Topic
Reverse Transcription Loop-mediated Isothermal Amplification
About: Reverse Transcription Loop-mediated Isothermal Amplification is a research topic. Over the lifetime, 625 publications have been published within this topic receiving 16340 citations.
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TL;DR: The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP, which should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.
1,699 citations
TL;DR: An improved simple visual detection system for the results of the LAMP reaction that enables visual discrimination of results without costly specialized equipment should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
Abstract: As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
1,521 citations
TL;DR: Real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity, which indicated an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized.
1,505 citations
TL;DR: The results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.
Abstract: A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 104 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.
434 citations
TL;DR: The results demonstrate that the RT-LAMP assay will be a powerful tool for SARS-CoV-2 identification, and for monitoring suspected patients, close contacts, and high-risk groups.
433 citations