Chapter 3.5. Loop-mediated isothermal amplification (lamp): furtherance of assorted detection systems towards point-of-care settings


Nurul Najian Aminuddin Baki, Engku Nur Syafirah Engku Abdul Rahman, and Chan Yean Yean

Art work
Oliver Reinhardt
Cultural issues are of capital importance in the design and introduction of diagnostic tools for poor regions.
Aspects such as the need of blood sampling and the name and the colour of the device could have a great
influence in the successful implementation.
Real generosity towards the future 
lies in giving all to the present.
Albert Camus, 


Significant developments in molecular detection have been reported over the years. Practical tools for nucleic acid amplification are invaluable in a wide range of applications, including human clinical tests, veterinary molecular tests, and identity and forensic tests. Nucleic acid amplification is widely used in the diagnosis of infectious diseases, because it can be carried out more rapidly than classical diagnostic methods [1]. Moreover, the process generates several target copies, which significantly increases assay sensitivity.

The development of molecular diagnostic tools has considerably improved accuracy and reliability. The ability to detect specific nucleic acid (DNA/RNA) sequences promotes accuracy, sensitivity, and specificity in clinically-based research, forensics, and other fields. Rapid, accurate, and readily available point-of-care diagnostic tests remain an urgent medical need. To this end, various molecular detection tools have been devised. Each of these amplification methods applies different synthesis principles, with specific advantages and disadvantages.

To date, various isothermal nucleic acid amplification protocols have been developed, including transcription-mediated amplification or self-sustained sequence replication, nucleic acid sequence-based amplification, signal-mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single-primer isothermal amplification, and circular helicase-dependent amplification [2]. Loop-mediated isothermal amplification (LAMP) is potentially one of the best options for the development of point-of-care devices. Upon combination with the appropriate detection methods, LAMP can be effectively utilized as a diagnostic tool whenever and wherever necessary, especially in resource-limited countries. This unique, outstanding tool isothermally amplifies nucleic acids, resulting in 109 copies of the target using only one enzyme. Bst DNA polymerase and specific primer sets are critical for strand displacement activity, leading to auto-cycling strand displacement DNA synthesis under isothermal conditions. The use of four specifically designed primers allows highly specific detection of six distinct sequences in the target DNA. Inner primers, designated “forward inner primers” (FIP) and “backward inner primers” (BIP), are designed to detect two regions each. Forward and reverse outer primers, F3 and B3, detect a single region each. Detection time can be reduced using loop primers acting as accelerators that amplify sequences in the stem-loop region [3]. The products are stem-loop DNA structures with several inverted repeats of target and cauliflower-like structures with multiple loops [4].

The major advantage of LAMP is that it can be performed in the field or anywhere outside the laboratory because it only requires a heat block or a water bath set at a single temperature, providing a low-cost and simple way to amplify DNA targets of interest. Moreover, large amounts of DNA can be amplified within one hour. By comparison, the polymerase chain reaction (PCR) requires special equipment (thermocyclers), and the cycles of denaturation, annealing, and extension are time-consuming. Furthermore, LAMP shows higher specificity because problems such as the formation of primer dimers, false priming, and false-negative results often arise in PCR [5]. As with other molecular methods, precautions to avoid carryover contamination are required. The manufacturer (Eiken Chemical Co., Ltd., Tokyo, Japan) has reported that carryover contamination of LAMP is significant owing to its high sensitivity. Therefore, the recommendation is that either LAMP reaction vessels should not be opened at all or should be opened in separate rooms using discrete equipment. Despite this limitation, various detection methods have been developed in combination with LAMP, with the aim of enhancing results and preventing contamination. From simple visual turbidity, detection methods for LAMP have expanded to involve turbidimeters, fluorescent agents, colorimetric agents, lateral flow dipsticks, and lab-on-a-chip devices. LAMP, in combination with an appropriate detection system, has significant potential as a diagnostic tool in resource-limited countries.

Chronology of LAMP

Loop-mediated isothermal amplification (LAMP), originally developed by Notomi et al. (2000) [4], is a novel gene amplification protocol that allows rapid and sensitive detection. The method is highly specific, and is therefore a feasible alternative to robust PCR. LAMP has been modified in various ways to achieve robustness. Its development over the years is detailed below.

