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

Arch Virol. 2019 Jul;164(7):1843-1850. doi: 10.1007/s00705-019-04230-z. Epub 2019 May 3.

Abstract

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.

MeSH terms

  • Child
  • Child, Preschool
  • Female
  • Humans
  • Infant
  • Male
  • Molecular Diagnostic Techniques / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • RNA, Viral / analysis*
  • Real-Time Polymerase Chain Reaction
  • Recombinases
  • Respiratory Syncytial Virus Infections / diagnosis*
  • Respiratory Syncytial Virus, Human / genetics*
  • Respiratory Syncytial Virus, Human / isolation & purification
  • Respiratory Tract Infections / diagnosis*
  • Respiratory Tract Infections / virology
  • Reverse Transcription
  • Sensitivity and Specificity

Substances

  • RNA, Viral
  • Recombinases