Loop-mediated isothermal amplification

Loop mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA. This may be of use in future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.

LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler.

In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Typically, 4 different primers are used to identify 6 distinct regions on the target gene, which adds highly to the specificity. An additional pair of "loop primers" can further accelerate the reaction. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification.

Detection of amplification product can be determined via photometry for turbidity caused by an increasing quantity of magnesium pyrophosphate precipitate in solution as a byproduct of amplification. This allows easy visualization by the naked eye, especially for larger reaction volumes, or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity or by fluorescence using intercalating dyes such as SYTO 9. Dyes such as SYBR green, can be used to create a visible color change that can be seen with the naked eye without the need for expensive equipment, or a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. In-tube detection of DNA amplification is possible using manganese loaded calcein which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis.Moreover, visual detection of the LAMP amplicons by the unaided eye was based on their ability to hybridize with the complementary gold-bound ss-DNA and thus prevent the normal red to purple-blue color change that would otherwise occur by salt-induced aggregation of the gold particles. So, a LAMP method combined with amplicon detection by AuNP has advantages over previously other methods in terms of reduced assay time, amplicon confirmation by hybridization and use of simpler equipment (i.e., no need for a thermocycler, electrophoresis equipment or a UV trans-illuminator.

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