[Rapid detection and identification of human adenovirus directly from conjunctival scrapings by polymerase chain reaction and restriction fragment length polymorphism analysis].
Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were combined for detection and identification of adenovirus (Ad), a common agent of conjunctivitis in Japan. Nested-PCR with two primer sets that hybridize to the conserved region for hexon protein of 14 prototypes of Ad serotype 1 to 8, 11, 14, 19, 37, 40, and 41, amplified 956 bps DNA fragment. The amplified fragments from 14 prototypes were completely differentiated with the combination of three restriction endonucleases, Eco T14I, Hae III, and Hin fI. We applied this new method to 70 conjunctival scrapings from patients with conjunctivitis, and compared the results with those of the combination of culture isolation and neutralization test. PCR was positive in 38 out of 70 samples (54.3%), whereas 33 of 70 samples (47.1%) were positive by cell culture. Compared with cell culture isolation, the PCR method had a sensitivity of 100% (33 of 33). Positive PCR samples were further classified into Ad 37 (44.7%), 3 (39.5%), 11 (7.9%), 8 (5.3%), and 4 (2.6%) by PCR-RFLP analysis. Of five samples that were PCR positive and cell culture negative, three samples were Ad 37 and two were Ad 8 by PCR-RFLP analysis. These differentiations of cell culture positive samples were identical to the results of the neutralization test. It took only about three days to detect and identify Ad by PCR-RFLP analysis, whereas it took at least two weeks by culture isolation and neutralization test. Our newly developed method of detecting and typing human Ad by PCR-RFLP analysis is more sensitive, accurate, and prompt than the conventional cell culture isolation and neutralization test.
Saitoh
W
W
Department of Opthalmology, Yokohama City University School of Medicine.
Itoh
N
N
Isobe
K
K
Ohno
S
S
Oshima
A
A
Nagamoto
Y
Y
Nakajima
H
H
Hata
K
K
Ishiko
H
H
Aoki
K
K
D000258:Adenovirus Infections, Human / Q000821:virology*; D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003228:Conjunctiva / Q000821:virology*; D003236:Conjunctivitis, Viral / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length