Detection of Nucleic Acids by Enzyme-Linked Immuno-Sorbent Assay (ELISA) Technique: An Example for the Development of a Novel Nonradioactive Labeling and Detection System with High Sensitivity

Keßler, Christoph

doi:10.1007/978-3-642-74955-1_5

Cited by 9 publications

(1 citation statement)

References 157 publications

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“…In a 1.5-ml tube, 50,u of CSF was added to 150 [lI of a solution consisting of 20% (wt/vol) Chelex 100 resin (Bio-Rad Laboratories, Richmond, Calif.) in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA-0.1% sodium azide. After vortexing for 10 s, the tube was incubated at 56°C for 20 min; this was followed by vortexing for another 10 s. After incubation at 100°C for 10 min, the tube was allowed to cool to room temperature.…”

Section: Materlals and Methodsmentioning

confidence: 99%

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique

et al. 1994

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A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutofwas determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.

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Section: Materlals and Methodsmentioning

confidence: 99%