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August 01, 2015 (Vol. 35, No. 14)

PCR Shows Off Its Clinical Chops

Thanks to Advances in Genomics, PCR Is Becoming More Common in Clinical Applications

  • SNP Analysis

    The Rapid Cycle In-line PCR instrument, a research-use-only platform from Canon U.S. Life Sciences, has been shown to streamline testing of multiple single nucleotide polymorphisms (SNPs) on a set of genomic DNA samples. By utilizing liquid-handling robots, the instrument automates serial PCR reaction preparation and dispensation to a microfluidic-based reaction cartridge, decreasing hands-on time and potential handling errors. The small reaction volumes significantly reduce the time required to complete PCR amplification and DNA-melting analysis.

    “To date, the instrument has been used successfully in genotyping SNPs, and we are investigating other genetic targets including, but not limited to, small insertions and deletions,” said Hanyoup Kim, Ph.D., senior scientist, Canon U.S. Life Sciences.

    In contrast to PCR assays for gene expression, SNP genotyping assays must precisely target a specific position within a gene. This spatial restriction limits the nucleotide sequences that PCR primers and/or probes will target, and in turn this impacts the cycling parameters (annealing temperatures and times) that will yield robust amplification. Therefore, users will often require different PCR conditions to effectively test all of their loci of interest.

  • Lot-to-Lot Consistency

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    Promega, which produces reaction components for PCR-based molecular assays, notes that the quality controls implemented by manufacturers can affect molecular diagnostic assay design and performance. The company adds that as multiplexed assays and panel tests become more common, lot-to-lot reagent consistency will become more important.

    PCR-based molecular diagnostic assays rely on high-quality reaction components to yield robust, reliable, and reproducible results. Assay components must perform the same, lot to lot.

    For clinical applications that require additional assurances, Promega has added multiple levels of control to their processes. The manufacturing process and quality control methods are validated and supplied with appropriate documentation to demonstrate repeated supply of a consistent product. Robust assays test for factors that can negatively impact an assay, such as DNA contamination, enzyme concentration, and activity. Detailed certificates of analyses dovetail with users’ quality systems.

    “We have custom manufacturing abilities that can adapt to the specific needs of clinical customers and can involve anything from format to very specific formulations or bulk product manufacture,” asserted Terri McDonnell, senior program manager, Promega. “Customers may need a specific enzyme concentration, formulation, or volume; custom documentation; [or] a specific QC assay—or [they may] even [need] help on assay design.”

    “Building upon the base of controls that we have in place around the manufacture of our products is really important for our molecular-diagnostic customers,” McDonnell continued. “It gives us the ability to customize products while maintaining control.”

    As assays are being pushed out closer to POC, such as in field work, temperature and humidity control can be a problem. Reagents need to be stable under very diverse conditions, and requests are growing more frequent for glycerol-free reagents that can be lyophilized. In the future, as the use of more complex multiplexed assays and panel testing becomes more common, lot-to-lot consistency along with supporting documentation will become more important.

  • Live Sentinel Mice vs. PCR Testing

    Most lab animal facilities monitor mouse colonies for pathogens that can negatively impact research. The current health monitoring program at Oregon Health and Science University (OHSU) consists of exposing three sentinel mice to soiled bedding from 70 colony cages during biweekly cage changes.

    Hoping to improve detection methods and reduce the number of mice necessary for sentinel testing, OHSU, working with Charles River Laboratories, decided to evaluate whether PCR testing using swabs collected from a single location on the exhaust plenum of each individually ventilated cage rack would be equivalent to results obtained from testing sentinel animals.

    The team’s results demonstrated that Exhaust Air Dust (EAD) sampling is a viable option for collecting samples and obtaining results indirectly from colony animals, according to Kim Saunders, D.V.M., who heads OHSU’s department of comparative medicine.

    “Eliminating the use of live sentinel mice would result in a substantial savings in costs for monitoring the health of colony animals,” says Dr. Saunders. “EAD testing appears to have great promise in our goal of refinement, reduction, and replacement of live animals and could potentially save thousands of rodents’ lives.”

    Ken Henderson, Ph.D., director of R&D at Charles River Labs, also agrees that EAD sampling appears to be cost-effective. “Yearly costs for the Oregon sentinel program ran around $191,000 compared to around $27,000 for EAD samples without the use of live sentinels,” he says.  

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