Syzygy Biotech™ Launches rEVAlution qPCR Master Mix

Syzygy Biotech™, a company that produces high-grade PCR reagents and enzymes, announced today that it has introduced rEVAlution qPCR Master Mix, an environmentally-safe, ready to use hot-start qPCR mixture specifically formulated with high quality components to offer superior safety, speed, and signaling capabilities.

EvaGreen® dye was purposely selected for this formulation because it is cell membrane impermeable making it a non-mutagenic, non-cytotoxic PCR dye that’s safe for aquatic life, safe for handling, and can be directly disposed of down the drain. Similar master mixes typically utilize SYBR® green dyes that are mutagenic. EvaGreen® also helps rEVAlution produce signals that are stronger than the SYBR® green dyes. As a Syzygy Biotech customer noted “I compared rEVAlution [to] a competitor SYBR®, and found CT values that are very similar, but [the] signals are four times stronger.”

In addition, Syzygy FlashTaq HotStart, a chemically-modified hot-start Taq DNA polymerase recognized for its rapid activation and high level of compatibility was selected for use in the formulation.

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BIO SPACE: Empirical Bioscience Launches rEVAlution qPCR Master Mix

GRAND RAPIDS, MICHIGAN – Empirical Bioscience™, a company that produces high-grade PCR reagents and enzymes, announced today that it has introduced rEVAlution qPCR Master Mix, an environmentally-safe, ready to use hot-start qPCR mixture specifically formulated with high quality components to offer superior safety, speed, and signaling capabilities.

EvaGreen® dye was purposely selected for this formulation because it is cell membrane impermeable making it a non-mutagenic, non-cytotoxic PCR dye that’s safe for aquatic life, safe for handling, and can be directly disposed of down the drain. Similar master mixes typically utilize SYBR® green dyes that are mutagenic. EvaGreen® also helps rEVAlution produce signals that are stronger than the SYBR® green dyes. As a Empirical Bioscience customer noted “I compared rEVAlution [to] a competitor SYBR®, and found CT values that are very similar, but [the] signals are four times stronger.”

In addition, Empirical Bioscience FlashTaq HotStart, a chemically-modified hot-start Taq DNA polymerase recognized for its rapid activation and high level of compatibility was selected for use in the formulation.

The Empirical Bioscience FlashTaq HotStart enzyme in the Master Mix can be activated in just two minutes versus the ten minutes of activation time required by many competitors’ formulations.

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Target Enrichment for Clinical and Molecular Diagnostics

By Anar Murphy, Ph.D., GEN Genetic & Biotechnology News

Target-enrichment technologies offer increasingly powerful and cost-effective ways to detect various pathogens, disease biomarkers, and toxins by molecular amplification or direct capture in clinical diagnostics, basic research, and industrial applications.

The latest developments in the field were discussed earlier this year at two conferences—CHI’s “Sample Prep and Target Enrichment in Molecular Diagnostics” and the Knowledge Foundation’s “Sample Prep”.

Mark Eshoo, Ph.D., director of new technology at Ibis Biosciences, a subsidiary of Abbott Molecular, talked about an isothermal amplification, broad-range PCR, electrospray ionization mass spectrometry (IA/PCR/ESI-MS) assay that allows early diagnosis of Lyme disease. The assay works by detecting the tick-borne pathogen Borrelia burgdorferi directly from whole blood, cerebrospinal fluid, or other clinical specimens. To achieve the necessary sensitivity, the assay selectively enriches the target Borrelia sequences in large sample volumes.

“The Centers for Disease Control and Prevention reports that there are an estimated 300,000 new Lyme disease cases a year in the United States. If left untreated, this pathogen can lead to chronic long-term infection,” said Dr. Eshoo.

“The optimal time to treat Lyme disease is at the onset of symptoms,” said Dr. Eshoo. “Currently, serological tests have low sensitivity early in the infection due to the biologically delayed immune response, and as a result, the tests cannot distinguish active infections from prior exposures.”

“Abbott has been working to develop a method for the direct detection of the bacteria that causes Lyme disease, Borrelia burgdorferi. To detect the bacteria, Abbott has developed methods for using large volumes of blood and an isothermal amplification technique that we used to increase our assay sensitivity,” added Dr. Eshoo.

The isothermal amplification, an effective and simple non-PCR DNA enrichment technique, was added to the existing PCR/ESI-MS assay. The study targeted eight Borrelia loci by using 50 primers per target (400 primers in total) in conjunction with a strand-displacing DNA polymerase to increase the target enrichment efficiency in the sample DNA extracted from 1.25 mL of whole blood.

The IA/PCR/ESI-MS assay takes eight hours and can detect Borrelia burgdorferi prior to an active infection. These capabilities allow doctors to diagnose erythema migrans and acute Lyme disease early in disease progression before seroconversion.

Next-Generation Sequencing

Jude Dunne, Ph.D., vp of product development at WaferGen, described the SmartChip TE system, which uses next-generation sequencing (NGS) to provide a target-enrichment solution for clinical and translational research.

