HIGHLY SENSITIVE AND SPECIFIC DETECTION OF SNPs AND DNA METHYLATION FROM BLOOD AND PLASMA USING SPECIMEN-SPECIFIC qPCR MIXES.
HIGHLY SENSITIVE AND SPECIFIC DETECTION OF SNPs AND DNA METHYLATION FROM BLOOD AND PLASMA USING SPECIMEN-SPECIFIC qPCR MIXES.
Evelyn Tait, Ph. D. ; William Ferreira, Ph. D. ; Rosa Maria Porreca, Ph. D. ; Danny Filer, Michele Amasio, Ph. D.
www.meridianbioscience.com/lifescience
Background
Liquid biopsy is a non-invasive procedure for the diagnosis and monitoring of oncology patients. The nucleic acids in this type of sample can be analysed through various PCR methods of particular relevance to oncology (Butler 2017). For example, the presence or absence of a particular single nucleotide polymorphism (SNP) can massively impact the prognosis for cancer patients. Similarly, hyper-methylation of a tumour-suppressor gene can confirm or refute a diagnosis for others. Individual genotypes can lead to a predisposition for certain types of tumour and affect the outcome of treatments for others e.g. CYP3A4 steroid hydroxylase may be involved in the aetiology of breast and prostate cancer, through the modulation of sex hormone metabolite levels. Inhibitors found in blood samples and carried through extraction can compromise such assays, whilst low yields of circulating nucleic acids mean that sensitivity and specificity are also significant challenges. We have demonstrated that blood inhibitor tolerant Air-Dryable Direct DNA qPCR Blood (MDX092) and Lyo-Ready ™ Genotyping Direct qPCR Blood Mix (MDX128) are perfectly suited for highly sensitive and specific discrimination of SNPs and accurate detection of methylation levels.
Sensitive Accurate, Reliable Results
Nucleic-acid Extraction
Standard qPCR
OR
Sample collection
Sensitive Accurate, Reliable Results
Direct qPCR
Compatibility with Direct and Indirect Workflows: High inhibitor tolerant formulations are compatible either with direct workflows or those that include extraction steps, making these chemistries suitable for point-of- care (POC) and high-throughput users.
1. SNP Detection - Specificity & Multiplexing
3. Methylation - Bisulfite-Treated Samples
We specifically detected “actionable” mutations in Epidermal Growth Factor Receptor (EGFR) in the presence of the competing, wild-type sequence using MDX092. Figure 1 shows the amplification of exon 21 SNP (L858R) and the wild-type sequence from an equal mix of synthetic template and wild-type human gDNA.
Following bisulfite treatment, the sensitivity and ability to resolve different levels of methylation were evaluated using a set of standards comprising 1%, 2%, 5% and 10% methylated human gDNA. These were used at a total input of 2000, 200 and 20 genome equivalents (ge) in a brcaI qPCR assay that was specific for methylated, bisulfite-converted sequence of the brca1 promoter region (Zhang, 2013). Using MDX092 we could clearly distinguish between 1-5% methylation using template concentrations that were 5 orders of magnitude less than that recommended by Supplier Q (Figures 5 & 6)
EGFR Exon 21 L858R SNP
EGFR Exon 21 wild-type Assay
brca1, 2000 ge
200 ge
20 ge
100 copies, wild-type
100 copies, mutant
100% Methylation 10% Methylation 5% Methylation
100% Methylation 10% Methylation 5% Methylation 2% Methylation
100% Methylation 10% Methylation 5% Methylation 2% Methylation
100 copies, mutant
100 copies, wild-type
1% Methylation
1% Methylation
Figure 5. Methylation Levels in brca1. Clear discrimination between 1%, 2%, 5% and 10% methylation and consistent detection of 1 copy (5% methylated DNA in 20 ge).
L858R Mutation, EGFR Exon 21 | wild-type EGFR Exon 21
Multiplexing L858R alongside the drug-resistant SNP, T790M in exon 20 and a deletion in exon 19 gave a LoD of 10 for all 3 markers in the presence of 10% plasma confirming that sensitivity and specificity were not compromised by inhibitors. Furthermore, the SNP T790M was detected in 25% plasma (Figure 2). Figure 1 MDX092 - Specificity of SNP Detection. A pairwise set of qPCR assays, using specific forward primers was used to amplify mutant and wild-type alleles in EGFR from an equal mix of synthetic template (mutant SNP: L858R) and wild-type human gDNA.
Total Input: 200 ge 2% Methylation
Total Input: 200 ge 1% Methylation
brca1, 20 ge 5% Methylation
Multiplex + 10% Plasma
T790M with & without 25% Plasma; Monoplex & Multiplexed with 2 Other Markers*
40.0 35.0 30.0 25.0 20.0 15.0
MDX092 | used in brca1 qPCR following bisulfite treatment of methylated, human gDNA standards.
Figure 6. Performance Comparison with Specialist Methylation-Detection Master Mix.
