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<p>The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has triggered an extraordinary global disturbance. According toWHO epidemiological reports (June 2025) indicate the COVID-19 pandemic has resulted in approximately 778&#xa0;million confirmed infections and 7&#xa0;million attributable fatalities globally. The swift mutation of SARS-CoV-2 and the arrival of multiple variants highlight the critical need for rapid, accurate diagnostic assays to monitor and mitigate public health risks. This study presents the development and validation of a multiplex reverse transcription-PCR (RT-PCR) assay designed for sensitive and specific SARS-CoV-2 detection. The assay simultaneously targets two highly conserved viral genes (ORF1ab and N) along with human RNase P as an internal control. Comprehensive analytical validation using plasmid standards (1.5–150,000 copies/µL) confirmed a limit of detection (LOD) of 1.5 copies/µL, high amplification efficiency (97.7–102.2%), and excellent reproducibility (coefficient of variation &lt; 5%). Clinical evaluation with 193 patient samples (<i>N</i> = 193:102 positive, 91 negative) demonstrated 100% concordance with commercial assays (diagnostic sensitivity/specificity: 100%), with results achievable in &lt; 90&#xa0;min. This rigorously validated, platform-independent assay provides a cost-effective and efficient diagnostic option, meeting WHO target product profile requirements and is suitable for use in both high-throughput and resource-limited laboratory settings.</p>

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Development and Validation of a Streamlined Multiplex RT-PCR Assay for Accurate SARS-CoV-2 Detection in Diverse Healthcare Environments

  • 期刊论文

  • 发表日期：

    2026年2月27日

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  • Kauser Banu,
  • Bhairab Mondal,
  • K. G. Lekhana,
  • N. Raviteja,
  • C. Sai Sudha,
  • S. Madan Kumar,
  • H. Raju

摘要

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has triggered an extraordinary global disturbance. According toWHO epidemiological reports (June 2025) indicate the COVID-19 pandemic has resulted in approximately 778 million confirmed infections and 7 million attributable fatalities globally. The swift mutation of SARS-CoV-2 and the arrival of multiple variants highlight the critical need for rapid, accurate diagnostic assays to monitor and mitigate public health risks. This study presents the development and validation of a multiplex reverse transcription-PCR (RT-PCR) assay designed for sensitive and specific SARS-CoV-2 detection. The assay simultaneously targets two highly conserved viral genes (ORF1ab and N) along with human RNase P as an internal control. Comprehensive analytical validation using plasmid standards (1.5–150,000 copies/µL) confirmed a limit of detection (LOD) of 1.5 copies/µL, high amplification efficiency (97.7–102.2%), and excellent reproducibility (coefficient of variation < 5%). Clinical evaluation with 193 patient samples (N = 193:102 positive, 91 negative) demonstrated 100% concordance with commercial assays (diagnostic sensitivity/specificity: 100%), with results achievable in < 90 min. This rigorously validated, platform-independent assay provides a cost-effective and efficient diagnostic option, meeting WHO target product profile requirements and is suitable for use in both high-throughput and resource-limited laboratory settings.