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Nonradioactive digoxigenin DNA labeling and immunological detection of HSV PCR products

A nonradioactive molecular assay for detection of Herpes Simplex Virus (HSV) amplification products was evaluated. After phenol-chloroform DNA extraction from green monkey kidney (GMK) cell cultures infected with HSV-1, specific DNA sequence was amplified by polymerase chain reaction (PCR), using specific oligonucleotide primers (OPERON, Operon Technologies, Inc., Alameda, CA). Amplification products were visualized by agarose gel electrophoresis, and if present, specific DNA HSV probe (OPERON, Operon Technologies, Inc., Alameda, CA) was labeled. DNA probe is labeled by random primed incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG DNA labeling and detection kit, Boehringer Mannheim GmbH). The dUTP is linked via a spacer-arm to the steroid hapten digoxigenin (DIG-dUTP). Southern transfer to positively charged nylon membrane (Boehringer Mannheim Nylon Membran) or Dot blot were performed. DIG-labeled DNA is used according to standard hybridization protocol. After hybridization to the target DNA hybrids were detected by enzyme-linked immunoassay using an antibody-conjugate (antidigoxigenin-alkaline phosphatase conjugate) and subsequent enzyme-catalyzed color reaction with 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and nitro blue tetrazolium salt (NBT).

nonradioactive labeling; digoxigenin DNA labeling; immunological detection; HSV; PCR products

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