[Detection of hepatitis A virus by riboprobe labeled with digoxigenin: comparison of methods].
A riboprobe (RNA probe), corresponding to the 5' end of the HM175 hepatitis A virus (HAV) genome, was synthetized in vitro and was digoxigenin-labeled. Then the riboprobe was used to detect the CF53 HAV strain. Conditions of virus denaturation (with or without SDS and proteinase K, timing of assay) to release viral RNA were tested by dot-blot hybridization on a ten fold dilution of HAV suspension. Densitometric measures of dot-blot spots allowed to appreciate optimization of the method. Sensitivity of hybridization was compared with sensitivity of radioimmunoassay (RIA) and cell culture methods. Hybridization signals and scale of HAV suspension were consistent when 0.05% SDS, 0.17 micrograms/ml Proteinase K, 37 degrees C, 30 mn or 3 hours are used. 8.10(2) TCID50 HAV was detected by hybridization with digoxigenin-labeled RNA probes. Detection threshold was the same as radioimmunoassay and lower comparatively to cell culture.
van Cuyck-Gandré
H
H
Unité de Biologie Moléculaire, CRSSA, La Tronche, France.
Gratier
D
D
Burckhart
M F
MF
Job
A
A
Crance
J M
JM
Deloince
R
R
D003720:Densitometry; D004076:Digoxigenin; D017957:Hepatitis A Virus, Human / Q000235:genetics / Q000302:isolation & purification*; D066298:In Vitro Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015347:RNA Probes; D012367:RNA, Viral / Q000235:genetics*
D015347:RNA Probes; D012367:RNA, Viral; D004076:Digoxigenin
Détection du virus de l'hépatite A par ribosonde marquée à la digoxigénine: comparaison de méthodes.