Polymerase Chain Reaction

Polymerase chain reaction, PCR (from the Greek polymeres — consisting of many parts) is a highly accurate molecular biology method that can detect even single fragments of the genetic material (DNA or RNA) of a specific infectious agent in biological material (blood, saliva, or a swab).

Etiology and pathophysiology

The PCR mechanism mimics the natural process of DNA replication in a living cell, but it occurs in a test tube (in a thermal cycler). The process consists of repeated thermal cycles:

• the sample is heated to 95°C to separate the double helix of the original DNA molecule into two single strands (denaturation);
• the temperature is lowered, and primers—artificially synthesized short markers that “recognize” a unique region of the target virus genome and bind to it—come into play;
• a specialized heat-resistant enzyme, DNA polymerase, begins synthesizing the complementary strand by copying the viral fragment.

The amount of viral DNA doubles during each cycle. After 30–40 cycles, one viral molecule yields billions of exact copies that are readily detected by the instrument’s laser optics.

If the virus is an RNA virus (such as rotavirus or coronaviruses), the process is preceded by a reverse transcription step—the conversion of RNA into DNA (RT-PCR).

Clinical significance

PCR is the gold standard for diagnosing acute infections. Unlike antibody tests, PCR detects the pathogen itself rather than the body’s response to the disease.

Detection of a pathogen by PCR (a positive result) is evidence of active infection in the presence of relevant symptoms or viremia. The method is indispensable in the following cases:

• during the incubation period or the first days of illness—before antibodies have had time to develop;
• for quantitative assessment of viral load—for example, in HIV infection or viral hepatitis (to determine how actively the virus is replicating and whether the therapy is effective).