Target Enriched Multiplex Polymerase Chain Reaction

Diatherix Laboratories utilizes its proprietary technology, TEM-PCR™ (Target Enriched Multiplex Polymerase Chain Reaction), to deliver cutting edge diagnostic services. TEM-PCR™ is a unique, multiplex amplification platform designed to overcome the challenges that exist with conventional laboratory methods.

Background

TEM-PCR™ (Target Enriched Multiplex Polymerase Chain Reaction) is a novel multiplex PCR amplification strategy invented by Dr. Jian Han   in 2003. Dr. Han had been in China combating the epidemic SARS outbreak at the time and became frustrated with the limitations of traditional PCR. This led him to investigate ways of multiplexing PCR to detect multiple targets. On a flight back to the U.S. from China, Dr. Han conceived of the basic process for achieving true multiplex PCR by implementing nested primers at lower concentrations with the addition of a universal primer set for the large-scale amplification. This process was proven later that year and Dr. Han named it TEM-PCR™.

Diatherix founder Dennis Grimaud became involved with TEM-PCR™ soon after its inception. Dr. Han recruited Grimaud to become the CEO of his company, Genaco Biomedical Products, after the invention of TEM-PCR™. From 2005-2008 Genaco participated in multiple studies such as this correlation study   demonstrating TEM-PCR™ to be “… both accurate and robust enough to be used in a routine clinical setting.” Also during that time, TEM-PCR™ was named both the Frost and Sullivan award for Technology Innovation and Leadership of the Year and runner up for the Wall Street Journal Medical Technology Innovation of the Year Award. Since 2008, Diatherix has been using TEM-PCR™ to develop and perform multiplex PCR panel testing for infectious disease diagnostics. Diatherix was the first CLIA certified laboratory to be awarded an Emergency Use Authorization (EUA) from the FDA for H1N1-09 during the 2009 pandemic. Diatherix Laboratories holds all associated rights and patents for TEM-PCR™.

Awards & Recognition

Process Overview

The process of performing TEM-PCR™ diagnostic panel testing consists of three major steps: extraction, amplification and detection. Prior to extraction, the specimens must be received, sorted, batched, and entered into the tracking software. Each day of operation begins in the early morning as Diatherix receives the specimens. Then our sorting team goes to work processing and sorting each specimen into panel specific batches. Batching specimens allows for high throughput, maximizing efficiencies, and reducing costs of operation. After sorting, each specimen and batch is entered into our proprietary specimen tracking software to ensure accurate result reporting. Typically, results are reported within 8 hours from the time of receipt of specimens at the laboratory.

Extraction

Nucleic acid extraction methods used in TEM-PCR™ are comparable to extraction methods used in traditional PCR, with variations among the panels depending upon the specimen types involved. Extraction begins with transferring volume from batched specimens into 96-well plates. Special lysing reagents are then added to each sample to expose the nucleic acids by breaking through and degrading unnecessary cellular structures and components. Lastly, magnetic beads are used to capture, wash, and elute purified nucleic acids into a final solution for use in downstream TEM-PCR reactions.

Amplification

Amplification begins by adding the panel-specific Diatherix primer mixes (shown below) to the extracted nucleic acids (template) in a new 96-well plate. After the primers have been added to the template, the plate is then sealed and placed in an instrument for thermocycling. Each sample goes through customized cycles of heating and cooling to cause the PCR reactions to take place, replicating the targeted sequences of DNA for detection. To learn more about basic PCR visit this website or watch this animation .

The key to making TEM-PCR™ occur successfully lies in these primer mixes and how they allow the enrichment of multiple targets. The use of target-specific nested primers (shown below) at low concentrations at the initial enrichment step allows high specificity of multiplexing amplification. After initial target enrichment is complete, the SuperPrimers (shown below) within the reaction carry out the exponential amplification and produce tagged PCR products for subsequent detection.

Nested target-specific primers are used during the initial target enrichment step.

A pair of SuperPrimers are then applied to amplify all targets.

Detection

The detection phase of the process is accomplished by measuring the fluorescence of special tags that are bound to the target sequences, which have been attached during amplification. For detection, each target sequence has a complementary sequence, or probe, bound to a specific location on an array plate(chemically-modified solid surface). Tagged PCR products are added to the array plate and undergo hybridization, a step in which DNA fragments will anneal to their specific, bound, complementary sequences. Unbound nucleic acids and reagents are then washed away. Lastly, the array is illuminated within a highly calibrated light reading instrument, which measures and records the RFUs (Relative Fluorescence Units) present for each target. If the RFUs are above the threshold for detection, then the target DNA is reported to be detected within the specimen.