2000. Invention of the novel LAMP method. The use of a single temperature and Bst DNA polymerase, which performs single-strand displacement, leads to the generation of up to 109 copies of the target sequence DNA in less than an hour [4]

2002Introduction of loop primers as accelerators of LAMP. These primers are designed between F1 and F2 (forward loop primer) or B2 and B3 (backward loop prime) to speed up reactions by hybridizing to stem-loops [3].

2004Development of the turbidimeter, an apparatus capable of simultaneously measuring the turbidity of multiple samples. This equipment maintains a constant temperature to facilitate real-time LAMP measurements [6]

Introduction of an integrated microsystem for isothermal amplification that analyzes specific gene fragments on poly(methyl methacrylate) microchips. Using this system, amplification and analysis can be performed in 15 minutes, leading to high specificity, good reproducibility, and rapid gene detection [7].

2012. Adaptation of standard LAMP primers to contain a quencher–fluorophore duplex region that generates a fluorescent signal for amplification. This method employs novel real-time multiplex LAMP [8]

2013. Combination of LAMP and nucleic acid lateral flow immunoassay (NALFIA) to produce a compact and low-cost device. This technique allows fully-integrated, sample-in to answer-out diagnosis at the point-of-care in low-resource settings [9]

LAMP principle

The LAMP reaction is carried out with a set of four primers designed specifically for the target DNA and designated F3, B3, FIP, and BIP. This set of primers recognize six distinct target sequence regions. Each F3 and B3 primer recognizes single sites of the target sequence at the 3ʹ and 5ʹ ends, whereas the FIP and BIP primers recognize two sites each at the 3ʹ and 5ʹ ends, respectively. Theoretically, this auto-cycling strand displacement activity is divided into two steps: non-cyclic and cyclic. In the non-cyclic step, the starting structure for the cyclic step is produced. FIP initiates DNA synthesis, by which the F2 primer of FIP recognizes the F2c region at the 3ʹ end of the target sequence, as shown in Figure 1. Next, the F3 primer anneals to the F3c region, followed by strand displacement DNA synthesis. Consequently, the strand elongated from FIP is replaced and released. The F1c of FIP forms a loop owing to its complementarity to the F1 region. The B2 primer anneals to the B2c region and DNA elongation takes place. As with F3, B3 anneals to the B3c region, and again strand displacement DNA synthesis causes release of strands elongated from BIP. B1c also forms a loop by attaching with the B1 region. This released strand thus forms a dumbbell-like structure.

In cyclic amplification, the dumbbell-like structure serves as a template. A process known as self-primed DNA synthesis occurs in which the 3ʹ end F1 region is initiated. In this step, only FIP and BIP play a role. Thus, in the LAMP assay, the amounts of FIP and BIP are greater than those of F3 and B3 primers. Various elongated structures are formed through several amplification steps. Ultimately, stem-loop DNA with several inverted repeats and cauliflower-like structures with multiple loops is produced. The LAMP product can be further detected using various methods. Details of the LAMP principle are presented in Figure 1.

Figure 1. I) Primer sets F3, FIP, B3, and BIP are included in the loop-mediated isothermal amplification (LAMP) reaction. The target DNA sequence with a specific primer-annealed region is ready to be amplified. The F2 primer of FIP anneals to the F2c region at the 3ʹ end to initiate DNA synthesis. The F3 primer anneals and synthesizes the complementary strand, which is released. The released strand forms a loop because at the 5ʹ end, F1c complements the F1 region. At the 3ʹ end, B2 of BIP anneals to the B2c region and DNA synthesis is initiated. B3 anneals to the B3c region and the resulting strand is released. Again, B1c, which is complementary to the B1 region, forms a loop. Consequently, a dumbbell-like structure is formed, which serves as the template for the next cycle. II) FIP and BIP primers recognize the F2c and B2c regions, and initiate the cyclic amplification step. III) Elongation and recycling steps occur with exponential amplification of the template with a dumbbell-like structure. At the end of the cycle, LAMP products with cauliflower-like structures are formed with inverted repeats of the target sequence on the same strand

