Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis

Abstract

We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.

Epidemiology and clinical features of gastroenteritis in hospitalised children: prospective survey during a 2-year period in a Parisian hospital, France

“…The Rotavirus RT-PCR used the primer sets Beg9/End9 [15] and Con2/Con3 [16], The astrovirus RT-PCR used the primers Mon 244 and Mon 245 [17] and the adenovirus PCR used the primers Hex1DEG/Hex2DEG [18]. The norovirus and sapovirus RT-PCR used primer sets in separate reactions.…”

“…The sex ratio was 1.26. The mean Vesikari score was 11.7 (standard deviation, 3.16; range, [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. Among the 95 sets of data rejected in this study, 13 were rejected for the lack of clinical data and 82 for the absence of a faecal sample.…”

No link between viral findings in the prostate and subsequent cancer development

“…Nested PCR assays were used for all the assays except HPV and C. albicans PCRs. Primers and PCR protocols for adenovirus (Allard et al, 2001), CMV (Brytting et al, 1991), EBV (Meyohas et al, 1996), HSV1 and 2 (Aurelius et al, 1991) and HPV (de Roda Husman et al, 1995) were used with minor modifications. Primers for the polyoma viruses JCV and BKV and C. albicans were designed according to published sequence information (Table 1).…”

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

“…Polymerase chain reaction (PCR), real-time PCR and immunochromatographic kits have recently become available for this purpose (Fujimoto et al 2004). Typing of Ads isolates can be achieved by neutralization, hemmaglutination inhibition, restriction endonuclease analysis (REA), sequencing and, PCR combined with REA (Allard et al 2001, Sarantis et al 2004. Until recently, the interest in serotype and genotype determination of Ad isolates was mainly epidemiological, however, this has changed due to the increasingly clear association between specific serotypes and genotypes and more severe disease presentation (Kajon & Wadell 1994, Larrañaga et al 2000, Carballal et al 2002.…”