engineering

Reverse Transcription Loop-mediated Isothermal Amplification

Reverse Transcription Loop-mediated Isothermal Amplification is a engineering topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Reverse Transcription Loop-mediated Isothermal Amplification rather than just read about it. In short: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses.

Key takeaways

Reverse Transcription Loop-mediated Isothermal Amplification belongs to engineering; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Reverse Transcription Loop-mediated Isothermal Amplification to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Reverse Transcription Loop-mediated Isothermal Amplification from memory before moving on to harder problems.

Reference excerpt

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses. It combines LAMP DNA-detection with reverse transcription, making cDNA from RNA before running the reaction. RT-LAMP does not require thermal cycles (unlike PCR) and is performed at a constant temperature between 60 and 65 °C. RT-LAMP is used in the detection of RNA viruses (groups II, IV, and V on the Baltimore Virus Classification system), such as the SARS-CoV-2 virus and the Ebola virus.

Applications RT-LAMP is used to test for the presence of specific RNA-samples of viruses for the specific sequence of the virus, made possible by comparing the sequences against a large external database of references.

Detection of the SARS-CoV2-Virus The RT-LAMP technique is being supported as a cheaper and easier alternative to RT-PCR for the early diagnostics of people that are infectious for COVID-19. There are open access test designs (including the recombinant proteins) which makes it legally possible for anyone to produce a test. In contrast to classic rapid tests by lateral flow, RT-LAMP allows the early diagnosis of the disease by testing the viral RNA. The tests can be done without previous RNA-isolation, detecting the viruses directly from swabs or from saliva.

Detection of non-human viruses One example of use case of RT-LAMP was as an experiment to detect a new duck Tembusu-like, BYD virus, named after the region, Baiyangdian, where it was first isolated Another application of this method was in a 2013 experiment to detect an Akabane virus using RT-LAMP. The experiment, done in China, isolated the virus from aborted calf fetuses.

Detection of body fluids RT-LAMP is also being used in Forensic Serology to identify body fluids. Researchers have done experiments to show that this method can effectively identify certain body fluids. Knowing there would be limitations, Su et al, come to the conclusion that RT-LAMP was only able to identify blood.

Methodology

Reverse transcription A specific sequence of the cDNA is detected by 4 LAMP primers. Two of them are inner primers (FIP and BIP), which serve as base for the Bst enzyme copy the template into a new DNA. The outer primers(F3 and B3) anneal to the template strand and help the reaction to proceed. As in the case of RT-PCR, the RT-LAMP procedure starts by making DNA from the sample RNA. This conversion is made by a reverse transcriptase, an enzyme derived from retroviruses capable of making such a conversion. This DNA derived from RNA is called cDNA, or complementary DNA. The FIP primer is used by the reverse transcriptase to build a single-strand of copy DNA. The F3 primer binds to this side of the template strand as well, and displaces the previously made copy.

Amplification This displaced, single-stranded copy is a mixture of target RNA and primers. The primers are designed to have a sequence that binds to the sequence itself, forming a loop. The BIP primer binds to the other end of this single strand and is used by the Bst DNA polymerase to build a complementary strand, making double-strand DNA. The F3 primer binds to this end and displaces, once again, this newly generated single-stranded DNA molecule. This new single strand that has been released will act as the starting point for the LAMP cycling amplification. This single-stranded DNA has a dumbbell-like structure as the ends fold and self-bind, forming two loops. The DNA polymerase and the FIP or BIP primers keep amplifying this strand and the LAMP-reaction product is extended. 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 less an hour, under isothermal conditions between 60 and 65 °C.

Read out The read out of RT-LAMP tests is frequently colorimetric. Two of the common ways are based on measuring either pH or magnesium ions. The amplification reaction causes pH to lower and Mg2+ levels to drop. This can be perceived by indicators, such as Phenol red, for pH, and hydroxynaphthol blue (HNB), for magnesium. Another option is to use SYBR Green I, a DNA intercalating coloring agent.

Advantages and disadvantages

This method is specifically advantageous because it can all be done quickly in one step. The sample is mixed with the primers, reverse transcriptase and DNA polymerase and the reaction takes place under a constant temperature. The required temperature can be achieved using a simple hot water bath. PCR requires thermocycling; RT-LAMP does not, making it more time efficient and very cost effective. This inexpensive and streamlined method can be more readily used in developing countries that do not have access to high tech laboratories. A disadvantage of this method is generating the sequence specific primers. For each LAMP assay, primers must be specifically designed to be compatible with the target DNA. This can be difficult which discourages researchers from using the LAMP method in their work. There is however, a free software called Primer Explorer, developed by Fujitsu in Japan, which can aid in the selection of these primers.

See also Loop-mediated isothermal amplification

References

External links LAMP Primer Explorer MorphoCatcher, a tool for design of species-specific primers Scholia page for RT-LAMP Open access protocols for RT-LAMP to detect SARS-CoV-2

Illustrations

Worked examples

Example 1 — a first encounter with Reverse Transcription Loop-mediated Isothermal Amplification

Start with the simplest possible case. Write down what Reverse Transcription Loop-mediated Isothermal Amplification claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In engineering, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Reverse Transcription Loop-mediated Isothermal Amplification before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Reverse Transcription Loop-mediated Isothermal Amplification ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Reverse Transcription Loop-mediated Isothermal Amplification

In research: Reverse Transcription Loop-mediated Isothermal Amplification appears in engineering research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Reverse Transcription Loop-mediated Isothermal Amplification in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Reverse Transcription Loop-mediated Isothermal Amplification is common in secondary-school and first-year university syllabi. It links to neighbouring topics Molecular biology techniques, RNA, so understanding it makes those chapters shorter.
In everyday life: Look for Reverse Transcription Loop-mediated Isothermal Amplification outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study Reverse Transcription Loop-mediated Isothermal Amplification in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Reverse Transcription Loop-mediated Isothermal Amplification means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
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Frequently asked questions

What is Reverse Transcription Loop-mediated Isothermal Amplification in simple terms?

Why does Reverse Transcription Loop-mediated Isothermal Amplification matter?

Because it connects several engineering ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Reverse Transcription Loop-mediated Isothermal Amplification?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Reverse Transcription Loop-mediated Isothermal Amplification.