ABSTRACT
Objective To establish a specific,sensitive,simple human respiratory adenovirus detection method to put a good experimental foundation for developing universal and specific diagnostic reagents of human respiratory adenovirus.Methods The nucleotide sequences of 10 serotype of human respiratory adenovirus were obtained from GenBank.Highly consewed five pairs of universal and specific adenovirus primers were designed on the evaluation of multiple sequence alignment of the 10 full genomic sequences with the software DNAMAN 5.2.2,Gene Runner 3.05,BLAST,and to ensure that the polymerase chain reaction(PCR)products were type-specific.NP-40 sample lytic method was employed to prepare the template.The effectiveness,specificity and sensitivity of primers were evaluated.And PCR was carried out to test 64 samples of throat swabs of acute respiratory infection children.The positive PCR products were sequenced directly to identify the adenovirus serotypes.The positive specimens was inoculated on HeLa cells to observe cytopathie effect(CPE)under light microscope,and virus morphology were observed under electron microscope.Results BLAST results indicated that the five pairs of primers were specific adenovirus primers with low homology to the others.The primers were identified as PCR positive fragments obtained by using five pairs of primers to amplify the human adenovirus type 3 DNA,which did not react to the respiratory syncytial virus and Coxsackie virus.the effectiveness and specificity of the primer were thus indicated.PCR sensitive results showed 10-5 dilutin of adenovirus culture and DNA sample could be deteeted which meant the method was sensitive and stable.Two PCR positive specimens were detected in 64 clinical samples.the positive rate was 3.13%(2/64).Using the two PCR positive specimens to inoculate the HeLa cells,the typical adenovirus CPEs of rounded and aggregated,detatched cells were observed under light microscope.And a large number of adenovirus typical particles with characteristic lattice arrangement were observed in the infected cells under electron microscope.PCR product sequencing results showed that these two isolated adenoviruses were typed in human adenovims group B.Conclusions Universal and specific adenovirus PCR primers with the serotype specificity of the PCR products were designed successfully.The PCR primers was sensitive and specific and could be routinely applied in clinical adenovirus diagnosis for respiratory specimens.