[Determination of Herpes simplex virus DNA with the use of solid-phase methods for the detection of PCR products].
To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.
Rar
V A
VA
Terekhova
D A
DA
Pyshnyĭ
D V
DV
Pyshnaia
I A
IA
Morozova
O V
OV
D019921:Biotinylation; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics*; D004076:Digoxigenin; D004586:Electrophoresis; D006561:Herpes Simplex / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D004076:Digoxigenin