Single-Tube Multiplex PCR for Rapid and Sensitive Diagnosis of Subgenus B and Other Subgenera Adenoviruses in Clinical Samples

Abstract

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non‐nested polymerase chain reaction (PCR). Sequences of the conserved hexon‐coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single‐tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non‐Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty‐seven out of 71 (94%) Sub B Ad culture‐positive samples, and 15 out of 19 (79%) Sub C or E‐positive samples amplified products of the expected size. Two of 20 (10%) culture‐negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non‐Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.