Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method

Viruses. 2023 Apr 17;15(4):983. doi: 10.3390/v15040983.

Abstract

The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.

Keywords: R294K mutation; RT-droplet digital PCR; absolute quantitation; avian influenza virus H7N9.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antiviral Agents / pharmacology
  • Antiviral Agents / therapeutic use
  • Birds / genetics
  • Humans
  • Influenza A Virus, H7N9 Subtype* / genetics
  • Influenza in Birds*
  • Influenza, Human*
  • Mutation
  • Neuraminidase / genetics
  • Neuraminidase / metabolism
  • Oseltamivir / pharmacology
  • Oseltamivir / therapeutic use
  • Polymerase Chain Reaction
  • Reverse Transcription

Substances

  • Oseltamivir
  • Antiviral Agents
  • Neuraminidase

Grants and funding

This work was supported by the Key Laboratory of Public Health Detection and Etiological Research of Zhejiang Province; Zhejiang Health Leading Talents Training Project ((2018)22); National key R&D Program of China (2021YFC2301200); The Key Research and Development Project of Zhejiang Province (2021C03044).