The use of hybridization probes with melting curve analysis provides significant benefits for diagnostic virology. The approach adds an extra element of specificity to any viral PCR assay. This is because mismatches with the probes will decrease the melting temperature of the hybridization complex formed between the probes and the PCR product during melting curve analysis. The extent of this decrease is dependent on the number and position of the mismatches with the probes. This may serve as a quality control mechanism, where shifts in observed melting temperature from that of expected melting temperature are indicative of sequence variation within the target and may warrant further investigation of the assay performance. On the other hand, such shifts in melting temperature can be advantageous, enabling characterization and typing of two closely related viruses. Briefly, this involves designing a set of hybridization probes capable of hybridizing to both viral types, but having a limited and defined number of mismatches with the DNA of one type and no mismatches with the other type. It is these mismatches and subsequent difference in melting temperature that then enable the two viruses to be distinguished during melting curve analysis.
To date, we have used hybridization probe protocols coupled with melting curve analysis for the routine detection and differentiation of herpes simplex virus (HSV) types 1 and 2 and human polyomaviruses JC and BK (JCV and BKV). Overall, the assays have high clinical sensitivity and specificity. However, two limitations have become evident. First, sequence variation within the viral types can interfere with virus characterization, and second, the assays can fail to detect one viral type when both are present in a single sample (Whiley and Sloots, 2005a).
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