Multiplex polymerase chain reaction for subgenus-specific detection of human adenoviruses in clinical samples

The polymerase chain reaction (PCR) has been used previously for the detect ion acid typing of adenoviruses directly in clinical samples. Since under c linical conditions subgenus-specific identification is often sufficient, we extended the genus- and type-specific PCR by a subgenus-specific PCR. By s equencing several loop I, gene regions of the hexon (major adenovirus coat protein) and comparing them to published sequences, subgenus-specific seque nces were identified in this region. By using primers targeted to this regi on and to a conserved hexon gene region, a multiplex, nonnested PCR for the detection and subgenus-specific identification of adenoviruses could be es tablished. The six subgenus-specific amplimers are distinguishable by agaro se gel electrophoresis, and subsequent restriction analysis is not necessar y. The specificity of the subgenus-specific primer pairs was tested on 23 a denovirus prototypes, representing all six subgenera, on 9 subgenus B and D intermediate strains, and on 16 subgenus C genome types. Furthermore, mult iplex, subgenus-specific PCR was performed directly with 100 clinical speci mens, including stool samples, ocular swabs, and throat swabs. Adenoviruses of all subgenera could be detected. Especially for clinical application, t he rapid, one-step differentiation between subgenus D adenoviruses, causing the severe and highly contagious epidemic keratoconjunctivitis, and subgen us B and E adenoviruses, causing relative harmless ocular infections, is of great importance. The subgenus-specific PCR could also facilitate the prim ary classification of unknown virus isolates. J. Med Virol. 58:87-92, 1999. (C) 1999 Wiley-Liss, Inc.