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Recent Studies Demonstrate Bio-Rad Droplet Digital PCR for Diverse Array of Applications

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A flurry of recently published peer-reviewed papers has demonstrated the growing breadth and diversity of applications for Bio-Rad's QX100 Droplet Digital PCR system.

Specifically, scientists from a variety of universities, research institutions, healthcare organizations, and government entities in the US and Europe published research establishing Bio-Rad's digital PCR platform as a potentially useful tool for breast cancer screening, measuring latent HIV reservoirs in patients, diagnosing hospital-acquired and sexually-transmitted infections, and analyzing food and feed samples for the quantification of genetic modifications.

Bio-Rad acquired the QX100 system along with its original developer, QuantaLife, for $162 million in October 2011. Although the early benefits of the technology were obvious for applications such as copy number evaluation and rare event detection, it was widely regarded as a technology without a killer application (PCR Insider, 10/6/2011).

Bio-Rad quickly set out to address this issue, forging collaborations with numerous academic institutions, government entities, and companies, and establishing a "digital biology center" near its headquarters in Hercules, Calif.

The recent influx of publications using the QX100 provides some indication that Bio-Rad's investment is beginning to pay off.

HER2 Amplification

First, in a study published last month in Clinical Cancer Research, researchers from the Royal Marsden Hospital in London described their design and use of a droplet digital PCR assay to non-invasively analyze HER2 copy number in plasma.

Because HER2 copy number in digital PCR is assessed relative to a reference gene, the researchers first assessed a pair of genes on chromosome 17, near the HER2 locus, that had been suggested by the literature and microarray comparative genomic hybridization as potential references. However, they found that these reference genes were suboptimal for a variety of reasons, and instead settled on EFTUD2, a gene within the ERBB2 locus that they deemed a superior reference because it was found not to co-amplify with HER2 and was not subject to normal copy number variations.

Then, they conducted digital PCR using the QX100 system and custom primers against HER2 and EFTUD2, first on a development cohort of 65 patients and then in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The team used a sequential probability ratio test to assign the plasma DNA digital PCR assay as being HER2-positive or –negative in the validation cohort.

In that cohort, the researchers found that 64 percent of patients with HER2-amplified cancers were classified as digital PCR HER2-positive and 94 percent of patients with HER2-nonamplified cancers were classified as HER2-negative by the assay, giving a positive and negative predictive value of 70 percent and 92 percent, respectively.

The authors concluded that "digital PCR of plasma DNA has high accuracy in the determination of HER2 status," and that the approach "could be adapted to the assessment of any amplified locus in cancer." They also noted that, in particular, the technique shows promise as a screening strategy "for potentially rare acquisition events in response to therapy, such as acquisition of MET amplification following EGFR-targeting therapy in lung cancer."

Latent HIV Infection

In another study published in April in PLoS One, a group comprising researchers from the University of California, San Diego, and the Veterans Affairs San Diego Healthcare System applied droplet digital PCR to measure the frequency of total HIV DNA (as measured by pol copies per millions cells) and episomal 2-LTR circles in cells isolated from infected patients.

The usefulness of this application of droplet digital PCR has been challenged by others, most notably scientists from Brigham and Women's Hospital and Harvard Medical School, who in December published a paper in the Journal of Virological Methods providing evidence that droplet digital PCR was no better than real-time PCR at detecting and quantifying low levels of cell-associated HIV-1 genetic material from samples taken from patients both on and off antiviral therapy (PCR Insider, 10/11/2012).

However, in the recent PLoS One study, the researchers analyzed more than 300 clinical samples, including more than 150 samples assayed in triplicate by ddPCR and by real-time PCR. They found that ddPCR yielded a significant increase in precision, with an average five-fold decrease in the coefficient of variation of pol copy numbers and a more than 20-fold accuracy improvement for 2-LTR circles.

Additionally, the researchers noted that ddPCR had the added benefit over qPCR of providing absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences.

