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Impact of Capsid and Genomic Integrity Tests on Norovirus Extraction Recovery Rates.

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Summary

This study assessed methods to distinguish infectious from non-infectious human norovirus (HuNoV). RNase and PMAxx treatments effectively reduced detection of inactivated HuNoV using RT-qPCR, minimizing false positives.

Keywords:
ISO 15216-1:2017RNasecapsid integritychelating agentintercalating dyelong RT-qPCRnorovirus

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Area of Science:

  • Foodborne Pathogen Detection
  • Molecular Virology
  • Food Safety Science

Background:

  • Human norovirus (HuNoV) is a major cause of foodborne illness, frequently detected by RT-qPCR.
  • Current methods can detect both infectious and non-infectious HuNoV particles, leading to potential overestimation of contamination.
  • Distinguishing between infectious and non-infectious norovirus is crucial for accurate risk assessment.

Purpose of the Study:

  • To evaluate capsid integrity treatments and long-range RT-qPCR for reducing the detection of inactivated human noroviruses and fragmented RNA.
  • To assess the efficiency of RNase, PMAxx, and PtCl4 treatments in combination with standard extraction protocols.
  • To compare the effectiveness of RT-qPCR and long-range RT-qPCR for detecting norovirus RNA after treatment.

Main Methods:

  • Spiking lettuce with heat-inactivated and non-inactivated HuNoV and murine norovirus (MNV).
  • Applying capsid integrity treatments: RNase, Propidium monoazide bromide (PMAxx), and Platinum tetrachloride (PtCl4).
  • Utilizing RT-qPCR and long-range RT-qPCR for viral RNA detection following ISO 15216-1:2017 extraction.

Main Results:

  • RNase and PMAxx treatments significantly reduced heat-inactivated HuNoV recovery by 2 and >3 log, respectively, when used with RT-qPCR.
  • PtCl4 treatment reduced both inactivated and non-inactivated norovirus recovery, indicating non-specificity.
  • Long-range RT-qPCR reduced inactivated HuNoV and MNV recovery by 1.0 and 0.5 log, respectively, offering a complementary detection approach.

Conclusions:

  • RNase and PMAxx treatments are effective in reducing the detection of non-infectious HuNoV particles by RT-qPCR.
  • Long-range RT-qPCR can aid in verifying RT-qPCR results, thereby reducing the risk of false-positive norovirus detection.
  • These methods improve the accuracy of norovirus detection in food safety applications.