Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization.
A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy.
Hurst
J W
JW
Viral and Rickettsial Disease Laboratory, California State Department of Health Services, Berkeley 94704.
Forghani
B
B
Chan
C S
CS
Oshiro
L
L
Darai
G
G
D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D006801:Humans; D015151:Immunoblotting / Q000379:methods* / Q000706:statistics & numerical data; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D008976:Molluscum Contagiosum / Q000175:diagnosis; D008977:Molluscum contagiosum virus / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral