engleski

Non-radioactive digoxigenin DNA labeling and immunologic detection of HSV PCR products

Background Herpes simplex virus (HSV) is a common cause of human skin and mucous membrane infections, and also causes sporadic meningoencephalitis. As a new method for rapid HSV diagnostics, polymerase chain reaction (PCR) has been introduced in clinical laboratories. Radioactive labeling of DNA probes has become a common practice in experimental laboratories. To avoid radioactive labeling of HSV oligonucleotide probes or PCR products, nonradioactive compounds, which are easily detected by enzyme or immunoassay techniques, are introduced. Objectives The aim of our study was 1. To introduce nonradioactive labeling of HSV DNA probe by digoxigenin-labeled dUTP; 2. to establish a rapid and reliable laboratory method for rapid HSV diagnostics; 3. to compare the PCR method with the standard virology techniques, such as cell culture virus isolation and HSV direct fluorescent antibody test (DFA). Study design We have tested the efficiency of PCR method and nonradioactive labeling of HSV DNA probe for detection of HSV from 30 clinical specimens (skin and mucous membrane swabs). HSV was detected in the specimens by standard virology techniques and PCR. Replicated HSV DNA was nonradioactively labeled by random incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP), and the hybrids were detected by the antibody conjugates and the appropriate enzyme-mediated staining reaction (DIG DNA labeling and detection kit nonradioactive, Boehringer Mannheim GmbH). Results Nonradioactive labeling of hybridization DNA probes with digoxigenin-dUTP was obtained. HSV DNA was successfully multiplied and detected in the HSV-infected cell culture supernatant; however, it was not detected in the clinical specimen supernatant or sediment. HSV DNA was detected by direct PCR method in noncentrifugated clinical specimens. Conclusions The PCR method could be successfully used for diagnoses of HSV infections. Since the sensitivity of this method is partly limited by the virus quantity in the specimen, we recommend cultivating the virus in the cell culture at least 24 h prior to PCR. The use of nonradioactive labeling of hybridization DNA probes, such as random primed DNA labeling with digoxigenin-dUTP, has proven both sensitive and specific, and more appropriate for diagnostic purposes than radioactive DNA labeling to be used until standardized commercial tests appear. Keywords:

Herpes simplex virus; Polymerase chain reaction; Digoxigenin-dUTP DNA labeling

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