“The system uses a singleplex PCR enrichment strategy, with a single reaction occurring in each of several thousand wells on a SmartChip TE panel in less than three hours,” said Dr. Dunne. “At the end of the enrichment, all the amplicons are extracted into one pool ready for sequencing. In situ addition of the required sample-identifying barcodes and sequencer-specific adapters … further simplifies the workflow and reduces cost.”

Singleplex PCR can allow the user to optimize individual assays on the chip, with the possibility of monitoring each reaction in real time. According to Dr. Dunne, WaferGen’s custom assay designs have a higher probability of working on the first iteration, unlike some multiplex technologies for which several rounds of optimization have become a common practice.

The SmartChip TE system consists of custom SmartChip TE panels, a desktop sin-gle-sample dispenser, and a PCR cycler. The system’s level of sensitivity with routine coverage is >99% of the targeted regions, and its uniformity of coverage is >98% at >10% of the mean.

“We are adding to our primer design pipeline by including designs for smaller am-plicons suitable for FFPE samples, which are critical for cancer-related clinical tests,” said Dr. Dunne. “In addition, a higher throughput SmartChip TE system with a multisample dispenser will be offered in the near future to enable enrichment of multiple samples on one chip. Those will be two main product improvements that will allow us to address the needs of the vast majority of clinical and CLIA-certified labs.”

“We feel that our technology gives our customers an edge in detecting all the genetic variants, obtaining the best uniformity and most complete coverage compared to other technologies, which is of critical importance for clinical tests that will be used by doctors to make patient-care decisions,” said Dr. Dunne.

Microbiome Analysis

According to Fiona Stewart, Ph.D., product development manager at New England Biolabs, customers asked the company to develop a better method for separating target microbial DNA samples from host DNA, the main contaminant. The method needed to be good enough to enable an accurate microbiome analysis.

“What we came up with was an idea to separate those two types of DNA based on methylation pattern. Eukaryotic DNA is generally methylated, and microbial DNA is generally not,” said Dr. Stewart. The separation is achieved by capturing the methylated host DNA on methyl-CpG-binding domain protein 2 (MBD2), immobilized on magnetic protein beads.

Erbay Yigit, Ph.D., applications and product development scientist, used the kit to analyze the microbiome of human saliva. “Once we pull down the methylated host DNA, what remains is the supernatant,” said Dr. Yigit. “Most of the host DNA goes in the beads fraction. We are primarily interested in sequencing prokaryotic DNA.” The prokaryotic DNA sequences are verified against the Human Oral Microbiome Database (HOMD).

The host DNA fraction can be released from the beads and analyzed separately. “We are not only enriching the supernatant, which contains the prokaryotic DNA, but we are also enriching the host DNA,” said Dr. Yigit.

The company’s NEBNext Microbiome DNA Enrichment kit has been used to analyze human, fish, plant, and arthropod microbiomes. It also selectively enriches the organellar DNA from mitochondria and plant chloroplasts, which enables a more efficient genome sequencing and mutational analysis. Dr. Stewart said that the company plans to develop the viral enrichment technology in response to demand in the research community.

“The variety of applications seems pretty broad, but what all of these people have in common is the same problem—they want to be able to sequence the microbial DNA in their sample, and previously they had not been able to do that,” said Dr. Stewart. “Being able to separate microbial DNA is bringing real changes.”

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Multitasking Multiplex Assays

By MaryAnn Labant, GEN Genetics & Bioengineering News

The development of multiplex companion diagnostics is driven by the need to understand efficacy and safety at both the molecular and phenotypic levels. In the future, personalized therapeutic regimens will be tailored to each patient’s genomic, transcriptomic, and epigenomic profiles to optimize individual outcomes.

For personalized (or precision) medicine to become a sustaining reality, all stakeholders—patients, healthcare professionals, drug development companies, regulatory agencies and payers—must work together.

Advancing multiplex companion diagnostics technology was among the topics discussed at CHI’s “Next Generation Diagnostics Summit,” an event held recently in Washington, DC.

“Pharmaceutical companies are more comfortable today with the value of using a personalized medicine approach in their drug discovery and development programs,” explained Jeremy Bridge-Cook, Ph.D., senior vp of research and development at Luminex.

“Reimbursement changes and inconsistencies constrained the market in 2013 and will need to be sorted out, or personalized medicine and its ability to deliver substantial healthcare savings will be slowed down dramatically.”

Rather than being the exception, companion diagnostics are becoming the norm in oncology. In other clinical areas, such as chronic and infectious diseases, advancements in biomarker programs are also being made. Many of these programs are at an earlier stage, however, with some just entering clinical trials.

Multiplex assays mitigate patient risk. For example, approximately a quarter of the drugs used clinically today are metabolized by cytochrome P450 2D6. The Luminex xTAG® CYP2D6 Kit v3 can be used as a clinical aid in determining therapeutic strategy. The qualitative genotyping assay analyzes 20 different cytochrome P450 2D6 variants and identifies metabolic differences, which can be used to help evaluate the risk of adverse events.

Another challenging syndrome is Alzheimer’s disease. Most of the damage caused by Alzheimer’s disease happens subclinically, before clinical symptoms emerge. Already a big problem, Alzheimer’s disease will become a larger problem as baby boomers age and become more susceptible.

