Nonenzymatic Signal Amplification

3.1.1. Hybrid Capture The hybrid capture kits from Digene Inc. (Gaithersburg, MD) can be used to detect HPV, CT/GC, and HSV. Full-length RNA probes are used for the detection of the HPVs and to determine whether they have a

Dna Signal Amplification
Fig. 6. Nonenzymatic signal amplification.

high or low risk for oncogenic potential. Full-length probes negate the potential for false negatives because SNPs do not inhibit hybridization. The hybrid consists of the viral DNA with the RNA probe. An antibody specific to the DNA/RNA hybrid that is attached to the microtiter plate well wall captures this hybrid. Following washing steps, only the hybrid is retained. Another DNA/RNA-specific antibody that is conjugated to alkaline phosphatase detects the captured hybrid following addition of a chemiluminescence substrate. A luminometer measures the light produced from the reaction. An advantage to this particular system is that thin prep and autocyte specimens obtained during routine pelvic examination can be used later to identify infectious agents in specimens determined by cytopathologists to be suspicious or indeterminate (29-31). For example, many Pap smears are not clearly normal or dysplastic/neoplastic, but are designated ASCUS (atypical squamous cells of unknown significance). Without viral typing, the clinical significance of ASCUS is not clear. Using the hybrid capture assay, however, a reading of ASCUS in the absence of HPV DNA or with HPV DNA from a nononcogenic type would be viewed as benign (32). If high-risk HPV DNA is found, however, the patient would be more likely treated/followed as if her cytopathology results were dysplastic. Unfortunately, there are no universal kits available for "home-brew" assays using the Digene system. However, using currently available reagents and user-supplied probes, a "home-brew" assay can be developed as is illustrated by the development of an in-house Parvovirus B19 test (33).

3.1.2. Branched Chain DNA Clinical assays from Bayer Diagnostics utilize branch chain DNA (bDNA) technologies to detect HIV and HCV using numerous subtype-specific probes to capture the target molecule (Fig. 6B) (34,35). The use of numerous subtype-specific probes negates genetic variations that could result in false-negative reactions or inaccurate quantification, as has been observed when using various target amplification methods (36). Synthetic oligomers that are complementary to the target molecule and to an adaptamer are then hybridized to the captured target. Adaptamers are then hybridized to this bridging molecule and these are extended in a branch formation using additional alkaline-conjugated oligomers. All of these reactions are carried out using different hybridization conditions to ensure appropriate branching reactions from the initial target. Ultimately, a substrate is added to generate a detectable reaction product. The QuantiGene kit can be adapted by the user to develop "home-brew" assays. The limiting step for setting up "home-brew" tests using this system is that the equipment for the various incubations needs to be purchased and is not readily available in most clinical laboratories.

3.1.3. In Situ Hybridization In situ hybridization is the only method that allows the identification of infectious agents in the context of the tissue's morphology. Although both target and signal amplification methods can be used for in situ hybridization, signal amplification using the tyramide

Fig. 7. In situ hybridization for aneuploidy.

Chromosome specific probes

Fig. 7. In situ hybridization for aneuploidy.

Hpv Testing With Well Plates
Fig. 8. Enzymatic signal amplification-digestion-cleavase.

amplification method and even bDNA applications are common (37-39). Epstein-Barr virus has been detected in subcutaneous panniculitis and in hypersensitivity reactions to mosquito bites and has been specifically localized to the infiltrating lymphocytes (40,41). The use of in situ hybridization was also useful in definitively identifying HPV-containing cells adjacent to mollus-cum contagiosum bodies (42). Localizing infectious agents to the site of inflammation provides pathologists with the context of the infection that no other method allows. Often infectious agents can contaminant the lesion without actually causing the infection. This caveat is addressed by in situ hybridization.

Additionally, different fluorescent probes specific to various chromosomes can also be used to detect aneuploidy (Fig. 7) in cases where cancer is suspected. In this case, different types of "stars" represent the number of different probes. In the normal example, the number of each star is two. Because the normalized probe or star is two, then the ratio is 1 (2 : 2) for each chromosomal probe. However in the test case, this ratio is greater than 1, which would indicate aneuploidy.

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