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Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). [1] It is primarily used to measure the amount of a specific RNA.
An RNA ladder is often run alongside the samples on an electrophoresis gel to observe the size of fragments obtained but in total RNA samples the ribosomal subunits can act as size markers. [11] Since the large ribosomal subunit is 28S (approximately 5kb) and the small ribosomal subunit is 18S (approximately 2kb) two prominent bands appear on ...
Viral disease testing is the use of a variety of testing techniques for a variety of purposes, including diagnosing conditions, assessing immunity and understanding disease prevalence. The primary approaches include DNA / RNA tests, serological tests and antigen tests.
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription.Reverse transcriptases are used by viruses such as HIV, COVID-19, and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes.
Viruses are often isolated from the initial patient sample. This allows the virus sample to be grown into larger quantities and allows a larger number of tests to be run on them. This is particularly important for samples that contain new or rare viruses for which diagnostic tests are not yet developed. [citation needed]
Nucleic acid amplification is a technique used to produce several copies of a specific segment of RNA/DNA. [3] Amplified RNA and DNA can be used for a variety of applications, such as genotyping, sequencing, and detection of bacteria or viruses. [4] There are two different types of amplification, non-isothermal and isothermal. [5]
TMA produces RNA amplicon rather than DNA amplicon. Since RNA is more labile in a laboratory environment, this reduces the possibility of carry-over contamination. TMA produces 100–1000 copies per cycle (PCR and LCR exponentially doubles each cycle). This results in a 10 billion fold increase of DNA (or RNA) copies within about 15–30 minutes.
The extent of proofreading in DNA replication determines the mutation rate, and is different in different species. [4] For example, loss of proofreading due to mutations in the DNA polymerase epsilon gene results in a hyper-mutated genotype with >100 mutations per Mbase of DNA in human colorectal cancers. [5]