Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control
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.
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.
- 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. https://doi.org/10.1016/S1473-3099(18)30292-5 Google Scholar
- 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
- 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
- 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
- 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
- 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
- 26.Rådström P, Löfström C, Lövenklev M, Knutsson R, Wolffs P (2003) Strategies for overcoming PCR inhibition. CSH Protoc 2008:pdb.top20Google Scholar
- 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