Investigative ophthalmology & visual science · 1994
Use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens.
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Kinchington P R, Turse S E, Kowalski R P, Gordon Y J
Аннотация
PURPOSE: To evaluate the application of polymerase chain reaction (PCR) methodology as a potential diagnostic tool for the detection of adenovirus DNA in ocular swab samples.
METHODS: Oligonucleotides derived from the adenovirus hexon gene were used to amplify a 306-base pair (bp) product by PCR. Radiolabeled oligonucleotides derived from sequences within the amplified product were used as specific probes. Specificity was determined against DNA of 13 adenovirus serotypes (types 1 to 11, inclusive, and types 19 and 37) and from nonadenoviral DNAs. Limits of detection were determined by PCR amplification of known amounts of purified adenovirus serotype 2 DNA. The assay was tested on 107 ocular swab samples and correlated to results obtained from tissue culture and a commercial immunoassay (Adenoclone).
RESULTS: The 306-bp PCR product was amplified from all adenovirus serotypes tested, but not from negative control DNAs. As little as 15 fg of adenovirus type 2 DNA could be detected by PCR and ethidium bromide stain. Using a simplified sample preparation procedure, 46 of 58 adenovirus culture-positive but Adenoclone-negative swabs were positive by PCR (79% sensitivity). All (11 of 11) Adenoclone-positive clinical eye swabs tested were positive by PCR (100% sensitivity). Only 1 of 38 nonadenoviral ocular swab samples was positive by PCR (97% specificity).
CONCLUSIONS: PCR appeared to be highly suitable for the diagnosis of adenovirus in ocular swabs, offering important improvements in speed over tissue culture isolation and in sensitivity over immunoassay.
METHODS: Oligonucleotides derived from the adenovirus hexon gene were used to amplify a 306-base pair (bp) product by PCR. Radiolabeled oligonucleotides derived from sequences within the amplified product were used as specific probes. Specificity was determined against DNA of 13 adenovirus serotypes (types 1 to 11, inclusive, and types 19 and 37) and from nonadenoviral DNAs. Limits of detection were determined by PCR amplification of known amounts of purified adenovirus serotype 2 DNA. The assay was tested on 107 ocular swab samples and correlated to results obtained from tissue culture and a commercial immunoassay (Adenoclone).
RESULTS: The 306-bp PCR product was amplified from all adenovirus serotypes tested, but not from negative control DNAs. As little as 15 fg of adenovirus type 2 DNA could be detected by PCR and ethidium bromide stain. Using a simplified sample preparation procedure, 46 of 58 adenovirus culture-positive but Adenoclone-negative swabs were positive by PCR (79% sensitivity). All (11 of 11) Adenoclone-positive clinical eye swabs tested were positive by PCR (100% sensitivity). Only 1 of 38 nonadenoviral ocular swab samples was positive by PCR (97% specificity).
CONCLUSIONS: PCR appeared to be highly suitable for the diagnosis of adenovirus in ocular swabs, offering important improvements in speed over tissue culture isolation and in sensitivity over immunoassay.