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In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction

Ray, R., Smith, M., Sim, R., Bruce, Ian J., Wakefield, A. (1995) In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction. Journal of Virological Methods, 52 (3). pp. 247-263. ISSN 0166-0934. (doi:10.1016/0166-0934(94)00117-Y) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1016/0166-0934(94)00117-Y

Abstract

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and ?-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of ?-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For ?-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80 of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.

Item Type: Article
DOI/Identification number: 10.1016/0166-0934(94)00117-Y
Uncontrolled keywords: alpha tubulin, animal cell; article; controlled study; cytomegalovirus; cytomegalovirus infection; dna probe; gene amplification; human; human cell; in situ hybridization; nonhuman; oligonucleotide probe; polymerase chain reaction; priority journal, Animal; Base Sequence; Blotting, Southern; Body Fluids; Cell Line; Cercopithecus aethiops; Cytomegalovirus; DNA, Viral; Electrophoresis; Feasibility Studies; Human; In Situ Hybridization; Molecular Sequence Data; Polymerase Chain Reaction; Preservation, Biological; Rats; Tubulin; Vero Cells, Animalia; Cucumber mosaic virus; Cytomegalovirus
Subjects: R Medicine > RB Pathology
Divisions: Faculties > Sciences > School of Physical Sciences
Faculties > Sciences > School of Physical Sciences > Functional Materials Group
Depositing User: I. Bruce
Date Deposited: 30 Jan 2019 16:32 UTC
Last Modified: 30 May 2019 08:50 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/71986 (The current URI for this page, for reference purposes)
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