One important limitation of the QX100 system, they noted, is its dynamic range, which meant that "HIV DNA copy numbers per cell cannot be measured in a single well by ddPCR" — an issue they circumvented by combining dilution series with the ddPCR assay. Another issue they noted is that the QX100 resulted in some unexplained false positives, events that they chalked up to potential limitations in the system's software. This latter issue was also cited in the JVM paper published last year.

Nevertheless, the San Diego team concluded that "despite these limitations, droplet digital PCR was found to be a practical method for highly accurate measurement of HIV DNA targets in clinical specimens," and is "well-suited to measurement of the size of the HIV latent reservoir," suggesting that the assay could prove useful for clinical studies aimed at eradication of HIV from infected patients.

HAIs and STDs

Two other recently published studies, both in the Journal of Clinical Microbiology, have demonstrated the potential of droplet digital PCR on the QX100 as a tool for infectious disease detection and diagnosis.

The first study, conducted by scientists from the University of Mississippi Medical Center and Bio-Rad, validated a ddPCR assay for high-precision analysis of methicilllin-resistant Staphylococcus aureus.

In their study, the researchers compared a QX100 assay and a real-time PCR assay run on the Roche Light Cycler to detect genes for the staphylococcal protein SA0140 and the methicillin resistance gene, mecA, using standard TaqMan chemistries. Both the qPCR and ddPCR assays correctly identified culture controls for MRSA, methicillin-sensitive S. aureus, and confounder organisms with 100 percent sensitivity and specificity.

Then, the team analyzed clinical samples (211 negative and 186 positive) to directly compare the qPCR and ddPCR assays to Cepheid's MRSA Gene Xpert assay. They analyzed a total of 397 clinical samples and used the Gene Xpert values to define negative and positive samples.

Both the qPCR and ddPCR assays were in good agreement with the reference assay, with both demonstrating sensitivities of 96.8 percent, and with qPCR demonstrating a specificity of 91.9 percent and ddPCR demonstrating a specificity of 91 percent.

In the second JCM study, a team comprising scientists from the London School of Hygiene and Tropical Medicine, the Medical Research Council Unit of The Gambia, and the Programa Nacional de Saude de Visao of Guinea Bissau developed a ddPCR assay for ocular Chlamydia trachomatis infections.

Using 1,509 clinical conjunctival swab samples from a trachoma-endemic population in Guinea Bissau, the team evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing its performance to the Roche Amplicor CT/NG test.

The sensitivity of ddPCR against Amplicor was 73.3 percent, specificity was 99.1 percent, and negative and positive predictive values were 94.6 percent and 94.5 percent, respectively.

Based on these results, the authors concluded that ddPCR has "great utility as a diagnostic," and unlike other nucleic acid amplification tests it "requires no external or internal calibration yet delivers a highly accurate estimation of target load." Further, they wrote that their assay could "rapidly be adapted for use in the detection of sexually transmitted C. trachomatis infections, where a higher sensitivity that which we have estimated would be desirable."

GMOs

Finally, a study published last month in PLoS One has demonstrated the potential of ddPCR as a suitable tool for routine analysis of genetically modified organisms in food, feed, and seeds.

In the study, scientists from the National Institute of Biology in Slovenia and Lawrence Berkeley National Laboratory used a ddPCR duplex assay to measure the absolute numbers of MON810 — a well-characterized transgene used in corn production — and hmg maize reference gene copies in DNA samples.

The researchers discovered that their assay achieved a wide dynamic range of close to five orders of magnitude with an upper limit of quantification of about 118,000 target copies, and showed "very good sensitivity" suitable for GMO testing.

"The excellent performance of the tested parameters enables the quantification of samples from different matrices, using DNA extracted with common methods without up-front DNA quantity estimation," the researchers wrote. "The limits of quantification, trueness, and repeatability of the duplex assay comply with international recommendations."

Further, the team noted that its data, in the context of GMO quantification, suggest that "ddPCR running costs are lower than those of standard qPCR technology, given the superior throughput, and especially when numerous samples are handled simultaneously."

In a statement accompanying publication of the paper, Dany Morisset, lead author and a researcher at Slovenia's National Institute of Biology, concluded that "droplet digital PCR could replace or be a good alternative to qPCR, the current benchmark in GMO quantification."

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