Comparison between microwell and bead supports for the detection of human cytomegalovirus amplicons by sandwich hybridization.
In this study, we compared the efficiency of capture DNA probes covalently bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene supports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times higher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes were optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target, with faster kinetics of hybridization obtained on beads than on microplates. Sensitivity of the hybridization assay was then determined with a nonisotopic method and the detection limit found was 30 amol of HCMV amplicons on both supports. HCMV DNA extracted from clinical samples were amplified by PCR. The resulting amplicons were then analyzed using the optimized sandwich hybridization assay discussed here. The results perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel.
Zammatteo
N
N
Laboratoire de Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, Namur, B-5000, Belgium.
Alexandre
I
I
Ernest
I
I
Le
L
L
Brancart
F
F
Remacle
J
J
D003124:Colorimetry; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification*; D015342:DNA Probes / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis / Q000097:blood / Q000134:cerebrospinal fluid; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D011137:Polystyrenes; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D011137:Polystyrenes