In Situ Hybridization ISH Probes for Virus Detection Identification

ISH uses labeled nucleic acid probes to detect specific DNA or RNA targets in tissue sections and intact cells. ISH combines the specificity and sensitivity of nucleic acid hybridization with the ability to obtain histological and/or cytological information. Probes for ISH were originally labeled radioisotopically with 35S, 32P, or 125I. Newer techniques using nonisotopic hapten digoxigenin are equally as sensitive and exhibit lower background and provide greater resolution than radiolabeled probes. The use of no isotopic labels eliminates the health hazards and disposal problems associated with radioactive probes. Digoxigenin-labeled probes are detected enzymatically with antigoxigenin antibodies conjugated with alkaline phosphatase or horseradish peroxidase. These enzymes convert soluble substrates into insoluble precipitates that appear as dark, localized cellular or subcellular staining. Biotin is another popular nonisotopic label that can be detected with enzyme conjugates of avidin, streptavidin, or antibiotin antibodies.

ISH is performed by transferring a small aliquot of a solution containing labeled probe (single-stranded or denatured double-stranded probes) to protease-digested tissue section. A coverslip is then placed over the specimen to prevent evaporation. Double-stranded targets must be denatured prior to hybridization, and denaturation may enhance hybridization in mRNA or rRNA by eliminating secondary structures. The stability of the hydrogen bonds between probe and target nucleic acid molecule is dependent on temperature, salt and formamide concentration, length, and GC (Guanine-Cytosine) content of the hybrid. Optimum conditions for a successful ISH should be developed by the laboratory performing the test; when a commercial ISH kit is used, the laboratory may still need to modify the procedure to obtain optimum results.

ISH is an important technique for identifying and localizing viral nucleic acids associated with infectious disease and cancer. ISH has been used to determine the intracellular localization of the hepatitis viruses, human papillomaviruses, and herpes simplex viruses, and to detect these viruses. ISH has also been used to detect adenovirus, cytomegalovirus (Wu et al., 1992), JC virus, Epstein-Barr virus (Prange et al., 1992), and HHV-8 (Li et al., 1996). Human papilloma virus (HPV) is accepted as the primary causative agent in the development of cervical cancer. Although there have been approximately 100 HPV genomic types identified, most of these are not oncogenic and therefore do not lead to the development of cervical cancer. Those HPV genotypes that have been identified as types that contribute to the development of cervical cancer are categorized into intermediate and high risk HPV. ISH has been widely used to detect and differentiate HPV in cervical specimens. Dako Corporation (Carpinteria, CA, USA) provides biotinylated DNA probes for HPV ISH, including probes for high-risk group or type-specific probe.

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