Dot-blot hybridisation assay for detection of parvovirus B19 infections using synthetic oligonucleotides.
Pools of 10 synthetic oligonucleotides with sequences derived from the genome of parvovirus B19 and of 30 bases in length were made and labelled at the time of synthesis with digoxigenin (DIG) or dinitrophenyl (DNP) at the 5' end. They were used in a dot-blot hybridisation assay to detect parvovirus B19 DNA in sera submitted for routine virological diagnosis where parvovirus infection was suspected. Detection down to 10-100 fg DNA (equivalent to 10(3)-10(4) copies of parvovirus B19 genome) was obtained with both probe cocktails and colorimetric or chemiluminescent detection systems. Of 141 clinical samples examined from 126 patients presenting with rash and/or joint pains, 107 were clearly negative with both probes, 20 were clearly positive and the remaining 14 samples gave discrepant results. Of these 34 samples, 33 contained parvovirus B19 specific IgM. The parvovirus oligonucleotide probe cocktail produced and labelled with either DIG or DNP provided a useful diagnostic reagent for the detection of specific DNA in clinical specimens using a simple and sensitive dot-blot assay.
Cubie
H A
HA
Regional Virus Laboratory, City Hospital, Edinburgh.
Molyneaux
P J
PJ
Shearman
M J
MJ
Gryzbowski
J
J
Brown
T
T
D000328:Adult; D016375:Antisense Elements (Genetics) / Q000138:chemical synthesis; D001483:Base Sequence; D016731:Erythema Infectiosum / Q000175:diagnosis*; D005260:Female; D016679:Genome, Viral; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007202:Indicators and Reagents; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D016375:Antisense Elements (Genetics); D007202:Indicators and Reagents; D015345:Oligonucleotide Probes