Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Abstract

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

A comparison between Mini-loop mediated isothermal amplification and polymerase spiral reaction for selective amplification of short template DNA

“…Within the context of our previous efforts, it is clear that the simplified 4-primer AS-Mini-LAMP (demonstrating sensitivity of 1,000 mutant copies in ∼ 60 min and > 10 minutes ahead of non-selective WT amplification) or PSR approach (no amplification) offered no advantages over our previously described 8 and 6 primer reactions (8, 14). False positives (the result of non-specific amplification) remained a challenge, although largely driven by polymerase choice in this case.…”

“…In summary, selective ESR1 E380Q detection within short template DNA is possible using either USS-sbLAMP (designed over ∼200 bp template DNA) (8) or the more compact AS-Mini-LAMP + USS design strategy (155 bp) (14). Amplification performed across shorter templates are likely to be less limited by strand displacement polymerase activity compared to reactions performed on longer templates (9), possibly underpinning the reason behind AS-Mini-LAMPs enhanced limit of detection.…”

“…Moreover, a significant sacrifice in amplification velocity resulted. Our equivalent 6-primer ‘AS-Mini-LAMP + USS’ alterative assay, also spanning 155 bp, provided 2-fold enhanced reaction sensitivity and velocity (500 mutant copies in ∼ 25 minutes) and greater allele selectivity (> 20 minutes ahead of non-selective WT amplification) (14). However, it is important to note that these AS-Mini-LAMP assays target overlapping but subtlety different (a few base pairs) template annealing locations surrounding E380Q, so primer thermodynamic properties may factor here.…”

“…AS-Mini-LAMP (absence of additional USS primers) is compared to an alternative isothermal amplification strategy termed polymerase spiral reaction (PSR) (16), which to date has only been demonstrated for the detection of infectious diseases where reaction template span is not of concern (17, 18). Targeting identical template annealing sequences surrounding the ESR1 E380Q missense mutation (and target of our previous investigations for comparison (8, 14)), this study directly compares the ability of AS-Mini-LAMP and PSR to amplify across a short stretch of synthetic DNA, optimal for cfDNA workflows.…”

“…Of challenge, classic LAMP reactions (9) and our own USS-sbLAMP reactions detailed above (7, 8), typically span greater than 200 bp of template DNA, outside the range of cfDNA (∼ 167 bp) and ctDNA (< 167 bp) fragments (10-13). To overcome this, we developed a more compact allele selective LAMP plus USS reaction termed AS-Mini-LAMP + USS (14) spanning only 155 bp of template DNA, within the range of cfDNA and ctDNA. However, in each of these cases (classic LAMP, USS-sbLAMP and AS-Mini-LAMP + USS) the number of primers required (6, 8 and 6 respectively) opens up the possibility of non-specific amplification, a key challenge for any LAMP optimisation (15).…”