Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus

J Virol Methods. 2017 May:243:31-34. doi: 10.1016/j.jviromet.2017.01.019. Epub 2017 Jan 30.

Abstract

The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains. A new Real-Time PCR, specific for influenza D virus (IDV), was developed and evaluated. The target sequences of primers and probe are highly conserved among IDV strains currently known. The specificity of the method was demonstrated in silico by BLAST, and in vitro with a huge panel of common swine and bovine respiratory pathogens. The analytical sensitivity of the Real-Time PCR was estimated through synthetic RNA molecules and the limit of detection was about 20 copies/μL. The assay was assessed in field and proved to be a valuable tool for the detection of IDV strains.

Keywords: Cattle; IDV; Influenzavirus D; Real-Time RT-PCR; Swine.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Cattle
  • DNA Primers / genetics
  • Humans
  • Influenza, Human / virology
  • Oligonucleotide Probes / genetics
  • Orthomyxoviridae Infections / veterinary
  • Orthomyxoviridae Infections / virology
  • Real-Time Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Swine
  • Thogotovirus / genetics
  • Thogotovirus / isolation & purification*

Substances

  • DNA Primers
  • Oligonucleotide Probes