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Novel design of nucleic acid standards for hydrolysis probe-based PCR with melting analysis

Gene Therapy volume 29, pages 425–430 (2022)Cite this article

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Abstract

Probe-based polymerase chain reaction (PCR), a popular method in genetic testing, could be prone to false positive or positively biased results if standards used for positive controls or as calibrants accidentally contaminate samples during analysis. To prevent this, a unique design strategy for nucleic acid standards has been developed. Several in-house designed synthetic standards and corresponding test targets were analysed in specific probe-based PCR assays in the presence of SYTO 82™, an intercalating dye compatible with a probe-labelling FAM (6-Carboxyfluorescein) fluorophore. PCR was followed by melting and fragment size analyses. We showed that a standard can be designed to allow discrimination from the test target in post-PCR melting analysis based on differences in melting temperature (Tm). A good predictor of Tm differences for the paired amplicons was the software package uMelt, but not the length of the amplicons nor guanine-cytosine (GC) content. Tm-based determination can be complimented by electrophoresis to measure differences in amplicons’ length. Designing genetic standards using the described method for tests that utilise probe-based PCR will prevent false positive and inaccurate results, while also simplifying the test and reducing its cost.

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Fig. 1: Design strategy for DNA standards for multiple assays.
Fig. 2: Discrimination of amplicons from paired WT and STD targets based on size.
Fig. 3: MCA results of amplicons generated from paired WT or STD.
Fig. 4: Relation between the experimentally measured ΔTm for the paired WT and STD amplicons and their difference in length, GC content or predicted ΔTm.

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Acknowledgements

We thank Leo Pinheiro (NMI) and Mark Van Asten (Diagnostic Technology) for supplying MCYE/NDAF and CYRA PCR assays, and the STD and WT materials. The authors thank Leo Pinheiro and Daniel Burke (NMI) for critically reviewing the manuscript.

Funding

This work was supported by the World Anti-Doping Agency (grants 14C13AB and 16E05AB) and the Australian Government’s Department of Health.

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  1. National Measurement Institute, Lindfield, NSW, 2070, Australia

    Anna Baoutina & Somanath Bhat

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  1. Anna Baoutina
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  2. Somanath Bhat
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Contributions

AB conceived and designed the study, participated in experimental work and analysis, and wrote the paper. SB contributed to standards design, experimental work including optimising data acquisition parameters, data analysis, formatting the figures for publication and reviewing the manuscript.

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Correspondence to Anna Baoutina.

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Baoutina, A., Bhat, S. Novel design of nucleic acid standards for hydrolysis probe-based PCR with melting analysis. Gene Ther 29, 425–430 (2022). https://doi.org/10.1038/s41434-021-00288-0

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