Polymerase Chain Reaction (PCR)

General Description

The polymerase chain reaction (PCR) is a molecular biology technique used in laboratories to amplify specific DNA sequences. Within a few hours, it is possible to generate millions of copies of a specific segment for detailed analysis.

PCR is a highly sensitive test that allows the study of DNA from minimal sources, including unicellular organisms. Thanks to this feature, it is useful for clinical diagnostics—particularly for infections—performing forensic studies, or conducting genetic research, enabling, among other things, the detection of genetic changes associated with hereditary diseases.

Kary Banks Mullis was awarded the Nobel Prize in Chemistry in 1993 for the development of PCR, as it represented one of the most significant scientific advances of the 20th century.

When is it indicated?

PCR is used in medicine for a wide range of applications:

• Diagnosis, study, and monitoring of multiple pathologies, such as:
• Viral infections: influenza, COVID-19, respiratory syncytial virus (RSV), adenovirus, HIV, human papillomavirus (HPV), sexually transmitted diseases.
• Trauma/burns: to detect infections, pathogens, and other infectious organisms that may arise as complications in patients with trauma or burns.
• Chronic diseases: lupus, rheumatoid arthritis, inflammatory bowel disease.
• Certain types of cancer.
• Hereditary conditions.
• Genetic mutations.
• Forensic analysis: PCR allows amplification of DNA samples, no matter how minimal, to create a genetic profile for victim identification, linking or excluding crime suspects, or conducting paternity testing.
• Genetic studies.
• Vaccine development: PCR is used to analyze the genetic material of a pathogen and identify sequences that encode bacterial or viral proteins. It is particularly useful in mRNA vaccines.

How Is It Performed?

PCR testing involves two main procedures. First, a fluid sample is collected:

• Blood: drawn from a peripheral vein, usually in the arm, in the same manner as a routine blood test.
• Saliva: collected by swabbing the inside of the cheeks.
• Mucus: a swab is inserted through the nasal cavity to the nasopharynx.
• Urine: the patient collects the sample in a sterile container.
• Cerebrospinal fluid (CSF): obtained via lumbar puncture, where a fine needle is inserted between two vertebrae in the lower back to extract a small amount of the fluid surrounding the spinal cord.

Once the sample reaches the laboratory, different types of PCR can be performed depending on the method and equipment used:

• Standard PCR: the simplest and most efficient method for amplifying DNA segments. Two primers (short single-stranded synthetic DNA fragments) are used to isolate a specific fraction and amplify it through three steps:
• Denaturation: heating above 95°C to break hydrogen bonds between DNA strands.
• Annealing: cooling to 50–65°C to allow primers to bind to the separated template sequences.
• Extension: raising the temperature to around 72°C to enable Taq polymerase to bind to the primer and synthesize a complementary DNA strand.
  This process is repeated 20–40 times to duplicate the original DNA sequence as needed.
• Real-time PCR (qPCR): provides quantitative information in real time using fluorescence as amplification occurs, particularly useful for assessing viral load or gene expression.
• Reverse transcription PCR (RT-PCR): uses reverse transcriptase to convert RNA into complementary DNA (cDNA) and then amplifies it. It is especially useful for detecting RNA viruses and gene expression studies.
• Real-time reverse transcription PCR (RT-qPCR): enables real-time quantification of RNA converted to cDNA, commonly used in virology and gene expression studies.
• Multiplex PCR: multiple DNA sequences are amplified and selected in a single reaction using various primer sets. Useful for detecting multiple pathogens and genotyping studies.
• Nested PCR: more sensitive and specific, employing two sets of primers and two successive PCR reactions. The first primer set targets regions upstream of the selected DNA fraction. This product serves as the template for the second PCR phase.
• End-point PCR: the simplest version, providing no quantitative data, but confirming the presence or absence of a DNA fragment.

Risks

Performing PCR in the laboratory poses no health risk to the patient. However, sample collection may occasionally cause minor side effects:

• Blood draw: bruising, bleeding, or fainting.
• Lumbar puncture: headache, discomfort at the puncture site, bleeding, and rarely, infection or neurological injury.
• Mucus collection: rhinorrhea, headache, or nosebleed.

What to expect from a PCR test

Patients are only present during sample collection, which varies depending on the type:

• Blood draw: the patient sits with the arm extended, clenching the fist before collection. Only a brief prick is felt upon needle insertion. To reduce bruising, apply pressure to the puncture site for a few minutes without bending the arm.
• Lumbar puncture: the patient lies on their side with knees bent toward the chest or sits slightly leaning forward. Remain completely still during needle insertion. Local anesthesia prevents pain, but pressure may be felt. Rest is recommended afterward to avoid headaches.
• Mucus collection: the patient usually sits, but may stand, with the head slightly tilted back. Mild discomfort, tingling, coughing, tearing, or nausea may occur but resolves after swab removal.
• Saliva collection: the patient slightly opens the mouth and may need to stimulate saliva production if the initial sample is insufficient.
• Urine collection: the patient collects the sample at home and brings it to the laboratory.

PCR result turnaround depends on the sample type and laboratory procedure:

• Nasopharyngeal mucus: 2–6 hours
• Saliva: 2–8 hours
• Urine: 4–8 hours
• Blood: 4–12 hours
• Cerebrospinal fluid: 1–4 hours

Specialties in which PCR is requested

PCR is routinely requested in Genetics, Microbiology and Parasitology, Medical oncology, and infectious diseasesInfectious diseasesInfectious Diseases , but other specialists may order it depending on clinical circumstances.