Nucleic acid hybridization in the diagnosis of viral infections.
Recombinant DNA technology, including molecular cloning and nucleic acid hybridization, is now being applied to problems in clinical virology. Although viral isolation in cell culture remains the most sensitive and specific diagnostic test for many viruses, for some viruses, isolation in cell culture is lengthy or difficult or has not yet been achieved. Utilization of hybridization techniques has already resulted in important new information concerning the pathogenesis of a number of viruses, such as Epstein-Barr virus, hepatitis B virus, and human papillomavirus. In addition, time to diagnosis for viruses such as cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus can be significantly shortened to 36 to 48 hours, a great improvement over standard isolation with obvious importance for patient management. Hybridization techniques have also been applied to screening of antiviral agents. Although results of studies to date have been encouraging, significant problems remain to be solved before these techniques can be applied in a routine diagnostic laboratory. First, more sensitive assays must be developed. One approach is the generation of probes with higher specific activities. Synthesis of single-stranded probes using recombinant M13 bacteriophage as a template results in probes of higher specific activities that also cannot re-anneal to themselves because they are not complementary. Thus, more probe is available to anneal to sample DNA. Synthesis of cRNA probes that form more stable hybrids with DNA is another approach that is receiving attention. A second problem is reagent safety and stability. The most sensitive and commonly used label in the studies reviewed in this article has been 32P. With its half-life of 2 weeks, potential hazards to personnel, and disposal problems, it is probably not suitable for clinical laboratories. A major step in the development of nonradioactive, stable probes has been synthesis of biotinylated nucleotide analogues that can be efficiently incorporated into DNA or RNA. Biotinylated probes are stable for 1 to 2 years at -20 degrees C, and their use obviates the need for autoradiography, thus shortening reaction times. In addition, very high concentrations of probes can be used without the background problems encountered with radiolabels. To date, biotinylated probes have been significantly less sensitive than those labeled with 32P, but continued efforts to improve sensitivity have yielded promising results.(ABSTRACT TRUNCATED AT 400 WORDS)
D001710:Biotin; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005819:Genetic Markers; D006801:Humans; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000032:analysis; D013997:Time Factors; D014771:Virion / Q000032:analysis; D014777:Virus Diseases / Q000175:diagnosis* / Q000382:microbiology; D014780:Viruses / Q000032:analysis
D004274:DNA, Recombinant; D004279:DNA, Viral; D005819:Genetic Markers; D012367:RNA, Viral; D001710:Biotin