Detection of herpes simplex virus type 1 in herpetic ocular diseases by DNA‐DNA hybridization using a biotinylated DNA probe

Abstract

A diagnostic hybridization assay for detecting herpes simplex virus type 1 (HSV-1) in ocular specimens was developed using cloned viral DNA as a probe. This hybridization assay is based on visualizing a biotinylated probe that is hybridized to the target DNA by a streptavidin/alkaline phosphatase system. The time required for performing this assay system is only two days. This assay system could detect a probe which had been hybridized to as little as 1 pg of homologous DNA and did not cross-react with DNA of other human herpes viruses except that of herpes simplex virus type 2 (HSV-2) which showed weak cross-reactivity. The assay system was applied to experimental keratitis in albino rabbits and clinical specimens. In experimental keratitis in rabbits it was possible to detect HSV-1 DNA in the eye swab samples at least until the ninth day after virus inoculation. Five clinical specimens collected from patients with corneal ulcer or blepharitis contained HSV-1 DNA in spite of the failure of demonstration of viral antigen and/or virus isolation in two cases.

Effects of Gangliosides on the Growth of Herpes Simplex Virus Type 1-Infected Cells Derived from Neurons and on Viral Replication

“…Slot blot hybridization experiments were carried out using a biotin-labeled Bam\\\ K fragment as described elsewhere (19]. For Southern blot hybridization, DNA was digested with BanM I and the digestion was subjected to 0.8% agarose gel electrophoresis.…”

Effect of Protein Kinase C Inhibitors with Different Action Mechanisms on Epstein-Barr Virus Replication

“…The cells were resus pended at lO^cells/ml lysis buffer (10 mMTris-HCl, pH 7.5, 1 m lf EDTA, 150 m M NaCI, and 0.6% Nonidet P-40) and sonicated for 30 s. After centrifugation (1,500 g, 10 min), the supernatant was sequencially diluted, boiled for 10 min, and applied to a slot blot apparatus (HYBRI-SLOT® manifold; BRL, City Md" USA). A BamHl fragment V of EBV (B95-8) DNA [20], labeled with biotin-11-dUTP (BRL) by nick translation [21,22], was used as a probe for the hybridization experiments. DNA-DNA hybridization and detection of virus-spe cific DNA on nitrocellulose filters were carried out as described elsewhere [21].…”

“…A BamHl fragment V of EBV (B95-8) DNA [20], labeled with biotin-11-dUTP (BRL) by nick translation [21,22], was used as a probe for the hybridization experiments. DNA-DNA hybridization and detection of virus-spe cific DNA on nitrocellulose filters were carried out as described elsewhere [21]. The calibration curve was pre pared from the known amounts of viral DNA applied and the height of peak determined with a densitometer (Clini Scan®; Helena Laboratories, City Tex., USA).…”

Rapid diagnosis of herpetic keratitis using direct immunoperoxidase technique

“…Various methods, including tissue culture, DNA hybridization, latex agglutination, immunofluorescent assays, ELISA, and immunoperoxidase techniques are being used for diagnosis of herpetic keratitis. Although tissue culture and DNA hybridization techniques are specific and sensitive, they require high technology and the results are 4 ActaOphthaL 72.1 not available before 24-48 h (Richman et al 1984;Nago et al 1988). The sensitivity of latex agglutination has been reported to be low (Lee & Pepose 1990;Storch et al 1988;Kowalski & Gordon 1989).…”