The use of a biomarker test for earlier diagnosis is thought by many to be a better way to demonstrate efficacy of a drug for halting Alzheimer’s progression. Luminex and Merck recently announced a partnership around a companion diagnostic for an Alzheimer’s disease compound in development.

Reimbursement remains an issue. Increasingly, evidence based on healthcare economic trials will be required to prove that a precision medicine approach reduces healthcare system costs.

Clinical trials demonstrate clinical outcomes. Healthcare economic trials are essentially the same activity, except that outcomes are measured in economic, not clinical, factors. Standard cost of care is compared to the cost of a personalized approach, that is, a model in which a companion diagnostic is followed by a precision therapeutic.

“Healthcare economic trials have been implemented for some time—just not for companion diagnostics. The industry as a whole is still learning, and although there is a pilot program in its infancy between the FDA and CMS, there is no set protocol,” concluded Dr. Bridge-Cook.

A Multiplex Microfluidics Platform

A fully integrated microfluidics platform for molecular diagnostics is currently available from Rheonix. Intended for research use, the platform is composed of the EncompassMDx™ instrument and a disposable CARD® Consumable cartridge. The cartridge contains all necessary pumps, valves, and reagent and reaction reservoirs.

Software delivers pneumatic signals to the cartridge to move fluids throughout the device and perform all processing steps including cell lysis, DNA or RNA extraction and purification, multiplex PCR amplification, and detection in either an end-point or real-time manner. FDA approval is expected in 2014.

A variety of multiplex assays can be performed on the platform, including pharmacogenomics tests for warfarin and Plavix sensitivity, a test for four common sexually transmitted infections, and a PCR assay to detect and distinguish 20 clinically relevant human papillomavirus (HPV) subtypes.

In addition, a “dual assay” can simultaneously perform both an immunoassay and PCR-based assay for the detection of HIV-1 in saliva samples. An aqueous-based system allows processing and analysis of FFPE samples.

“FFPE tumor blocks are a valuable resource. Now, you can take a curl, put it into the CARD, push ‘go,’ and get results. The CARD introduces the aqueous-based buffer to remove the paraffin and reverse the cross-linking. The cells are lysed, and the rest of the steps are the same as our other assays,” said Richard A. Montagna, Ph.D., senior vp for corporate business development and scientific affairs, at Rheonix.

“That would have taken a lot of expense and work on the benchtop. Results show that we are now detecting seven different somatic cell mutations involving the KRAS colon cancer marker in FFPE tumor blocks,” added Dr. Montagna. “Our mandate is not to just do good science, but to do it in a manner that permits larger-scale, cost-effective manufacturing to help reduce healthcare costs.”

The microfluidics system achieves both high multiplex and high throughput, and processes a range of sample types and volumes, ranging from 5 µL to 5 mL.

Rheonix has partnered with Life Technologies to use the microfluidics platform in the applied markets of food/beverage testing and animal health. The platform was selected based on cost, ease-of-use, and the ability to rapidly migrate existing benchtop assays to a fully automated system.

Next-Generation Sequencing

At Knight Diagnostic Laboratories, Ion Torrent™ technology is used for next-generation sequencing (NGS). This approach allows laboratory-developed tests (LDTs) to be performed that comprise oncology panels with 23–43 genes. The LDTs are focused on actionable mutations that are targetable with either FDA-approved drugs or therapeutics in clinical trials.

“We take a look at the tumor and then try to find a personalized cancer care option for patients who have failed standard of care. In addition to oncologists at our university, we serve a number of community practices around the country,” said Christopher L. Corless, M.D., Ph.D., surgical pathologist and CMO at Knight Diagnostic Laboratories, Oregon Health & Science University.

“A large pharmaceutical company told us if you can find us patients with mutations, then we will provide therapeutics. That started driving the testing. In the last four months we have performed tests on over 400 patients and identified treatment opportunities for a lot of them.”

The LDTs and potential observations are heavily biased toward experimental therapies. This means there is the chance that oncologists may not be able to get the needed therapeutic, or that it will not be reimbursed.

“It is a tough environment,” said Dr. Corless. “Labs like ours want to test more genes. So we are working to reduce the cost of NGS-based testing to the point where it is little more expensive than standard testing. Payers are technology agnostic, but if it is not standard of care, they want to see data that demonstrates the test is necessary. However, proving medical necessity is a slow process because we are trying to do this across many cancers at the same time.”

“A personalized approach does result in better care and better outcomes for many patients, but it is difficult to prove up front that broader-based testing is a good value proposition for all patients. That is why it is so important to further drive down the costs of NGS. In the future, NGS will allow us to become smarter on how to use and combine drugs and to understand what happens when a tumor becomes resistant to treatment,” added Dr. Corless.

NGS platforms are still relatively new technology and fairly labor intensive. Expertise is required to understand how the mutations should be interpreted and reported. As the technology matures and becomes easier to use, and as techniques are found for dealing with the vast amount of information generated, NGS will be implemented more broadly in the clinical setting.

Continue reading at GEN Genetics & Bioengineering News…