Detection and Localization of Viral Infection in the Pancreas of Patients With Type 1 Diabetes Using Short Fluorescently-Labelled Oligonucleotide Probes

Oncotarget. 2017 Feb 21;8(8):12620-12636. doi: 10.18632/oncotarget.14896.

Abstract

Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family.With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.

Keywords: Pathology Section; enteroviruses; islets; oligonucleotide probes; pancreas; type 1 diabetes.

MeSH terms

  • Diabetes Mellitus, Type 1 / virology*
  • Enterovirus
  • Enterovirus Infections / diagnosis
  • Fluorescent Dyes
  • Humans
  • Immunohistochemistry
  • In Situ Hybridization, Fluorescence / methods*
  • Oligonucleotide Probes*
  • Pancreas / virology*
  • Polymerase Chain Reaction
  • RNA, Viral / analysis
  • Sensitivity and Specificity

Substances

  • Fluorescent Dyes
  • Oligonucleotide Probes
  • RNA, Viral