Application of Melting Curves to Identify High Risk HPV Types

Robert Bruce

Product Development

Celera

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Objective and Methodology

● Objective: To try to simplify and reduce the costs of HPV variant detection we investigated the prospect of possible application melt curve analysis in determining HR HPV variants.

● Materials and Methods: The method consists of adding a double stranded specific fluorescent dye to the PCR reaction, post PCR thermal melting of the amplicon and reading of the fluorescence emitted by the dye at each temperature point. The resulting melting curves are subsequently analyzed by the software provided by manufacturer (Life Technologies). Identification was verified by sequencing the amplicons.In theory, melt curve analysis enables the distinction of PCR amplicons differing in even a single nucleotide, and as such should be capable of indicating HPV variantsIn this feasibility study, we tested the performance of melt curve analysis in a multiplexed PCR assay with our HPV primer set and HR HPV plasmids.

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Thermocycling Protocol

The following thermocycling protocol was used:

A 40 cycle program followed by a thermal gradient from 60ºC to 95ºC

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Amplification Curves of High Risk HPV Types

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Dissociation Curves of High Risk HPV Types

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Comparison Between HR HPV Sequences Showing Homology Between Variants (HPV 18 and 45) and (HPV 35 and 52)

HPV 18

HPV 45

HPV 35

HPV 52

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Comparison Between HR HPV Sequences Showing Homology Between Variants (HPV 31, 51 and 68)

HPV 31

HPV 51

HPV 68

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Results and Conclusion

● Results: Of the HPV variants in this study, HPV 16, and 33 yielded melt curves that were different from the other variants studied. HPV 45 yielded results identical to HPV 18, while HPV 52 was identical to HPV 35. HPV 51 and 68 yielded results identical to HPV 31. This may be due to the fact that these HPV types are more highly related to each other than the rest of the variants studied as illustrated on the previous slide.

● Conclusions: This approach has definite possibilities, but was limited due to the limitations of the instrument used. Using an instrument capable of providing the necessary precision needed to detect the few nucleotides that differ and separate the variants could result in the resolution of the remaining high risk HPV types in single or mixed infections.