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High-throughput Detection Method for Influenza Virus
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Evaluation of PCR-Based H5N1 Influenza Detection Methods in Milk from an Interlaboratory Comparison Study

Megan R Miller1, Kirstin Frost2, Emily L Smith3

  • 1Center for Veterinary Medicine, U.S. Food and Drug Administration, 8401 Muirkirk Road, Laurel, MD 20708, USA.

Journal of Food Protection
|May 8, 2026
PubMed
Summary

Highly pathogenic avian influenza (HPAI) H5N1 virus detection in U.S. dairy cattle milk shows significant variability. Method selection impacts real-time reverse transcription quantitative polymerase chain reaction (rRT-qPCR) assay sensitivity for H5N1 testing in milk.

Keywords:
H5N1Interlaboratory comparisonMilkrRT-qPCR

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Published on: November 22, 2017

Area of Science:

  • Veterinary Virology
  • Food Safety Science
  • Molecular Diagnostics

Background:

  • Highly pathogenic avian influenza (HPAI) H5N1 has been detected in U.S. dairy cattle since March 2024.
  • Infectious H5N1 virus shed in milk poses a risk to animal and human health.
  • Real-time reverse transcription quantitative polymerase chain reaction (rRT-qPCR) assays are crucial for H5N1 detection in milk for animal diagnostics and food safety.

Purpose of the Study:

  • To evaluate the performance and variability of PCR-based H5N1 detection assays in milk samples.
  • To compare the sensitivity of different rRT-qPCR methods used by various laboratories.
  • To identify factors influencing H5N1 detection rates in milk.

Main Methods:

  • An interlaboratory comparison exercise involving 45 participants from government, academic, and commercial laboratories.
  • Testing of blinded milk samples spiked with inactivated H5N1 virus at concentrations from 85 to 170,000 genome copies/100 μL.
  • Analysis of detection rate (ROD), Ct values, and limits of detection (LOD50) based on extraction and PCR kit combinations.

Main Results:

  • High detection rates (≥99%) for high H5N1 concentrations (17,000 and 170,000 copies/100 μL).
  • Significantly decreased detection at lower concentrations: 34% at 1,700 copies/100 μL and 24% at 85 copies/100 μL.
  • Substantial variability in H5N1 detection across laboratories and methods, with some methods showing 0% detection at low levels.

Conclusions:

  • Method selection significantly impacts the sensitivity of H5N1 detection in milk.
  • There is a critical need for protocol evaluation and quality control measures for H5N1 testing in milk.
  • Findings have implications for animal and human health surveillance and emergency response strategies.