Lamp Assay

The LAMP reaction mixture has a total volume of 25 µL. The assay constituents include FIP, BIP, F3, and B3 primers, Bst DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), betaine, Tris-HCl (pH 8.8), KCl, (NH4)2SO4, MgSO4, 0.1% Triton X-100, and target DNA. A loop primer (LB or LF) can also be included to increase the assay speed. The mixture is incubated at 60–65°C for 1 hour to yield products of various sizes. This step is followed by termination at 80°C for 10 min to inactivate the Bst DNA polymerase, which displays 5ʹ-3ʹ end polymerase and strand displacement activities but lacks 3ʹ-5ʹ exonuclease activity. Hence, the DNA polymerase allows strand displacement activity without the need for high temperatures for DNA double-strand denaturation. Normally, 8U Bst DNA is used in the LAMP assay [10-12]. The presence of betaine increases the overall rate of the LAMP reaction and target selectivity by significantly reducing the amplification of irrelevant sequences [4]. Betaine also plays an important role in reducing base stacking in the reaction [13-15].

Primer design

The design of appropriate primers for LAMP is crucial to optimize assay specificity and efficiency. LAMP primers can be designed either manually or using software, such as PrimerExplorer (http://primerexplorer.jp/e/). Four important criteria are considered: melting temperature (Tm), stability at the end of each primer, GC content, and secondary structure [16]. The recommended Tm value for the primer region is ~60–65°C for GC-rich and normal regions, and ~55–60°C for AT-rich regions. The nearest-neighbor method is used to determine the Tm because it provides the closest approximate to the actual value. The primers should be highly stable because they serve as the starting point of DNA synthesis. The stability of the primer ends (the 5ʹ end of F1C/B1C and the 3ʹ end of F3/F2, B3/B2, and LF/LB) should be −4 kcal/mol or less. The GC content should be ~50–60% for GC-rich and normal sequences, and 40–50% for AT-rich sequences. To avoid secondary structure formation, the 3ʹ end of the primer should not be AT-rich or complement other primers; this prevents the possibility of self-priming, which reduces amplification efficiency [17]. Moreover, the distance between the primers should be considered. The four-primer set that recognizes the six distinct DNA target sequences is the main feature of the LAMP reaction. The distance between the 5ʹ ends of F2 and B2 should be 120–180 bp, and that between F2 and F3, and B2 and B3 should be in the range 0–20 bp. The length of the loop-forming region between the 5ʹ end of F2 and the 3ʹ end of F1, and between the 5ʹ end of B2 and the 3ʹ end of B1 should be in the range 40–60 bp. Each F3 and B3 primer is specific to a single target sequence only. The F3 primer recognizes the F3c region at the 3ʹ end of the target sequence, whereas B3 recognizes the B3c region at the 5ʹ end of the target sequence. Unlike F3 and B3, FIP (forward inner primer) and BIP (backward inner primer) are hybrid primers consisting of two oligonucleotides each connected via a T-linker [4]. FIP is composed of F1C and F2 primers, whereas BIP comprises B1C and B2 primers. FIP recognizes F1 and F2c regions, and BIP is specific to B1 and B2c at the 5ʹ end of the target sequence. Notably, the authors of an earlier study reported that the linker disrupts the LAMP reaction [18]. This was attributed to primer self-hybridization owing to the design of coincidently similar sequences, and it was thus proposed that the T-linker can be omitted [19]. A number of LAMP detection studies without a T-linker between F1c and F2 and between B1c and B2 have been carried out [20-23]. The LAMP reaction is accelerated with the aid of loop primers (Loop forward/ Loop backward) [3] designed using the region between F2 and F1 (or B1 and B2) in the direction of F1 to F2 (or B1 to B2).

In 2012, electronic LAMP (eLAMP) was developed to simulate the LAMP reaction, where users could efficiently test the designed LAMP primers for target sequences [24]. This PERL script electronically predicts specific amplification of the target without the need to perform bench work. Hence, users can determine whether the designed primers work on known target sequences.

LAMP detection systems

The higher sensitivity and ease of carryover of amplicon contamination with LAMP, compared with other nucleic acid amplification methods, is discussed with respect to various product amplicon detection methods, including the advantages and disadvantages of these particular techniques; LAMP reduces the risk of contamination and prevents false-positive results. The methods vary from simple conventional protocols to detect nucleic acid amplification products, such as agarose gel electrophoresis, to methods of moderate complexity, including visual turbidity (assessed by the naked eye and/or using a turbidimeter), visual fluorescence or colorimetric methods, and further complex technologies, such as the nucleic acid lateral flow immunoassay (NALFIA) and biosensors.

