) Recombinase-aid amplification: a novel technology of in vitro rapid nucleic acid amplification. Sci Sin 40:983–988Google Scholar
  23. 23.
    Zhang X, Guo L, Ma R, Cong L, Wu Z, Wei Y, Xue S, Zheng W, Tang S (2017) Rapid detection of Salmonella with recombinase aided amplification. J Microbiol Methods 139:202–204CrossRefGoogle Scholar
  24. 24.
    Wang J, Xu Z, Niu P, Zhang C, Zhang J, Guan L, Kan B, Duan Z, Ma X (2014) A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea. Biomed Res Int 2014:648520Google Scholar
  25. 25.
    Sanghavi SK, Arlene B, Shahid H, Rinaldo CR (2011) Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections. J Med Virol 84:162–169CrossRefGoogle Scholar
  26. 26.
    Rådström P, Löfström C, Lövenklev M, Knutsson R, Wolffs P (2003) Strategies for overcoming PCR inhibition. CSH Protoc 2008:pdb.top20Google Scholar
  27. 27.
    Chen C, Li XN, Li GX, Zhao L, Duan SX, Yan TF, Feng ZS, Ma XJ (2018) Use of a rapid reverse-transcription recombinase aided amplification assay for respiratory syncytial virus detection. Diagn Microbiol Infect Dis 90:90–95CrossRefGoogle Scholar
  28. 28.
    Yan TF, Li XN, Wang L, Chen C, Duan SX, Qi JJ, Li LX, Ma XJ (2018) Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA. Arch Virol 163:1455–1461CrossRefGoogle Scholar
  29. 29.
    Duan S, Li G, Li X, Chen C, Yan T, Qiu F, Zhao L, Zhao M, Wang L, Feng Z (2018) A probe directed recombinase amplification assay for detection of MTHFR A1298C polymorphism associated with congenital heart disease. Biotechniques 64:211CrossRefGoogle Scholar
  30. 30.
    Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 42:1863–1868CrossRefGoogle Scholar
  31. 31.
    Higgins O, Clancy E, Forrest MS, Piepenburg O, Cormican M, Boo TW, O’Sullivan N, Mcguinness C, Cafferty D, Cunney R (2018) Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection. Anal Biochem 546:10–16CrossRefGoogle Scholar
  32. 32.
    Mu Y, Zeng J, Chen Q, Jia L, Wang L, Yao F, Meng C, He Z, Zhang C, Ming X (2014) New method for the visual detection of human respiratory syncytial virus using reverse transcription loop-mediated amplification. J Virol Methods 206:84–88CrossRefGoogle Scholar
  33. 33.
    Eugene-Ruellan G, Freymuth F, Bahloul C, Badrane H, Vabret A, Tordo N (1998) Detection of respiratory syncytial virus A and B and parainfluenzavirus 3 sequences in respiratory tracts of infants by a single PCR with primers targeted to the L-polymerase gene and differential hybridization. J Clin Microbiol 36:796Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Juju Qi
    • 1
    • 2
  • Xinna Li
    • 2
  • Yi Zhang
    • 2
  • Xinxin Shen
    • 2
  • Guowei Song
    • 3
  • Jing Pan
    • 3
  • Tao Fan
    • 1
    • 2
  • Ruihuan Wang
    • 2
  • Lixin Li
    • 1
    • 3
    Email author
  • Xuejun Ma
    • 2
    Email author
  1. 1.Hebei Medical UniversityShijiazhuangChina
  2. 2.Key Laboratory for Medical Virology, National Health and Family Planning CommissionNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and PreventionBeijingChina
  3. 3.Myasthenia Gravis Research InstituteThe First Hospital of ShijiazhuangShijiazhuangChina

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