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Arun Kumar Adhikary, Jiro Numaga, Toshikatsu Kaburaki, Hidetoshi Kawashima, Satoshi Kato, Makoto Araie, Kazunori Miyata, Hideaki Shimizu, Fumihiro Yagyu, Eiko Suzuki, Hiroshi Ushijima; Rapid Detection and Typing of Oculopathogenic Strain of Subgenus D Adenoviruses by Fiber-Based PCR and Restriction Enzyme Analysis. Invest. Ophthalmol. Vis. Sci. 2001;42(9):2010-2015.
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purpose. To develop a new detection and typing method of oculopathogenic strains
of subgenus D adenoviruses directly from conjunctival scrapings by a
combination of polymerase chain reaction (PCR) and restriction enzyme
methods. A new PCR method using primer pairs of AF2/AR2, which are specific for
the fiber genes, were developed to amplify 1150-bp products from nine
oculopathogenic prototypes of subgenus D adenoviruses. Amplicons were
cleaved with three restriction enzymes: DdeI, HinfI, and RsaI. Clinical specimens of
102 conjunctival scrapings were also evaluated by this PCR method.
Restriction patterns of prototypes were used for the typing of clinical
samples. Detection limit was determined by the PCR amplification of a
known amount of purified adenovirus serotype 8 DNA.
results. A novel PCR method based on the fiber genes allowed the amplification
of nine oculopathogenic serotypes of subgenus D (Ad8, Ad9, Ad15, Ad17,
Ad19, Ad22, Ad28, Ad37, and Ad39). As little as 38.4 fg of adenovirus
type 8 could be detected by this method. Positive results were obtained
from 48 of 102 samples (47%) by both hexon- and fiber-based PCR,
whereas only 29 of 102 (28.4%) yielded positive results by culture
isolation/neutralization test (NT). All positive specimens (29 samples)
of culture isolation and PCR-RFLP methods showed positive results by
our new fiber-based PCR method, and no positive products were detected
from other subgenus of adenovirus or nonadenoviral DNA.
conclusions. A newly developed fiber-based PCR-REA method for the detection and
typing of adenoviruses is faster than any former PCR methods. This
all-in-1-day detection and typing method will be quite useful to the
rapid diagnosis of subgenus D adenovirus
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