4. Methylation - MSRE Analysis (Methylation-Sensitive Restriction Enzyme)
10 copies
1000 copies
Figure 2 Multiplexing in Plasma. A triplex of EGFR mutations amplified from 10 copies of template in 10% plasma, while ct values for T790M assay were unchanged in 25% plasma. * Multiplexed with L858R SNP & Exon 19 Deletion.
Restriction Enzymes that are methylation-dependent (only cut methylated DNA) or methylation-sensitive (only cut UNmethylated DNA) can be used as an alternative to bisulfite treatment in determining levels of methylation. Simply by adding a heat-inactivation step at the end of digestion the methylated DNA was amplified directly using MDX092, discriminating between the same low levels of methylated target (brca1) as with the bisulfite treatment (Figure 7).
2. SNP Detection - Allelic Discrimination
brca1 1000 ge - Methylation-dependent Lpn I
brca1 1000 ge - Methylation-sensitive Ssi I (Aci I)
An Allelic Discrimination assay for CYP3A4 was used in evaluating tolerance of MDX128 of hemin, a common inhibitor in haemolysed blood samples, relative to leading Supplier mixes. Genotyping analysis from the Lyo-Ready ™ Genotyping Direct qPCR Blood Mix showed more accurate allelic discrimination clustering than other available master mixes in the presence of hemin that was approximately 100-fold higher than physiological concentrations, indicating substantially greater inhibitor tolerance using MDX128 (Figure 3).
100% UNMethylated 25% UNMethylated 10% UNMethylated 5% UNMethylated 1% UNMethylated 0% UNMethylated
100% Methylated 25% Methylated 10% Methylated 5% Methylated 1% Methylated 0% Methylated
Figure 7. MDX092 - MSRE Analysis. brca1 qPCR directly from Lpn I and Ssi I (Aci I) digests of human gDNA standards. Restriction digests were heat-inactivated at 80 °C for 20 min and 5 µ l from each digest was then added directly to a 20- µ l qPCR reaction in triplicate.
5. Maximising Sensitivity - Detection Down to a Single Copy
Figure 3. MDX128 - Allelic Discrimination in 2 mM Hemin. Blue: homozygous DNAs for VIC fluorescence ( C allele ). Green: heterozygous samples (A/C). Red: homozygous for FAM ® fluorescence ( A allele ). Black: NTCs or undetermined. Genotyping analysis using MDX128 was compared to mixes from Suppliers T and K in the presence of 2 mM Hemin.
We demonstrated that by air-drying MDX092 and maximising sample volume we could achieve sensitivity down to a single copy of an EGFR Exon 19 deletion (Figure 8).
100% Eluted Sample, MDX092 - EGFR Ex19 Deletion, 1 copy 100% Eluted Sample, MDX092 - EGFR Ex19 Deletion, 10 copies 100% Eluted Sample, MDX092 - EGFR Ex19 Deletion, 100 copies
45% Eluted cfDNA, Supplier K - EGFR Ex19 Deletion, 1 copy 45% Eluted cfDNA, Supplier K - EGFR Ex19 Deletion, 10 copies 45% Eluted cfDNA, Supplier K - EGFR Ex19 Deletion, 100 copies
Figure 4. MDX128 - Allelic Discrimination in Whole Blood & Plasma. Blue: homozygous DNAs for VIC fluorescence ( C allele ) Green: heterozygous samples (A/C). Red: homozygous for FAM ® fluorescence ( A allele ). Black: NTCs or undetermined. Allelic Discrimination using MDX128 was performed in the presence of 10% whole blood and 20% plasma. Similar results were observed in 10% whole blood and 20% plasma (Figure 4) demonstrating suitability for POC testing directly from liquid biopsy samples.
Figure 8. MDX092 - Comparison of Sensitivity & Sample Volume. Plasma extract was spiked with synthetic template that incorporated an EGFR exon 19 deletion. The maximum sample volume that could be used with Supplier K was 45% in a wet reaction. MDX092 was air-dried with primers and probe and therefore could be reconstituted in 100% plasma extract, spiked with the same quantities of template.
Conclusions
References
• Blood-specific inhibitor tolerant master mixes were perfectly suited to molecular analyses either from extracted samples or directly from liquid biopsy . • The optimised formulations showed maximum specificity and amplification efficiency in SNP detection and allelic discrimination . • The tested chemistries were able to resolve 1 and 2% methylation either from bisulfite-treated DNA or directly from MSRE restriction digests . • These qPCR mixes represent powerful tools for the development of oncological diagnostic applications whether these are high-throughput screening or closer to patients, with point-of-care testing due to their compatibility with dried formats.
Timothy M. Butler, Paul T. Spellman and Joe Gray (2017) “Circulating-tumor DNA as an early detection and diagnostic tool” Current Opinion in Genetics & Development 42 , 14-21. Qing Zhang et al. (2013) “A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA” Gynecologic Oncology 130 , 132-139.
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