Reverse Transcriptase-Polymerase Chain Reaction

The diagnosis of many infectious diseases, both viral and bacterial, may include the use of reverse transcriptase–polymerase chain reaction (RT-PCR).

Specimen - Serum

Container - Blue-top vacuum tube

Samples must be sent in sealed, leak-proof containers marked with biohazard stickers in order to comply with Occupational Safety and Health Administration (OSHA) safety requirements.

Description

Reverse transcription is the synthesis of a complementary DNA sequence from an RNA template using reverse transcriptase, which is an RNA-dependent DNA polymerase. ^[1] The resultant complementary DNA is amplified by polymerase chain reaction (PCR). Specific DNA present in small amounts in a clinical specimen are amplified by PCR so they become detectable. In PCR, a thermostable DNA polymerase is used to amplify target DNA 2-fold with each temperature cycle. ^[2]

The 3 steps of conventional PCR are denaturation, annealing, and primer extension. Initially, DNA is taken from the clinical specimen, as well as certain sequence-specific oligonucleotide primers, thermostable DNA polymerase, nucleotides, and buffer. The temperature of these is increased to 90-95°C in order to separate (denature) the 2 strands of target DNA. In the second step, the temperature decreases (45-60°C), depending on the primers, to permit annealing (strengthening) of the target DNA primers. Finally, nucleotides complementary to the target DNA are added extending each primer by the thermostable DNA polymerase.

The target DNA segment is amplified in the range of 10⁵ - to 10⁶ -fold by repeating this cycle no less than 30-40 times. The amplified segment may be observed using electrophoretic gel or can be identified by Southern blot analysis, using specific DNA probes for that segment. ^[2]

Reverse transcriptase (RT) PCR is PCR performed on RNA targets. These assays are commercially available for detection of bacterial and viral pathogens, including human immunodeficiency virus 1 (HIV-1), cytomegalovirus, enteric viruses, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that cause coronavirus disease 2019 (COVID-19).

Other "in-house" PCRs have been developed at individual laboratories to diagnose infections (eg, testing of cerebrospinal fluid [CSF] for herpes simplex virus to diagnose herpes encephalitis and testing of nasopharyngeal wash fluid to diagnose Bordetella pertussis infection), particularly if traditional culture and antigen detection techniques have failed. ^[2]

Contamination of reagents or specimens with target DNA from the environment must be avoided because such contamination can produce false-positive results. ^[2]

RT-PCR is extremely sensitive and can be performed using paraffin-embedded, fresh, or frozen tissues. Its high false-positive rate (low specificity) is its Achilles heel; therefore, meticulous care is necessary to prevent contamination.

Analysis of fresh or frozen tissue is preferred; however, RT-PCR can be performed on paraffin-embedded tissue. The recommendation is to freeze and store a portion of suspected sarcomas or poorly differentiated malignant lesions for molecular analysis. ^[3]

Whether coronaviruses cause neurologic diseases in humans remains controversial, although a causative link has been established in animals. ^[4] These viruses have been identified by culture, in situ hybridization, and RT-PCR in brain tissue from a few cases of multiple sclerosis. ^[4]

Indications/Applications

RT-PCR aids in the diagnosis of viral and bacterial infections, genetic diseases, and neoplasms; it is also used to prognosticate the recurrence of diseases, particularly malignancies.

Considerations

RT-PCR is commonly used in molecular biology and is a variant of PCR.

The use of real-time quantitative RT-PCR to quantify specific mRNAs allows for more rapid testing, higher sensitivity, increased simplicity, and more accuracy. Additionally, small amounts of input RNA are required. RT-PCR is the method of choice when monitoring minimal residual disease, such as in chronic myelogenous leukemia (CML).

CML comprises approximately 20% of all leukemias. In CML, a balanced (9;22) chromosomal translocation results in a chimeric BCR-ABL fusion gene. This gene codes a fusion protein with high tyrosine kinase activity, resulting in growth factor–independent proliferation. Current CML therapy targets this kinase; BCR-ABL fusion gene levels are monitored to determine the treatment efficacy. ^[5]

RT-PCR is also used in prognosticating disease recurrence. In gastric adenocarcinoma, CEA mRNA copy number in peripheral blood during the initial diagnosis is associated with cancer recurrence; thus, detection of CEA mRNA levels with real-time RT-PCR during initial diagnosis seems to be a promising technique for prediction of gastric adenocarcinoma recurrence. ^[6]

A literature review by Pecoraro et al reported that in the included studies, the false-negative rate for the detection of SARS-CoV-2 with RT-PCR ranged from 2% to 58%, with the “summary estimate of the overall false-negative rate” being 12%. A low false-negative rate (2%) was found in studies in which a second RT-PCR test was performed more than 3 days after the first. ^[7]