What is PCR Testing?

PCR: The Foundation of Syndromic Testing

Polymerase chain reaction technology is at the heart of our mission here at BIOFIRE: to provide syndromic infectious disease diagnostic solutions that help make the world a healthier place.

What is PCR?

PCR stands for polymerase chain reaction. It is a revolutionary chemical process that makes it possible to rapidly and exponentially amplify targeted nucleic acid. According to its inventor, American biochemist Kary Mullis, PCR "lets you pick the piece of DNA you're interested in and have as much of it as you want."¹

The potential to produce millions to billions of copies of a particular segment of DNA or RNA is useful for sequencing, analysis, and experimentation—especially when an initial sample is too small to utilize by itself. Since its discovery in 1984, PCR has become fundamental to the field of microbiology. It is a simple technique with myriad applications, from criminal forensics to the detection of pathogens.

How Does PCR Work?

A polymerase chain reaction requires two types of reagents, DNA polymerase and primers, and the process of thermal cycling. These ingredients make possible the chain reaction that exponentially copies the starting DNA sample.

DNA polymerase is an enzyme that synthesizes molecules from deoxyribonucleotides, the building blocks of DNA, in order to create two identical DNA segments from a single original.

Taq polymerase is the specific enzyme typically used in PCR because it can withstand the extreme temperature changes of thermal cycling, eliminating the need to add a new, non-temperature-stable DNA polymerase after every PCR cycle. Its source is the bacterium Thermus aquaticus, which was first isolated from geothermal springs in Yellowstone National Park around the time PCR was discovered.

Primers are short pieces of single-stranded DNA, pairs of which must be specifically designed to prompt the DNA polymerase to initiate amplification of the targeted DNA segment.

Thermal cycling refers to the repeated heating and cooling cycles that facilitate the temperature-dependent reactions of DNA amplification. A thermal cycler is a laboratory instrument that performs temperature-dependent chemical reactions, including PCR.

One cycle of PCR doubles the amount of DNA with which it began. Each cycle involves three steps: denaturation, annealing, and extension.

Denaturation - The first step in a PCR cycle heats the starting sample to almost boiling. The high temperature causes DNA melting, or denaturation—the process of physically breaking down the hydrogen bonds between the two complementary strands of the DNA double helix. The result is two single strands of DNA that will later be synthesized into two complete DNA double helixes.
Annealing - After denaturation, the temperature of the sample is reduced. This allows the primers to bind to their complementary sequences on each of the separated DNA strands. The primers set the stage for Taq polymerase to begin DNA formation in the final step.
Extension - In the final step, the sample is reheated to the temperature at which Taq polymerase is most active—about 162 °F (72 °C)—so that the enzyme can latch onto the base created by the primers during the annealing stage. Taq polymerase assembles two complete DNA segments from the original one.

A new cycle of PCR is then ready to begin with double the amount of DNA of the previous cycle. Cycles continue until the requisite quantity of DNA is achieved.

Gloved hand inserts a pouch into the BIOFIRE FILMARRAY TORCH.

How BIOFIRE's Syndromic Testing Using PCR

In 1990, BIOFIRE debuted its first instrument, a thermal cycler that was the first on the market to match the speed of biochemical reaction, completing PCR in record time.² Since then, BIOFIRE has built upon that revolutionary invention to lead the industry in syndromic infectious disease diagnostics.

Today, the BIOFIRE^® FILMARRAY^® System performs multiplex PCR syndromic tests. Multiplex PCR refers to the amplification of multiple DNA targets in a single polymerase chain reaction. Syndromic testing is the process of simultaneously testing a patient for multiple pathogens that cause overlapping signs and symptoms.

Each of the BIOFIRE^® FILMARRAY^® Panels targets a comprehensive grouping of pathogens associated with a specific syndrome—like an upper respiratory infection or gastroenteritis. A BIOFIRE syndromic test isolates, amplifies (using PCR), and detects the genetic material of targeted pathogens.

Preparing and inserting a panel pouch into a BIOFIRE System instrument requires just two minutes of hands-on time. After that, everything from sample integration to automated results analysis—with multiplex PCR in between—occurs automatically inside the pouch. The main feature of BIOFIRE's groundbreaking FILMARRAY technology is the array, which consists of individual wells grouped together on a single plate. This design allows the separate reactions of multiplex PCR to occur simultaneously without cross-contamination. Each well in the array includes its own set of primers designed to detect one specific target on the panel menu. After the cycles of PCR are all completed, organisms in the sample are identified based on which wells in the array confirm the presence or absence of their assigned organism's DNA. BIOFIRE's software then processes this data and makes a positive or negative call for each organism in one easy-to-read report. Take a look inside the pouch by watching our short "How it Works" video.

Because PCR is an extremely sensitive process, the potential for issues like false positive results and contamination—whether by organic materials from within the pouch, or by microorganisms from outside the pouch—is carefully considered at all stages of product development at BIOFIRE. Additionally, we thoughtfully design all products to be as automated and user-friendly as possible, maintain a library of ongoing research and clinical data, follow strict quality control and clean manufacturing protocols, and offer exceptional customer support.

Why Syndromic Testing?

The BIOFIRE System is fast, accurate, and comprehensive. It is more sensitive than culture and more efficient than using a variety of individual and send-out tests. By utilizing PCR technology to target a broad menu of pathogens and antibiotic resistance genes at the molecular level, syndromic testing by BIOFIRE can help streamline laboratory workflow and eliminate the guesswork so clinicians are empowered to choose the right test, the first time.