Peptide nucleic acid-based in situ hybridization assay for detection of parvovirus B19 nucleic acids.
BACKGROUND
Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens.


METHODS
We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA.


RESULTS
PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results.


CONCLUSIONS
The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic test for the detection of viral nucleic acids in clinical specimens.
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna, Italy.
Francesca
F
Bonvicini
Claudia
C
Filippone
Elisabetta
E
Manaresi
Giovanna Angela
GA
Gentilomi
Marialuisa
M
Zerbini
Monica
M
Musiani
Giorgio
G
Gallinella
D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D016679:Genome, Viral; D006801:Humans; D017403:In Situ Hybridization; D016732:Parvovirus B19, Human / Q000235:genetics*; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction; D011446:Prospective Studies
D004279:DNA, Viral; D020135:Peptide Nucleic Acids