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Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a technique for the amplification of RNA. Within the last 10 years of its development, applications of the LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification have been widely used. This method was then improved by taking it a step further and combining it with a reverse transcription phase to allow for the detection of RNA. RT-LAMP is a one step nucleic acid amplification method that is used to diagnose infectious disease caused by bacteria or viruses. Although it has not been formally recognised by NAT, the method has been developed into many commercial kits that can be used for the identification of pathogens.〔 The commonly used PCR method is able to generate millions of copies of the target strand. This process relies on thermal cycling, cycles of heating and cooling to facilitate the DNA replication. RT-LAMP does not require these cycles and is performed at a constant temperature between 60 and 65 °C. Similar to RT-PCR, RT-LAMP uses reverse transcriptase to synthesize complementary DNA (cDNA) from RNA sequences. This cDNA is then amplified using DNA polymerase, generating 10^9 copies per hour. RT-LAMP is used in the detection of viruses. This method can be very effective in detecting viruses with an RNA genome (Group II, IV, and V based on the Baltimorevirus classification system). ==Methodology== thumbthumb Four specially designed primers recognize distinct target sequences on the template strand. The primers bind only to these sequences which allows for high specificity. Out of the 4 primers involved, two of them are “inner primers” (FIP and BIP) which are designed to synthesize new DNA strands. The outer primers (F3 and B3) anneal to the template strand and also generate new DNA. These primers are accompanied by DNA polymerase which aids in strand displacement and releases the newly formed DNA strands. The BIP primer, accompanied by reverse transcriptase, initiates the process by binding to a target sequence on the 3’ end of the RNA template and synthesizing a copy DNA strand. The B3 primer binds to this side of the template strand as well, and with the help of DNA polymerase simultaneously creates a new cDNA strand while displacing the previously made copy. The double stranded DNA containing the template strand is no longer needed. The single stranded copy now loops at the 3’ end as it binds to itself. The FIP primer binds to the 5’ end of this single strand and accompanied by DNA polymerase, synthesizes a complementary strand. The F3 primer, with DNA polymerase, binds to this end and generates a new double stranded DNA molecule while displacing the previously made single strand. This new single strand that has been released will act as the starting point for the LAMP cycling amplification. The DNA has a dumbbell-like structure as the ends fold in and self anneal. This structure becomes a stem-loop when the FIP or BIP primer once again initiates DNA synthesis at one of the target sequence locations. This cycle can be started from either the forward or backward side of the strand using the appropriate primer. Once this cycle has begun, the strand undergoes self-primed DNA synthesis during the elongation stage of the amplification process. This amplification takes place in only an hour, under isothermal conditions between 60-65 °C. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Reverse Transcription Loop-mediated Isothermal Amplification」の詳細全文を読む スポンサード リンク
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