title: Assay optimization for molecular detection of Zika virus.
abstract: OBJECTIVE
  To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection.
  
  
  METHODS
  We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays' target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays.
  
  
  RESULTS
  Oligonucleotides of the published real-time RT-PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104RNA copies/mL of blood and 2 × 104RNA copies/mL of urine.
  
  
  CONCLUSIONS
  We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Victor M
authors.initials: VM
authors.lastname: Corman
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Andrea
authors.initials: A
authors.lastname: Rasche
authors.affiliation: UMR EPV Emergence des Pathologies Virales, Aix Marseille Université, Marseille, France .
authors.forename: Cecile
authors.initials: C
authors.lastname: Baronti
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Souhaib
authors.initials: S
authors.lastname: Aldabbagh
authors.affiliation: Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Hamburg, Germany .
authors.forename: Daniel
authors.initials: D
authors.lastname: Cadar
authors.affiliation: Erasmus MC, Department of Viroscience, Rotterdam, Netherlands .
authors.forename: Chantal Bem
authors.initials: CB
authors.lastname: Reusken
authors.affiliation: Erasmus MC, Department of Viroscience, Rotterdam, Netherlands .
authors.forename: Suzan D
authors.initials: SD
authors.lastname: Pas
authors.affiliation: Department of Infectious Diseases, University of Amsterdam, Amsterdam, Netherlands .
authors.forename: Abraham
authors.initials: A
authors.lastname: Goorhuis
authors.affiliation: Clinical Virology Laboratory, University of Amsterdam, Amsterdam, Netherlands .
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.affiliation: Clinical Virology Laboratory, University of Amsterdam, Amsterdam, Netherlands .
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Beate M
authors.initials: BM
authors.lastname: Kümmerer
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Tobias
authors.initials: T
authors.lastname: Bleicker
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brünink
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Monika
authors.initials: M
authors.lastname: Eschbach-Bludau
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Anna M
authors.initials: AM
authors.lastname: Eis-Hübinger
authors.affiliation: Erasmus MC, Department of Viroscience, Rotterdam, Netherlands .
authors.forename: Marion P
authors.initials: MP
authors.lastname: Koopmans
authors.affiliation: Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Hamburg, Germany .
authors.forename: Jonas
authors.initials: J
authors.lastname: Schmidt-Chanasit
authors.affiliation: Department of Infectious Diseases, University of Amsterdam, Amsterdam, Netherlands .
authors.forename: Martin P
authors.initials: MP
authors.lastname: Grobusch
authors.affiliation: UMR EPV Emergence des Pathologies Virales, Aix Marseille Université, Marseille, France .
authors.forename: Xavier
authors.initials: X
authors.lastname: de Lamballerie
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Sigmund Freud-Str. 25, 53127 Bonn, Germany .
authors.forename: Jan Felix
authors.initials: JF
authors.lastname: Drexler
mesh_terms: D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012680:Sensitivity and Specificity; D000071243:Zika Virus Infection / Q000175:diagnosis*