OBJECTIVETo develop a rapid, sensitive and specific method in identifying and typing on adenovirus from clinical specimens.

METHODSPrimers were designed using hexon gene of adenovirus as target. One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hexon gene of adenovirus from all types. Four primer pairs located within the region of this 1278 bp were specifically designed for amplifying types 3, 7, 11 and 21 of adenoviruses, which were used for multiplex nest-PCR in a single tube. The products from this multiplex nest-PCR were 502 bp (for type 3), 311 bp (for type 7), 880 bp (for type 11) and 237 bp (for type 21), respectively. Type of the adenovirus tested could then be determined after agarose electrophoresis analysis of the PCR products.

RESULTSPCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3, 7, 11 and 21, but not for other respiratory viruses, indicating that the technique was specific without cross reaction with other viruses. Out of the 118 clinical specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay, 76 belonged to adenovirus type 3 (76/118, 64.4%), 37 to adenovirus type 7 (37/118, 31.4%), 3 to adenovirus type 11 (3/118, 2.5%) but no adenovirus type 21 was detected. Two of the 118 positive specimens which were positive by both tissue culture and immunofluerescence could not be identified, suggesting that these 2 strains (1.7%) were with the types other than types 3, 7, 11 and 21. Out of the 33 specimens which were negative by both tissue culture and immunofluerescence, 3 showed positive by this multiplex PCR (2 of type 3 and 1 of type 7), suggesting this method was more sensitive than tissue culture and immunofluerescence.

CONCLUSIONThis multiplex nest-PCR method had the benefit of rapid,sensitive and specific nature so could be used for identifying types of adenoviruses in the clinical specimens.