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Multiplex and high fidelity: the potential of qPCR

Ahead of the qPCR & Digital PCR Congress, we sat down with David Zhang to talk about his work with PCR as a diagnostic platform.

Please tell us about your lab and your work involving PCR?

My lab has been working on DNA molecular diagnostics ever since we started up as an independent laboratory at Rice University. PCR is one of the better-established methods for diagnostics with Nucleic Acids.

Our goal is enabling precision molecular diagnostics which helps patients achieve better outcomes, primarily in cancer and infectious diseases.

I am passionate about preventing disease, rather than curing a disease expensively once it has already advanced to a stage where it has become difficult to control.

We are currently working on two PCR related projects.

The first involves developing qPCR-based methods for analysing rare mutations in cancer. These are mutations in genes which can cause drug insensitivity and resistance. Alternatively, they could be personalised tumour-specific markers that help oncologists identify cancer recurrence after treatment.

The other project is an instrument platform that my lab has been developing for infectious diseases. This ‘donut PCR’ enables 50- to 1000-plex qPCR readouts from a single sample in less than 30 minutes using one closed consumable.

What successes have you seen in these projects so far?

We have two start-ups commercialising each of these technologies.

In terms of the qPCR-based assays for cancer, we currently have the world’s best sensitivity for qPCR. We can reliably detect mutations with as low as 0.1% variant allele frequency (VAF) using qPCR, and we can do this in a multi-plex way. If you compare this to other commercial assays, their limit of detection is around 1-7% VAF. We currently have advantages in sensitivity over other players in the qPCR space.

With instrument platform for infectious diseases, the big success is the fact that we can detect fifty different DNA targets from a single closed system in 30 minutes. As far as I am aware, the closest technology can achieve 20-plex detection in an hour.

Please tell us more about the “donut PCR”?

This technology is a marriage of qPCR with microarrays. Fundamentally, it combines multiplex qPCR with micro-array based readout.

qPCR can multiplex up quite significantly, performing PCR simultaneously on 10s to 100s of target DNA sequences. The problem then is “how do you read it out?”

Commercial PCR instruments usually use spectrally distinct colour channels: green, red, blue channel. This approach can limit the readout to five or maybe six channels. Therefore, in a single sample, you can only detect about five to six different DNA targets.

Instead of trying to separate different target pathogens by colours, we have distinguished them by space: i.e. their positions on a DNA array. For example, at 1.1. might be e.coli, 1.2 might be MRSA, and 1.3 streptococcus. We can, therefore, achieve much higher multiplexing in terms of the readout for a closed system.

Why has no one done this already? Usually, micro-arrays are a slow process, requiring a 16-hour hybridisation time and wash process.

We have invented technology to overcome this restriction so we can get from DNA to answer in thirty minutes and do this in a closed system that does not require washes.

What do you imagine being the clinical application(s) of this technology?

A rapid turnaround time is critical with infectious diseases. Our idea is to apply this method to syndromic testing.

For example, if someone presents with respiratory symptoms or an inflamed urinary tract, we want to understand which pathogen is causing this syndrome. Based on this information, the doctor can provide a very targeted treatment.

This method would help stem the worldwide spread of antibiotic resistance. It would also help with antibiotic side-effects. Clinicians will often prescribe broad-spectrum antibiotics which can kill all the microbiota in the gut and lead to multiple other problems.

By narrowly pinpointing the pathogen, we can inform the clinician to take appropriate action in a narrow setting.

What are your hopes for future work and challenges you are hoping to tackle?

Our lab is also interested in higher-fidelity DNA polymerases, as most of the qPCR assays on the market use Taq based enzymes. These are the oldest enzymes used for PCR assays. The problem is they have quite limited fidelity, making an error roughly every 8000 bases copied. This error rate limits the accuracy of qPCR.

There are better assays out there with error rates that are about one in a million. The problem here is that they are not compatible with TaqMan.

My lab has been working on designing probe systems in a way that we can use the high-fidelity DNA polymerase assays in familiar multiplexed qPCR assays.

David Zhang is Associate Professor of Bioengineering at Rice University, USA.

The 7th qPCR & Digital PCR Congress meeting will focus on the underlying statistics of Digital PCR such as developing optimal workflow. Click here to see what else is on the agenda.

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