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.
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.
Authors: Elfateh M Elnifro; Robert J Cooper; Ian Dady; Sa'id Hany; Zolf M Mughal; Paul E Klapper Journal: J Clin Microbiol Date: 2005-11 Impact factor: 5.948
Authors: Meei-Li Huang; Long Nguy; James Ferrenberg; Michael Boeckh; Anne Cent; Lawrence Corey Journal: Diagn Microbiol Infect Dis Date: 2008-08-15 Impact factor: 2.803