In 1992, Prof. Morley and his lab at Flinders University and Medical Center in Adelaide, South Australia, published a general method called “limiting dilution PCR” for quantifying PCR targets. As a proof of concept, they used this method for the quantification of marker mutations in acute leukemia. By diluting DNA samples so that only one or two copies per well were present and then amplifying those copies with PCR, Morley’s team was able to detect two copies of leukemic DNA against a background of 160,000 normal genomes.
They subsequently reported in The Lancet that the outcome of acute leukemia can be predicted by measuring the response to treatment using limiting dilution PCR to quantify the leukemic cells at high sensitivity. In later work, the Morley Lab used real-time quantitative PCR (qPCR) to develop a highly sensitive method for isolating and quantifying the chromosomal translocation that is typically associated with CML.
Using droplet digital PCR to diagnose leukemia
Because the translocation point for each patient is different in CML, real-time PCR conditions may vary from patient to patient and may therefore produce different results. The lab has now returned to digital PCR.
“Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve,” Prof. Morley said.
Monoquant, a company associated with Flinders University, recently used Bio-Rad’s QX100 system to refine the new clinical test for CML. Not only does the instrument offer high sensitivity, it also removes variability in amplification efficiency that results from using patient-specific PCR primers, a traditional sticking point for the FDA. Monoquant hopes the results from the QX100 system will fast-track the FDA approval process for its test.
“It’s a great feeling knowing that something we helped create is propelling our work today,” Prof. Morley said. “We are hoping that this new test we’re developing will offer a better degree of monitoring and better disease management for patients by tracking the progression or remission of CML.”