Digital PCR – improving reproducible molecular methods in the clinic

Posted on Thu, Apr 06, 2017 @ 08:40 AM

A recent study coordinated by LGC and published in the journal Analytical Chemistry, has demonstrated the potential of digital PCR (dPCR) to improve the reproducibility of routine clinical testing between facilities.

Real-Time PCR has revolutionized the way clinical microbiology laboratories diagnose a whole range of diseases and conditions, from cancer to infectious diseases. However while the method is precise, ensuring reproducible measurements between laboratories can be challenging.

Jim Huggett, Principal Scientist at LGC“One of the biggest obstacles to the adoption of quantitative molecular methods in the clinical arena has been the inability of the methods to perform with high reproducibility,” states Jim Huggett, Principal Scientist at LGC. Which is why the recent study to examine the reproducibility of featuring 21 independent laboratories is such a big deal.

Not only is the study the first to examine the reproducibility of dPCR between such a large number of laboratories, it also highlights the ability of dPCR to detect relevant rare sequence variants.

In the study, the laboratories were asked to test for a KRAS point mutation – one of the most difficult types of cancer biomarkers to identify. All the test materials were prepared at LGC and the samples were then analysed at the individual laboratories.

“Three levels of mutation concentration were analyzed in a background of wild type sequence, from ~0.2% to 12% fractional abundances. The participants in the study had various levels of experience with dPCR, ranging from being highly experienced, to being relatively new to the technique” Huggett explains.

Huggett concludes that, “When present as low as 0.2%, these results demonstrate that fractional abundancies and absolute quantities of mutant sequence can be reproducibly quantified using dPCR without the need for calibration.”

So how does it do this? the technique minimizes the variability from common sources of error that influence qPCR results permitting precise and accurate quantification of nucleic acid concentrations.

One major source of variability in qPCR is establishing a standard curve – and this isn’t necessary in dPCR as it provides absolute quantification of target nucleic acids.

While reproducibility challenges have plagued the clinical adoption of PCR-based platforms in the past, with this study comes support for a new and “more robust approach to nucleic acid analysis”. Huggett predicts that dPCR will be “increasingly used as a reference method to improve the harmonization across laboratories”, and as it is continuously used more and more will “find they are more easily able to obtain comparable results”.

Topics: Real-time PCR

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