Rapid Fluorescence Detection of in Situ Hybridization with Biotinylated Bovine Herpesvirus-1 DNA Probes

Abstract

Abstract. The use of biotinylated DNA hybridization probes for clinical detection of bovine herpesvirus-1 was investigated. Biotinylated DNA hybridization probes were prepared from bovine herpesvirus-l DNA purified from infected cell cultures. The viral DNA was nick translated in the presence of biotin-dUTP with DNA polymerase to incorporate biotin into the newly synthesized strand. The probe was tested for specificity in in situ hybridization assays with bovine herpesvirus-l DNA. Hybridization was detected using avidinfluorescein single sandwich systems and an avidin-globulin with anti-globulin-fluorescein double sandwich system. Hybridization was detected by specific fluorescence of infected cells. Fluorescence was present only in bovine herpesvirus-1-infected cell culture and not in noninfected cell culture or cell cultures infected with several other viruses. The assay was performed in 6 hr.The diagnosis of viral diseases often is frustrating, time-consuming, and not always definitive. Virus isolation from clinical samples is not always successful because of the presence of antibody, lability of virus in the sample, fastidious requirements for propagation, and low concentrations or absence of virus (due to a low level of virus shedding or latency). The routine isolation of bovine herpesvirus-1 (BHV-1) from clinical samples may be complicated by the presence of nonspecific inhibitors such as interferon. Latent herpesvirus infections are difficult to identify with standard diagnostic methods. 12,24 The ability to construct and use DNA hybridization probes has improved the ability to diagnose and study the pathogenesis of viral diseases at the molecular leve1. 6,16,25,26 This technology has allowed the rapid detection of viruses with a high degree of specificity and sensitivity. Hybridization probes can detect levels of DNA as low as 1-2 pg. 6,24 Specimens include infected cell culture, tissues, secretions, and body fluids. l, l3 In situ hybridization has been used to detect 1 infected cell in 50 uninfected cells and is useful to localize viral genetic material within a ce11. 5,7 The diagnosis and pathogenesis studies of several viral diseases have been documented using gene p r o b e s . 4,8,9,19,28 Most of the early applications using DNA hybridization probes required the use of radionucleotides as reporter groups. Recently the availability of biotinlabeled nucleotides has allowed a broader application of DNA hybridization probes. 2,15,18 the latter is that they are not limited by the expense, equipment, personnel risks, and disposal regulations that plague the use of isotopically labeled probes. In addition, biotinylated probes have a longer shelf-life than radiolabeled probes, and the detection systems are faster than autoradiography. 11,17,18,22 The purpose of the study reported here was to investigate the use of biotinylated DNA hybridization probes for clinical detection of BHV-1, to determine the specificity of the hybridization probe in situ in infected and noninfected cell cultures, and to evaluate h...

Normalization of Dye Bias in Microarray Data Using the Mixture of Splines Model

“…For this reason intensely fluorescent dyes must be used which can unfortunately cause a high amount of background fluorescence. An alternative is to incorporate a non-fluorescent label, such as biotin, during transcription and to attach a fluorescent dye later in the process using molecules with an affinity for biotin (Brock and Potgieter, 1989;Warrington et al, 2000). This indirect labeling allows for the amplification of the signal but introduces a second step that can contribute variation to the process.…”