Measles ( rubeola) virus qualitative real-time reverse transcription–polymerase chain reaction assay: development and clinical validation on an automated workflow

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Abstract
Introduction: Detection of Measles virus (MeV) RNA is an important method for confirming infection. This study described the clinical validation of a qualitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) assay for MeV detection. Methods: A real-time RT-PCR assay was developed and validated using primers and probes previously reported in the literature. The assay was evaluated using reference materials, cultured virus, and previously tested clinical throat swab samples. An automated workflow was established for RNA extraction, RT-PCR amplification, and result reporting using robotic platforms and a real-time PCR instrument. The assay targets the measles nucleoprotein (N) and hemagglutinin (H) genes, with human RNase P gene used as an internal control. Results: The MeV RT-PCR assay has a limit of detection of 500 genome copies per milliliter. The assay was tested against 85 different samples: 50 positive clinical samples and reference materials and 35 negative clinical samples or reference materials. All expected results were correctly identified, giving 100% sensitivity (50/50) and specificity (35/35). The assay showed 100% interassay and intra-assay reproducibility. No cross-reactivity was observed with other respiratory viruses or common bacterial and fungal pathogens. Discussion: This automated measles RT-PCR assay demonstrated high accuracy, sensitivity, specificity, and reproducibility.
Funding Information
  • Professorship in Biomedical Science

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