(H-291) High resolution melt analysis using L-DNA internal calibrator: Detection of Isoniazid drug-resistant tuberculosis

Introduction::
High resolution melt analysis (HRM) characterizes PCR-amplified nucleic acid products based on their sequence-specific melting temperature (Tm). The melting profile of a double-stranded amplicon depends on its guanine-cytosine content, length, and sequence¹. HRM is a rapid and cost-effective manner for genotyping, mutation scanning, and detecting sequence variants down to the single base pair². Detecting small changes in PCR product sequences via HRM is challenging but has been shown to be possible for detection of drug resistance related mutations, such as in tuberculosis (TB)³.




Currently, melt analysis detection of single base differences is limited to high-resource settings with access to highly calibrated PCR instruments and technical expertise required for complex reaction preparation. Resource-constrained settings lack access to calibrated instruments capable of detecting the very small differences in Tm (2°C to less than 0.5°C) for two equal length sequences with a single base change in one nucleotide. An approach capable of high-resolution analysis with simpler PCR instrumentation would make HRM detection strategies more widely available in resource-constrained settings.




In this report, we have developed an internally calibrated melt analysis for PCR detection of drug resistance. This single tube melt analysis method includes an internal synthetic comparator with melt characteristics identical to the reference sequence PCR product. We demonstrate the use of internal L-DNA calibration in melt analysis to confirm drug susceptibility to isoniazid (INH), a first-line pro-drug therapeutic for TB, by detecting resistance-associated base changes in the katG gene. This approach does not require multiple tube comparisons or a calibrated instrument.




Materials and Methods::
The internal calibration HRM strategy was designed around the TB katG gene which is known to develop single base changes conferring INH drug resistance. Single-stranded D-DNA targets representative of drug-susceptible TB and nine clinically relevant INH-resistant katG mutants were used as target amplicons. Mutants included the most prevalent variant, S315T, which is found in 94% of INH-resistant clinical isolates^4,5, and other nucleotide polymorphisms in neighboring regions. A double-stranded L-DNA drug-susceptible katG comparator was designed from DNA enantiomeric bases such that it had identical melt characteristics to the reference drug-susceptible D-DNA target and did not participate or interfere with PCR. All D-DNA oligonucleotides were synthesized by Integrated DNA Technologies; all L-DNA oligonucleotides were synthesized by Biomers.




Detection of the amplicon product was performed by PCR. Each 20μL reaction contained 10μL of the SensiFAST™ HRM Kit containing EvaGreen® intercalating dye, 0.5μL of 10μM katG-specific primers, and 1μL of end-labeled forward and reverse L-DNA strands in a 1:1 ratio for a concentration of 7.5 x 10¹¹ copies/reaction. The workflow included preheat (95°C–2 min), melt analysis (65ºC to 95ºC at 1ºC/s), thermocycling (95°C–2 min, 40 cycles of 95ºC–5s and 59ºC­–20s), and melt analysis (65ºC to 95ºC at 1ºC/s). Melting temperatures determined with Rotor-Gene Q software analysis features were used for validation of our technique. To identify samples as mutant or wild-type, we performed a custom comparative analysis of the first derivative of fluorescence change over temperature using raw melt data generated by the Rotor-Gene Q before and after PCR.




Results, Conclusions, and Discussions::
Results and Discussion: L-DNA was successfully employed as an HRM internal calibrator to detect single base changes conferring INH drug resistance in the TB katG gene. The melt characteristics of the pre-PCR mixture (L-DNA alone) were compared to the melt characteristics post-PCR (both L-DNA and D-DNA) to assess the presence or absence of sequence variation in the D-DNA amplicon product as compared to the reference drug-susceptible wild-type L-DNA (Figure 1). Each D-DNA drug-susceptible target had no statistically significant difference in post-PCR Tm when compared to the pre-PCR Tm of wild-type katG L-DNA (p >0.05, ANOVA, paired t-test). Single base changes in D-DNA drug-resistant mutants were detected with resolution smaller than 0.2 ºC. Each D-DNA drug-resistant mutant had a statistically significant difference in post-PCR Tm when compared to the pre-PCR Tm of wild-type katG L-DNA (p< 0.05, ANOVA, paired t-test). The aforementioned results depend on the use of multiple tube comparisons, statistical analysis, and calibrated instruments. A custom analysis approach based on single tube internal comparison demonstrated correct detection of 3/3 drug-susceptible wild-type samples, 17/18 drug-resistant mutants, and 3/3 no template controls (N=3 per group). Notably, our custom detection technique does not rely on multiple tube comparisons, statistical analysis, or calibrated instruments.

Conclusions: In conclusion, we developed a single tube HRM strategy that effectively uses internal L-DNA calibration to confirm INH drug susceptibility with single base change resolution. This technique can be adapted and applied to variant scanning for many other mutation-induced diseases and disease variants, such as rifampicin-resistant TB in which any single nucleotide polymorphism in TB’s rpoB gene confers drug resistance. These proof-of-principle results demonstrate the first development of L-DNA-based HRM detection that does not depend on the calibrated instruments or multiple tube comparisons required by current gold-standard HRM approaches, and thus makes this approach particularly well-suited for resource-constrained settings.




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References (Optional): :

1. Farrar, J. S. & Wittwer, C. T. High-Resolution Melting Curve Analysis for Molecular Diagnostics. Molecular Diagnostics: Third Edition 79–102 (2017) doi:10.1016/B978-0-12-802971-8.00006-7.


2. Wittwer, C. T. High-resolution DNA melting analysis: Advancements and limitations. Hum Mutat 30, 857–859 (2009).


3. Keikha, M. & Karbalaei, M. High resolution melting assay as a reliable method for diagnosing drug-resistant TB cases: a systematic review and meta-analysis. BMC Infect Dis 21, (2021).


4. Vilchèze, C., Jr, W. R. J. & Author, M. S. Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis: Genes, Mutations, and Causalities HHS Public Access Author manuscript. (2019) doi:10.1128/microbiolspec.MGM2-0014-2013.


5. Zhang, Y., Heym, B., Allen, B., Young, D. & Cole, S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358, 591–593 (1992).