Adoption of digital PCR is slow at this time, but pathologists may want to track how researchers use this technology in the new clinical laboratory assays they use

Molecular pathologists will be interested to learn that there is a new contender in the polymerase chain reaction (PCR) arena. It is digital PCR, and it has specific advantages over traditional real-time PCR methods.

For example, digital PCR can target specific DNA sequences in just one molecule of DNA. This new tool allows researchers to isolate rare genetic mutations too difficult to segregate with real-time PCR, noted a report from Insight Pharma Reports.

Digital PCR Offers High Sensitivity and Specificity

In fact, digital PCR’s high sensitivity, specificity, and precision is facilitating breakthroughs in research and development because, among other things, it enables detection of rare mutant alleles, including single nucleotide polymorphisms.

Digital PCR also has the ability to copy number variations and to identify prenatal genetic defects,and rare cancer or viral mutations.

Digital PCR Enabled Micropharma Researchers to Develop a New Lab Test

At Montreal, Canada-based Micropharma Limited, researchers used digital PCR to develop a new lab test, called Microbiome SenseIT bsh.

This assay quantifies bile salt hydrolase (bsh) gene abundance in gut-derived samples, noted a press release from Micropharma Limited. bsh is a bacterial gene that produces an enzyme active in the human GI tract that modifies bile acid for the benefit of its host.

Individuals with inflammatory and metabolic conditions, such as inflammatory bowel disease, irritable bowel syndrome and Type 2 diabetes have low quantities of bsh gene.

Bio-Rad Offers Assay that Uses Digital PCR

Microbiome SenseIT bsh is the first test of its kind to use Bio-Rad droplet digital PCR technology, which allows absolute quantification of gene copies with 95% accuracy and produces results within a few hours at a fraction of the cost to sequencing DNA, according to the Micropharma press release.

The test involves about 20000 nanoliter-volume droplets filled with diluted DNA sample. Following droplet generation, digital PCR amplifies the droplets containing bsh gene, which becomes fluorescent, explained Jorge Ganopolsky, Ph.D., the lead research scientist for development of this digital PCR project at Micropharma. “The technique is so sensitive that it can detect only one copy of the gene without false positives,” he declared, noting that the accuracy of droplet digital PCR is up to 10 times greater than traditional PCR, and without this level of accuracy, analysis of bsh would not be possible.

Jorge Ganopolsky, Ph.D. (pictured) was the lead research scientist for Montreal, Canada-based Micropharma’s digital PCR project, which resulted in development of Microbiome SenseIT, a new lab test to quantify the level of bile salt hydrolase (bsh) gene in gut samples. Low levels of bsh have been linked to inflammatory and metabolic conditions, such as inflammatory bowel disease, irritable bowel syndrome and Type 2 diabetes. (Photo copyright Micropharma Limited.)

Jorge Ganopolsky, Ph.D. (pictured) was the lead research scientist for Montreal, Canada-based Micropharma’s digital PCR project, which resulted in development of Microbiome SenseIT, a new lab test to quantify the level of bile salt hydrolase (bsh) gene in gut samples. Low levels of bsh have been linked to inflammatory and metabolic conditions, such as inflammatory bowel disease, irritable bowel syndrome and Type 2 diabetes. (Photo copyright Micropharma Limited.)

 

Why PCR is Vital to Genetic Research

Traditional PCR, or real-time PCR, has been considered the gold standard of nucleic acid quantification. It is commonly used for gene expression analysis. A biosynthetic reproduction of DNA synthesis, PCR allows biomedical researchers to amplify genetic material, and its use has revolutionized research, particularly in studying genetic mutations, viruses, and cancers.

When combined with next-generation sequencing (NGS) methods, which sequence multiple samples of DNA simultaneously, digital PCR improves NGS’ efficiency and cost effectiveness, explained  the Insight Pharma report.

Why Switch From Traditional PCR to Digital PCR?

Real-time PCR lacks the high sensitivity needed to detect rare mutant alleles. When using traditional PCR, rare mutant DNA blends with wild-type DNA. That is different with digital PCR, as the wild-type DNA stands out because of the higher specificity and sensitivity of the technology.

Additionally, real-time PCR sequences must be repeated, usually 30 times, to amplify DNA and verify the sequence, explained the Insight Pharma report. Since fluorescence verifies target sequences, digital PCR eliminates the need for further sequencing. With real-time PCR, the DNA may also be subject to supercoiling, and degradation of DNA due to enzymatic activity, which does not happen with digital PCR.

 

This flowchart, published in an Insight Pharma Report on digital PCR, shows the steps involved in the Real-time, or traditional, PCR process. This process must be repeated about 30 times to verify a DNA sequence. (Copyright Watson, Giman, Witkowski & Zoller)

This flowchart, published in an Insight Pharma Report on digital PCR, shows the steps involved in the Real-time, or traditional, PCR process. This process must be repeated about 30 times to verify a DNA sequence. (Copyright Watson, Giman, Witkowski & Zoller)

Droplet vs. Chip-based Digital PCR

There are two digital PCR platforms available: droplet and chip-based.

Droplet digital PCR technology, which was employed by Micropharma, uses microfluidics to emulsify samples in oil, creating reproducible droplets to be processed and analyzed, stated a report published by Genetic Engineering News (GEN).

The goal of this technology, which is utilized in digital PCR products sold by RainDance Technologies  and Bio-Rad Laboratories, is one molecule of DNA per droplet, with each droplet subject to thousands or even millions of PCR reactions. When analyzing samples, this platform’s fluorescent detection allows molecules contained in the DNA sample to be counted.

This drawing, published in a report from Insight Pharma Reports on digital PCR, depicts the digital PCR process, which verifies target sequences in one step with fluorescence, so researchers can simply count the molecules. (Image copyright Sedlaka & Keith)

This drawing, published in a report from Insight Pharma Reports on digital PCR, depicts the digital PCR process, which verifies target sequences in one step with fluorescence, so researchers can simply count the molecules. (Image copyright Sedlaka & Keith)

 

Chip-based technology operates differently. Chip-based technology partitions samples across a disposable chip. The idea is similar to drop technology in that each DNA partition ideally has one molecule of DNA and detects specific sequences using fluorescence, noted the GEN article. Life Technologies and Fluidigm digital PCR products use this platform.

Unlike droplet technology, chip-based platforms are limited to synthesizing only up to 20,000 partitions, thus presenting a more limited dynamic range. An advantage of this platform, however, is that each partition holds a fixed volume and that prevents coalescence from occurring among samples. With droplet systems there is a slight chance of this occurring.

What Digital PCR Will Bring to Pathologists and Medical Laboratories

Despite the advantages of digital PCR over traditional PCR, at this time relatively few research institutions have fully converted to the new technology. Cost may be one reason, but the GEN report points out other factors, including unfamiliarity with the benefits offered by digital PCR technology or the fact that these PCR methods don’t apply to an instititution’s specific types of research.

That aside, expections are that wide adoption of digital PCR is inevitable. As this new technology spreads to research facilities worldwide, pathologists and medical laboratory managers can expect to see a plethora of new, highly accurate tests for all kinds of medical problems, from rare diseases and birth defects to more common health issues, such as cancers and inflammatory bowel disorders.

—By Patricia Kirk

Related Information:

Digital PCR Technology Report: An Insight to Vendors, Costs & the End User Community

Digital PCR Slow to Convert Users Only a Few Institutions Have Embraced dPCR

 

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