Accelerated detection of DNA on membranes by automated enzyme-linked immunofiltration assay.
Methods used to detect DNA after transfer to nitro-cellulose or nylon membranes are all based on slow incubation with agitation. We describe an application of the ELIFA technique (enzyme-linked immunofiltration assay) for rapid detection of DNA immobilized on a membrane by active filtration of the reagents across the membrane. The different steps (saturation, hybridization to a nonisotopically labeled probe, washing, and immunoenzymatic revelation) are automated and controlled by a microcomputer that determines the direction of flow and flow rates of the solutions through the membrane. We applied this method to the detection of Toxoplasma gondii DNA in 108 samples of amniotic fluid during antenatal tests for toxoplasmosis and compared the results with those obtained by the conventional method. In addition to a major time saving (2 h against almost 15 h), automation improves reproducibility and avoids manipulation of the membranes between the different steps, while keeping the same sensitivity and specificity as the standard method.
Aubert
D
D
Laboratoire de Parasitologie-Mycologie, UPRES EA2070, Equipe IV INSERM U314, IFR 53, Hôpital Maison-Blanche, Reims, France.
Toubas
D
D
Foudrinier
F
F
Villena
I
I
Gomez
J E
JE
Marx
C
C
Lepan
H
H
Lemaire
P
P
Jacquier
P
P
Bonhomme
A
A
Pinon
J M
JM
D000653:Amniotic Fluid / Q000469:parasitology; D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005260:Female; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008567:Membranes, Artificial; D051379:Mice; D009693:Nucleic Acid Hybridization; D011247:Pregnancy; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification; D014125:Toxoplasmosis, Congenital / Q000175:diagnosis / Q000469:parasitology
D016054:DNA, Protozoan; D008567:Membranes, Artificial; D004247:DNA