Evaluation of a measles virus multiplex, triple-target real-time RT-PCR in three specimen matrices at a U.S. academic medical center

M. Jana Broadhurst, Natasha Garamani, Zoe Hahn, Becky Jiang, Jenna Weber, Chun Hong Huang, Malaya K. Sahoo, Jason Kurzer, Catherine A. Hogan, Benjamin A. Pinsky

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Background: Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available. Methods: We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes. Results: The MeV triple-target rRT-PCR was tested against serial dilutions (7.0−2.0 log10 copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log10 copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus. Conclusions: The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions.

Original languageEnglish (US)
Article number104757
JournalJournal of Clinical Virology
StatePublished - Mar 2021
Externally publishedYes


  • Measles
  • Molecular methods

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases


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