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.

FIG. 1

Phylogenetic analysis of 27 different Ad types showing the subgenus relationship between the 253-bp sequence at positions 46 to 299 within the hexon gene. The view is unbalanced, and the scale beneath the tree measures the distance between sequences. Published sequences from the GenBank database of Ad2 (J01917), Ad3 (X76549), Ad5 (X02997), Ad7 (X76551), Ad12 (X73487), Ad16 (X74662), Ad40 (X51782), Ad41 (X51783), and Ad48 (U20821) are also included.

FIG. 2

Flowchart of REs for typing of PCR products framed by the general hexon primers hex1deg and hex2deg. The symbol of a pair of scissors indicates that the product will be cleaved by the enzyme in question, whereas no symbol indicates an uncleaved product. See Results for further information on the use of the flowchart.

FIG. 3

Flowchart of REs used for partial typing of prototypes and genome types of Ad3 and Ad7, subgenus B1. The symbol of a pair of scissors indicates that the product will be cleaved by the enzyme in question, whereas no symbol indicates an uncleaved product.

FIG. 4

Comparison of TaqI restriction profiles of amplified DNA of prototype strains of Ad types 21, 51, 40, and 41.

FIG. 5

RE digestion profiles of PCR products of viruses from six different clinical samples representing subgenera A, B1, C, E, and F. Sections: 1, Ad31, subgenus A, patient 10; 2, Ad7 variant, subgenus B1, patient 2; 3, Ad 4, subgenus E, patient 5; 4, Ad1, subgenus C, patient 39; 5, Ad40, subgenus F, patient 4; 6, Ad41, subgenus F, patient 34. See Table 1 for patient data. φX174 DNA digested with HaeIII was used as a molecular size marker. The symbol of a pair of scissors indicates that the product will be cleaved by the enzyme in question.

FIG. 6

BamHI restriction patterns of full-length Ad DNAs extracted from cells infected with viruses from different patient samples. The numbers above each lane indicate the patient number listed in Table 1. Patients 2, 3, 6, 8, 9, 11, 25, and 36 were infected with Ad7b. Patients 10, 12, and 33 were infected with Ad31, patient 38 was infected with Ad5 D2, patients 23 and 40 were infected with Ad3a, patient 5 was infected with Ad4a, and finally, patients 4 and 7 were infected with Ad40 and Ad41, respectively. Molecular size standards consisted of bacteriophage λ DNA digested with HindIII and φX174 DNA digested with HaeIII.