Agarose gel analysis

The most common detection method for LAMP products is agarose gel electrophoresis [20, 21, 25]. After amplification, ~10 µL of the product is electrophoresed through a 2–3% agarose gel, and the amplified bands are viewed under ultraviolet (UV) light. Positive amplification is observed as typically smeared ladder-like bands under the UV transilluminator at 302 nm, because the LAMP reaction produces stem-loop DNA and cauliflower-like structures with multiple loops of various sizes [4]. The intensity of the smeared band on the agarose gel is based on the quantity of the initial DNA template (decreasing the quantity of the initial DNA template leads to decreased band intensity). Specificity of amplification is confirmed from the approximate size of the smallest band [4]. Three methods can be applied to confirm that the correct target of interest has been amplified [26]. First, LAMP amplicons can be further digested by restriction enzymes and the predicted size of the target sequence can be visualized on the gel to determine the specificity of the LAMP assay [26-28]. Mitsunaga et al. (2013) improved LAMP assay specificity by including restriction enzyme digestion, especially for multiplex LAMP. The amplified product was purified by ethanol precipitation and digested with the restriction enzyme at 37°C for 1–3 hours before electrophoresis on a 2–3% agarose gel. Lin et al. (2009) inserted an AluI endonuclease site in the target segment. After amplification, the LAMP product was confirmed by digestion with AluI [27]. The two fragments of the electrophoresis procedure shown in Figure 2 can be used to estimate the specificity of the amplification.

Figure 2. Lane 1: DNA ladder, Lane 2: loop-mediated isothermal amplification (LAMP) product, Lane 3: AluI-digested LAMP product. Adapted from Lin, Chen [27] with permission.

The second method of confirmation involves obtaining DNA melting curves [29]. DNA melting curve analysis was carried out to evaluate the dependency on temperature of double-stranded DNA dissociation. After monitoring the detection channel at high temperature for a specific time, products with the expected sizes were analyzed and confirmed using gel electrophoresis [29]. In the third method of confirmation, LAMP bands are excised from the agarose gel, cloned into the appropriate vector, and transformed into competent cells. The sequence is aligned with that of the target sequence. The cloned sequence possessing 100% identity with the target sequence confirms the LAMP assay specificity of the purified DNA [26]. Figure 3 shows the undigested LAMP product, compared with that digested using restriction enzymes.

Although agarose gel electrophoresis is popular for LAMP amplicon detection, it involves a high risk of causing cross- and carryover contamination, leading to false-positive results. Contamination may occur when the LAMP reaction tubes are opened, which is necessary for gel electrophoresis. This problem does not occur in PCR gel analysis, but because LAMP produces high quantities of target products, aerosolized LAMP amplicons may be present in the laboratory environment if appropriate precautions are not taken.

He and Xu [30] showed that the uncontrolled products from previous LAMP runs are the major source of subsequent LAMP reactions. If large quantities of LAMP products are exposed to the environment, within a short time, the buildup of aerosolized amplicons contaminates the laboratory reagents, pipette sets, equipment, and even ventilation systems. Recommendations to prevent this include providing a different room or work area to add the DNA template from the workplace where the master mix is prepared, introducing ultraviolet treatment for LAMP premixes and decontamination of work areas, use of filtered pipette tips, wiping all equipment used for DNAse to prevent DNA contamination, and preparing the LAMP master mix in a laminar flow arrangement to avoid cross-contamination. Carryover contamination caused by LAMP amplification products can be overcome using modified primers and the uracil-DNAglycosylase/dUTP approach, which is effective in eliminating up to 109 copies of the product for PCR [31]. 

Careful handling of waste disposal is another way to counter contamination risk by preventing aerosol formation, as well as providing appropriate disposable clothing and/or frequently changing personal protection equipment (PPE) such as gloves, masks, and laboratory coats.

The presence of intercalating agent

DNA amplicon visualization under UV light is facilitated by staining with dyes such as ethidium bromide (EtBr), SYBR Gold, SYBR Green, Crystal violet, and Methylene blue [32]. EtBr is generally used in agarose gel analysis for the detection of DNA bands [33] owing to its sensitivity, ease of use, and low cost. EtBr intercalates within the DNA molecule, and when exposed to UV light, electrons in the aromatic ring of EtBr are activated, leading to fluorescence of the intercalating DNA segments. However, EtBr is not recommended for use because it has potent mutagenic properties; extreme caution and decontamination prior to disposal are imperative. The choice of any specific intercalating agent depends on sensitivity, toxicity, and cost. Methylene blue and Crystal violet do not require UV exposure, which is a major advantage. However, these agents lack the sensitivity of other dyes [32]. SYBR Green and SYBR Gold, which are UV-dependent agents, may be considered substitutes for EtBr because they are less toxic.

Visual Turbidity

Visual turbidity is the simplest, most cost-efficient detection method for LAMP because the visual presence or absence of a white precipitate determines whether target nucleic acid amplification has occurred. The turbidity of the LAMP reaction mixture after amplification is highly correlated with the amount of amplified DNA [34]. A turbid solution at the end of the process indicates a positive result.

Conversely, no turbidity in the reaction tube indicates a negative result. During the LAMP reaction magnesium pyrophosphate, a white precipitate byproduct, is produced. Theoretically, the pyrophosphate ion is released from substrate dNTPs in the LAMP solution [35]. Pyrophosphate strongly binds to metal ions and forms insoluble salts. During the LAMP reaction, pyrophosphate ions combine with magnesium ions (metal ions) to produce a white precipitate. After amplification, the reaction mixture is centrifuged for several seconds at 3,500 x g, and the accumulated white precipitate is easily visualized by the naked eye in ambient light [34], as shown in Figure 4.

Visualization of turbidity with the naked eye is considered an ineffective detection method because it does not produce a detectable color change. Hence, a real-time turbidimeter has been developed to monitor LAMP amplification [6]. The turbidimeter is capable of simultaneously and continuously measuring the turbidity of multiple samples, which makes LAMP outstanding for real-time detection.

The apparatus is designed to maintain a constant temperature, facilitating real-time measurement of changes in the turbidity during LAMP reactions. LAMP real-time turbidimetry can be applied to diagnostics in view of the correlation between the LAMP reaction and the amount of initial template DNA, and the ability to quantify template DNA [6]. The turbidimeter functions by emitting light (650 nm) through reaction tubes. Tubes in the turbidimeter are heated at a constant temperature within the range 60–65°C. This detection method is advantageous in diagnostics because the results can be obtained in real time. However, multiplex detection is impossible because turbidimetry does not depend on the sequence of amplification products, as in real-time PCR. Moreover, the method cannot be applied to samples that are turbid or contain materials that absorb light in the turbidimeter unless they are purified or suitably pretreated [6].

Visual Flourescence

Fluorescence spectroscopy is a well-known technique that is used for the visualization of macromolecules and macromolecular complexes such as nucleic acids and proteins [36]. Complex formation through interactions between fluorophores and nucleic acids leads to increased brightness. A number of studies on LAMP have used fluorescent agents for detection [27, 28, 35, 37]. The method is reasonably simple and can be assessed directly by the naked eye. Moreover, different agents display dissimilar sensitivities. The fluorescence of the LAMP reaction can be visualized using either intercalating dye or fluorescent-labeled probes.

Ethidium BromideEthidium bromide intercalates between DNA base pairs [38]. This intercalating agent speeds up sample screening without gel electrophoresis. Moreover, considering availability, sensitivity, and cost, ethidium bromide is a superior option [25]. Theoretically, electrons in the aromatic ring of ethidium bromide are activated when exposed to UV light, and release energy when they return to the ground state [32]. Positive amplification produces an orange color in the reaction solution under UV light [25]. However, one major shortcoming is that this detection agent is carcinogenic and requires additional care during handling.

SYBR Green. SYBR Green is a fluorescent double-stranded DNA-specific intercalating dye. It interacts with DNA in three different ways: intercalation between base pairs, electrostatic interaction, and extended contact with the DNA grooves [36]. Fluorescence is enhanced through association with or dissociation from DNA. SYBR Green has many applications including gel electrophoresis, double-stranded DNA quantification in solution, and real-time PCR [38]. The authors of recent studies using SYBR Green to detect LAMP amplification products [27, 28, 39] have reported that the dye is simple to use and clearly discriminates between positive and negative LAMP amplification.

