Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

Nat Commun. 2018 Apr 11;9(1):1391. doi: 10.1038/s41467-018-03772-1.


The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 103 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calibration
  • Chikungunya Fever / blood
  • Chikungunya Fever / diagnosis*
  • Chikungunya Fever / virology
  • Chikungunya virus / genetics*
  • Chikungunya virus / isolation & purification
  • Dengue / blood
  • Dengue / diagnosis*
  • Dengue / virology
  • Dengue Virus / genetics*
  • Dengue Virus / isolation & purification
  • Humans
  • Limit of Detection
  • Multiplex Polymerase Chain Reaction / methods*
  • Multiplex Polymerase Chain Reaction / standards
  • Reagent Kits, Diagnostic
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Zika Virus / genetics*
  • Zika Virus / isolation & purification
  • Zika Virus Infection / diagnosis*
  • Zika Virus Infection / urine
  • Zika Virus Infection / virology


  • Reagent Kits, Diagnostic