Molecular Biology

Loop mediated isothermal Amplification (LAMP) & Lateral Flow

Loop mediated isothermal Ampli­fi­cation – Intro­duction

If you are confronted with the topic of “isothermal DNA ampli­fi­cation” for the first time, the chances are very high that a method called LAMP is somehow involved. This has nothing to do with light bulbs or LEDs. LAMP stands for Loop mediated isothermal amplification, a DNA ampli­fi­cation method that is usually carried out at temper­a­tures between 60 and 70°C. LAMP is partic­u­larly charac­terized by its excellent sensi­tivity, ampli­fi­cation effiency and high reaction speed.

A liter­ature study containing more than 300 peer reviewed publi­ca­tions revealed that LAMP and RPA are the most frequently used DNA ampli­fi­cation methods combined with a rapid Lateral Flow readout. Approx. 28% of these publi­ca­tions used the LAMP method for nucleic acid ampli­fi­cation.

Loop mediated isothermal ampli­fi­cation is a versitile technique and without a doubt one of the most important tools for high-​​quality DNA analysis outside of specialized labora­tories.”

History of the Loop mediated isothermal Ampli­fi­cation

Inspired by existing isothermal DNA ampli­fi­cation methods, Notomi and colleagues initially described the loop mediated isothermal ampli­fi­cation method for the first time in 2000. They used existing knowledge and invented an ampli­fi­cation method that is highly efficient without the need for addtional enzmyes beside a DNA-​​Polymerase. The creation of a reaction inter­me­diate, which has terminal, single-​​stranded loop struc­tures, allows multiple annealing and elongation options. As a result, the reaction is extremely efficient and reaction products of various size are amplified. At that time, the authors were able to conclude that the (RT) LAMP works fast and efficient with a sensi­tivity compa­rable to that of PCR. It was pointed out that LAMP reaction products can be easily and quickly detected via antigen-​​antibody-​​interaction. From today’s perspective, this can already be under­stood as an indication for the potential of the combi­nation of loop mediated isothermal ampli­fi­cation and Lateral Flow. One of the first papers on this topic appeared in 2008. An RT-​​LAMP reaction product was specif­i­cally detected with the universal Lateral Flow Device, Milenia HybriDetect. The specific and sensitive detection of a viral shrimp pathogen was achieved in about an hour. Since then, this technology has been contin­u­ously developed, adapted and optimized, so that today the LAMP repre­sents one of the most promising tools for a new gener­ation of molecular Point-​​of-​​Care diagnostics.

Mechanism

The name “Loop mediated isothermal Ampli­fi­cation” is a very apt description of the molecular ampli­fi­cation mechanism. In general, LAMP is more complex than the other ampli­fi­cation methods, such as PCR or RPA. The use of 4 to 6 primers in combi­nation with a DNA polymerase with lacking exonu­clease activity and strong strand displacement activity is the basis for an isothermal LAMP reaction. The following figure shows the complex primer-​​/​ probe-​​arrangement which is neccessary for a well working LAMP assay.

Complex Primer-​​/​ Probe-​​Arrangement of Loop mediated isothermal Ampli­fi­cation

Complex Primer-​​/​ Probe-​​Arrangement of LAMP assay

Charac­ter­istic LAMP incubation temper­a­tures are between 60 and 70°C, which allows initial primer invasion into weakened doublestranded DNA (template). This can be improved in some cases by reaction additives such as betaine. In the initial phase of ampli­fi­cation, a dumbbell-​​shaped reaction inter­me­diate is formed due to the elongation of large modular primers and continuous strand displacement of primary amplicons. The terminal loops of this inter­me­diate structure are the starting point for the exponential DNA ampli­fi­cation. In contrast to other DNA ampli­fi­cation methods, signif­i­cantly more product is amplified during LAMP containing amplicons of many different sizes. The following figure is explaining the ampli­fi­cation mechanism in more detail.

General Amplification Mechanism_Loop mediated isothermal Amplification
Mechanism of a Loop mediated isothermal Ampli­fi­cation

How to Modify my LAMP Assay for Lateral Flow Readout

LAMP requires a relatively complex primer design. 4 to 6 primers are necessary to initiate efficient DNA ampli­fi­cation. Due to the complexity, it is not recom­mended to design LAMP-​​Primers by hand. Rather, it makes sense to use available online tools to find an appro­priate primer set and use this as a basis for further optimization. In order to visualize LAMP reaction products on a Lateral Flow Device, it is recom­mended to use a labeling strategy which allows incor­po­ration of Biotin– and FAM/​FITC–lables into amplicons. The easiest way to integrate neccessary labels is the usage 5‘ labeled LAMP primers. Therefore, it can be beneficial to test various labeling options (e.g. FIP & LF, FIP & BIP, LF & LB, etc.). Moreover it is possible to inter­grate an additional probe hybridization, in order to improve overall assay speci­ficity.

LAMP & Multi­plexing

Despite the complex primer design, LAMP is a promising tool for the devel­opment of multiplex DNA ampli­fi­cation assays. Once a suitable primer combi­nation has been found, duplex or triplex ampli­fi­ca­tions can also work with satis­fying accuracy and sensi­tivity under low resource settings. The hybridization of specific probes to amplicons is a frequently used methodical tool in LAMP appli­ca­tions in particular, which of course can also be an important facet for increased multi­plexing in the context of lateral flow. We at Milenia Biotec see the enormous potential of multiplex LAMP lateral flow assays. The future will show whether this actually proves to be true.

Learn more about (LAMP) Multi­plexing & Lateral Flow

Limita­tions

The LAMP is a versatile tool and offers numerous possi­bil­ities, especially in combi­nation with lateral flow. Like any other method, this technique has advan­tages, but also disad­van­tages. An important LAMP-​​characteristic limitation is the complex assay design. Of course, there are very good, available LAMP primer design tools, such as NEB‘s Primer Design Tool or the Primer Explorer (V5) from Eiken. When using less suitable sequence sections or multi­plexing is neccessary, the devel­opment is often compli­cated. In such cases, signif­i­cantly more energy, money and time has to be invested in the devel­opment compared to other DNA ampli­fi­cation methods. The induction of the so-​​called high dose hook effect or the problem of carryover conta­m­i­na­tions are LAMP-​​LFA charac­ter­istic pitfalls. But both phenomena are more connected to handing mistakes, rather than method­ological limita­tions.

Benefits

A LAMP reaction is able to detect low copy DNA targets in less than 30 minutes. In some publi­ca­tions ampli­fi­cation speed could be further accel­erated. The time and energy invested in assay design pays off. Once a suitable primer combi­nation has been found, the LAMP works with excellent sensi­tivity, which is often compa­rable to estab­lished RT-​​PCR appli­ca­tions. Very sensitive DNA analysis methods are often a blessing and a curse at the same time. Carryover conta­m­i­nation is a common phenomenon, especially for nucleic acid lateral flow assays. The very good compat­i­bility of the LAMP reaction with the uracil-​​DNA-​​glycosylase (UDG /​ UNG) for prevention of carryover conta­m­i­nation has been shown several times and thus has to be highlighted as an important advantage of this method.

Method Comparison Table
Method combined with LFASensi­tivitySpeci­ficitySpeedreverse Transcription (one pot)RobustnessMethod. VarietySimple Assay DesignMulti­plexing AbilityPrevention Carryover Cont.
PCR
LAMP
RPA
CRISPR (Label-​​Sep.)
CRISPR (Label-​​Inc.)