Detection and typing of human herpesviruses by multiplex polymerase chain reaction.
A new approach to simultaneous detection and typing of related agents by the multiplex polymerase chain reaction (PCR) is described. The reaction was been applied to human herpesviruses by nested amplification of fragments of the DNA polymerase genes. During the first amplification, primers were used as two equimolar mixtures of non-degenerate oligonucleotides, aligning the 3'-ends with selected consensus regions, and their 5'-ends with the non-related sequences of each herpesvirus to be amplified. The specific fragments obtained were the substrate for a second, multiplex reaction for which primers were designed to produce different-size fragments for each related virus. The results showed high specificity for the detection and typing of the human herpesviruses with known sequences and no amplification of human DNA, in spite of the presence of the same consensus regions within human DNA polymerase alpha. It is concluded that this new approach would be useful for the differential diagnosis of herpesviruses, as well as for other groups of agents with conserved regions in their genomes and causing similar syndromes.
Tenorio
A
A
Servicio de Microbiología Diagnóstica, C.N.M.V.I.S., Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
Echevarría
J E
JE
Casas
I
I
Echevarría
J M
JM
Tabarés
E
E
D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D016384:Consensus Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005814:Genes, Viral / Q000235:genetics; D006564:Herpesviridae / Q000145:classification* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000175:diagnosis*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D016375:Antisense Elements (Genetics); D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase