Rapid Identification of Human Adenovirus Types 3 and 7 from Respiratory Specimens via Multiplex Type-Specific PCR

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Abstract
The rapid diagnosis of human adenovirus (Ad) infection is crucial for the timely recognition of epidemics. Moreover, identification of the serotypes known to cause serious disease can be helpful in therapeutic intervention. A multiplex PCR assay was developed for the rapid detection of adenovirus type 3 (Ad3) and Ad7 directly from clinical specimens. For this assay, three primer pairs (primers were based on the conserved and hypervariable regions of the hexon) were designed in order to simultaneously amplify all adenoviral serotypes and discriminate between Ad3 and Ad7. In our preliminary analysis, this multiplex PCR assay generated amplicons of the consensus primers from all 106 adenoviral isolates of diverse serotypes and proved able to correctly identify Ad3 and Ad7. This assay was subsequently applied to the detection of Ad3 and Ad7 in respiratory specimens. Among the 127 nasal aspirates from which an adenovirus was grown, the sensitivity with which any serotype could be detected was 91% (115/127). Two of the 53 nasal aspirates which did not grow Ads yielded adenovirus-specific bands, which were confirmed by sequencing analysis. Among the 115 specimens which produced common adenoviral bands, the sensitivity with which Ad3 could be detected was 93% (26/28), and the sensitivity with which Ad7 could be detected was 100% (35/35). Five out of the 115 specimens were proved to harbor more than one type of Ad via sequencing analysis of the amplicons, suggesting mixed infection with at least two different serotypes. In conclusion, this multiplex PCR system can be utilized in the rapid identification of Ad3 and Ad7 directly from clinical specimens. Furthermore, this method constitutes a diagnostic strategy for the detection of coinfection by different Ad serotypes.