Investigative ophthalmology & visual science · 1999
Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.
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Cooper R J, Yeo A C, Bailey A S, Tullo A B
Аннотация
PURPOSE: To evaluate newly designed primers in a polymerase chain reaction (PCR) for the detection of adenovirus DNA in conjunctival swabs.
METHODS: Oligonucleotides were derived from the adenovirus hexon gene and modified such that a maximum of only two mismatches occurred with adenovirus types 2 through 5, 7, and 16. Specificity was determined against adenovirus types 2 through 4, 7, 8 through 11, 14, 19, 37, 40, and 41 and from non-adenoviral DNA and the sensitivity by PCR amplification of purified adenovirus type 2 DNA. The assay was compared retrospectively with cell culture and a PCR with different primers on 59 stored conjunctival swab samples. The new PCR also was used prospectively in comparison with cell culture on 2743 conjunctival swabs.
RESULTS: The 140-bp product was amplified from all the adenovirus serotypes tested except types 40 and 41, which have not been isolated from the eye. There were no amplified products from the non-adenoviral DNA tested. With adenovirus type 2 DNA, despite two deliberate mismatches, 40 copies of the target were detectable after PCR and ethidium bromide-staining. In the retrospective study, 51 of 55 (92.7%) were positive by this new PCR compared with 42 of 55 (76.4%) by the older PCR and 40 of 55 (72.7%) by cell culture. In the prospective study, the new PCR detected 386 of 415 (93%) adenovirus-positive specimens compared with 248 of 415 (59.8%) by cell culture. Of 167 specimens positive for herpes simplex virus by cell culture, none were positive by the adenovirus PCR.
CONCLUSIONS: PCR with the newly designed primers shows a much increased sensitivity over cell culture and previous PCRs for the detection of adenoviruses in conjunctival swabs.
METHODS: Oligonucleotides were derived from the adenovirus hexon gene and modified such that a maximum of only two mismatches occurred with adenovirus types 2 through 5, 7, and 16. Specificity was determined against adenovirus types 2 through 4, 7, 8 through 11, 14, 19, 37, 40, and 41 and from non-adenoviral DNA and the sensitivity by PCR amplification of purified adenovirus type 2 DNA. The assay was compared retrospectively with cell culture and a PCR with different primers on 59 stored conjunctival swab samples. The new PCR also was used prospectively in comparison with cell culture on 2743 conjunctival swabs.
RESULTS: The 140-bp product was amplified from all the adenovirus serotypes tested except types 40 and 41, which have not been isolated from the eye. There were no amplified products from the non-adenoviral DNA tested. With adenovirus type 2 DNA, despite two deliberate mismatches, 40 copies of the target were detectable after PCR and ethidium bromide-staining. In the retrospective study, 51 of 55 (92.7%) were positive by this new PCR compared with 42 of 55 (76.4%) by the older PCR and 40 of 55 (72.7%) by cell culture. In the prospective study, the new PCR detected 386 of 415 (93%) adenovirus-positive specimens compared with 248 of 415 (59.8%) by cell culture. Of 167 specimens positive for herpes simplex virus by cell culture, none were positive by the adenovirus PCR.
CONCLUSIONS: PCR with the newly designed primers shows a much increased sensitivity over cell culture and previous PCRs for the detection of adenoviruses in conjunctival swabs.