Discrimination of respiratory syncytial virus subgroups A and B by reverse transcription-PCR.
Reverse transcription (RT)-PCR with shared primers differentiating respiratory syncytial virus (RSV) subgroups A and B was developed for subtyping of RSV isolates. Results of RT-PCR were compared with those of an indirect immunofluorescence test using monoclonal antibodies. Viral RNA isolated from cell cultures infected with RSV served as a template for cDNA synthesis with random primers. For PCR, we used three synthetic oligonucleotides corresponding to the G protein mRNA sequence of subgroup A (bases 248 to 267; 3'ATGCAACAAGCCAGATCAAG), subgroup B (bases 314 to 333; 3'ACTCATCCAAACAACCCACA), or both (bases 511 to 530; 3'GGWACAAARTTGAACACTTC). PCR products of RSV subgroups A and B had molecular sizes of 283 and 217 bp, respectively. Specific cutting sites for RSV A and B in amplified cDNA were demonstrated by restriction fragment analysis with four restriction endonucleases. Our RT-PCR assay divided 68 RSV isolates into 47 strains of subgroup A and 21 strains of subgroup B in full agreement with subtyping by monoclonal antibodies. RT-PCR seems to be a good alternative to subtyping of RSV with monoclonal antibodies.
Gottschalk
J
J
Institute of Microbiology, Kantonsspital, Aarau, Switzerland.
Zbinden
R
R
Kaempf
L
L
Heinzer
I
I
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018113:Respiratory Syncytial Virus, Human / Q000145:classification* / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral