Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology

Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes

“…This enabled us to target single RNA molecules. To detect a positive signal, it was determined that at least 17 of the probes in a given set must bind to their target sequence with only one mismatch allowed with respect to the stringency parameters [25]. …”

“…The use of other sensitive techniques, which might match the sensitivity achieved here (such as PCR, either direct or nested), is limited because of the low accessibility and stability of nucleic acids [26]. By contrast, improvements in in situ hybridization (ISH) and fluorescent in situ hybridization (FISH) have increased both the sensitivity and specificity of RNA detection such that they now represent a valid alternative to PCR, with the advantage that they can spatially localize particular RNA molecules within fixed cells [25]. …”

“…Sensitivity and specificity of our probe set was tested on different virus strains as well as on various embedding procedures and was compared with indirect FISH on a set of previously analysed samples [25] as well as on newly prepared samples [30]. Our viral probes showed a sensitivity comparable to that of PCR analysis of cell lines when tested in infected cell lines.…”

“…A well-characterized cohort of human pancreatic donor tissue has been established by nPOD (Network for Pancreatic Organ Donors with Diabetes) and is available, similar to other cohorts, mainly as formalin fixed, paraffin embedded samples (FFPE) [25]. This method ensures preservation of the samples for many years, but a limiting factor is the relatively poor RNA integrity often associated with FFPE preservation which means that analysis of tissue samples by PCR can be difficult [26].…”

“…Thus, improved RNA FISH methods have been developed to overcome these hurdles. Enteroviral RNA was detected by a new generation of RNA probes (QuantiGene ® ViewRNA Assay), which depend on signal amplification [25]. This approach offers the advantage of signal amplification via the use of branched secondary probes but, theoretically, may be affected by several conditions.…”