PCR and Restriction Endonuclease Analysis for Rapid Identification of Human Adenovirus Subgenera

Abstract

Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.

Molecular identification of adenovirus causing respiratory tract infection in pediatric patients at the University of Malaya Medical Center

“…Viruses causing RTI are usually isolated in the laboratory from patients' nasal secretions and serotyped by immunological typing methods [ 11 - 13 ]. In recent years, molecular identification of the virus has become more common, where distinction of the virus types can be made through specific genomic sequence amplification by polymerase chain reaction (PCR) and determination of the partial hexon gene sequence [ 14 , 15 ]. Presently, there is no report of the prevalent HAdV types causing infections among Malaysians.…”

Molecular Diagnosis of Human Adenoviruses D and E by a Phylogeny-Based Classification Method Using a Partial Hexon Sequence

“…To improve on this time, PCR-and RFLP-based rapid identification methods were developed by several researchers, including us (11). To identify the serotype of HAdV that causes conjunctivitis, we developed PCR-RFLP based on 956 bp of the 3Ј one-third of the hexon gene digested with the REs EcoT14I, HaeIII, and HinfI (29).…”

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus®) for the detection and the species identification of adenoviruses in respiratory specimens

“…Several teams have designed other approaches to identify the Ad species involving enzyme restriction analysis (Elnifro et al, 2000) or multiplex PCR methods (Na et al, 2002;Pring-Akerblom et al, 1999;Xu et al, 2000). These techniques seemed to be very sensitive on isolates, but they were not evaluated on respiratory samples.…”

“…Non-nested and nested PCRs have also been developed for the detection of the six species of Ad. These methods are based on the comparison of the migration of amplimers (Kidd et al, 1996), the analysis of restriction enzyme profiles (Elnifro et al, 2000) and the use of multiplex PCR (Na et al, 2002;Pring-Akerblom et al, 1999;Xu et al, 2000). Finally, a method based on a combination of PCR and restriction endonuclease digestion has been developed for the rapid identification of species and serotypes of Ad (Allard et al, 2001).…”