Identification of human rotavirus strains with the P[14] genotype by PCR

Abstract

A seminested PCR typing assay has been extended to identify rotavirus strains with the P[14] genotype. The specificity of the method was confirmed by Southern hybridization and by restriction analysis with the enzyme AluI. One out of four human rotavirus (HRV) strains with unusual subgroup-electropherotype linkage but none out of 50 HRV strains with usual linkage was typed as P[14].

FIG. 1

Electrophoretic analysis of PCR products of HRV reference strains. Lane 1, DNA molecular weight marker VIII (a mixture of pUCBM21 DNA HpaII and DraI-plus-HindIII digest; Boehringer Mannheim); lane 2, strain Wa (G1, P[8]); lane 3, strain DS1 (G2, P[4]); lane 4, strain ST3 (G4, P[6]); lane 5, strain AU-1 (G3, P[9]); lane 6, strain PA169 (G6, P[14]). (A) Ethidium bromide-stained gel showing RT-PCR products after second amplification, visualized under UV light. (B) Results of Southern hybridization with biotinylated HRV P[14] genotype-specific PR-1 probe.

FIG. 2

Electrophoresis profiles of PCR-amplified gene 4 cDNA of strain PA169 (lanes 2 and 3) and of fecal isolate PA-5/89 (lanes 4 and 5), before and after, respectively, digestion with AluI, visualized by ethidium bromide staining of the gel. Lane 1, DNA molecular weight marker VIII (Boehringer Mannheim).