Addition of 1 µL (1:1000) SYBR Green I dye facilitates color differentiation between positive and negative results [40]. Positive and negative reactions can be assessed either in ambient or in UV light (320 nm). In ambient light, positive amplification leads to a change in the original orange of the dye to green, whereas the orange color is retained if no amplification occurs. Under UV light, positive amplification is visualized as a bright apple green color. This color is permanent, allowing storage for further reference.

Calcein. Calcein, a fluorescent metal indicator, forms complexes with divalent metal ions. A LAMP detection system that focuses on changes in metal ion concentration in the reaction solution throughout the amplification process was developed by Tomita, Mori [35]. In LAMP reactions, manganese is used as the metal ion. During amplification, pyrophosphate ions produced as a byproduct from deoxynucleotide triphosphates (dNTPs) strongly bind to various metal ions to form insoluble salts. Calcein is included in the LAMP reaction mixture preparation before incubation. Initially, calcein combines with manganese ions (Mn2+) and remains quenched. However, manganese ions are deprived of calcein by the pyrophosphate ions during amplification, resulting in fluorescence. Magnesium ions, another byproduct of the LAMP reaction, also combine with free calcein and increase fluorescence. Consequently, positive reactions fluoresce under UV light, as shown in Figure 3.

Figure 3. Left: Negative (-) and positive (+) amplification under UV light. Right: Negative (-) and positive (+) amplification under ambient light. Figure adapted from Tomita, Mori [35] with permission.

Propidium iodide. Propidium iodide is an intercalating fluorescent molecule. Its application leads to a color change of the positive mixture in LAMP. Propidium iodide is reported to be one the best agents for visual discrimination [37]. Using this intercalating dye, a positive result can be visualized in ambient light. During positive amplification, the color of the reaction solution changes from a deep red-orange to light pink. Visualization can be further enhanced with a UV transilluminator. A positive reaction leads to bright transillumination. This dye does not require freezing for storage, and is less expensive and more widely available than SYBR Green in developing countries. Moreover, for any color range (pale to yellow) of urine samples, propidium iodide has no effect on the final LAMP products [37].


Hydroxynaphthol blue. Hydroxynaphthol blue (HNB) is an indicator for simple colorimetric LAMP detection. The dye is used as a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions [41]. The use of HNB for the LAMP assay has a number of advantages, including reduction of cross-contamination due to pre-mixing and elimination of the necessity of opening the tube. Pre-addition of 120 µM HNB to the LAMP reaction solution does not inhibit amplification efficiency. In positive reactions, the HNB color changes from violet to sky blue. HNB colors in solutions with positive or negative reactions remaining stable, even after two weeks of exposure to ambient light [41]. The detection sensitivity is equivalent to that of the SYBR Green assay, and can be easily judged with the naked eye.

Gold nanoparticles. Gold nanoparticles can be broadly applied to various aspects of diagnostics. These include nanoparticle aggregation, combination with a wide variety of surface functionalities, and utilization in electrochemical-based methods [42]. The aggregation of gold nanoparticles leads to a color change from red to blue. Gold nanoparticles aggregate when they cross-link with target DNA or at high ionic strength [43]. In the LAMP reaction, gold nanoparticles have been used to detect amplified amplicons. The agents are mixed together prior to LAMP amplification. Theoretically, dNTPs bind to gold nanoparticles via ligand–metal interactions and enhance their stability [44]. During LAMP amplification, dNTPs are reduced, causing gold nanoparticle aggregation. Positive amplification leads to a change from the original red color of gold nanoparticles to blue. This detection method depends on the ionic strength of the solution and the initial concentration of the dNTPs [44]. LAMP detection using gold nanoparticles thus provides a rapid and simple method owing to its label-free colorimetric characteristics, which are easily observed by the naked eye, thus providing a specific and low-cost diagnostic method. Moreover, no expensive apparatus is required, and the procedure can be used in sites where adequate instrumentation is available.

Sequence-specific detection. Specific targets of LAMP products are detectable via a colorimetric method in the presence of low-molecular-weight polyethylenimine (PEI). The color change of the LAMP reaction solution is indicative of amplification. PEI forms an insoluble complex with high-molecular-weight DNA, such as LAMP DNA products of varying lengths, which yield a colored precipitate that can be observed by the naked eye. Theoretically, an oligonucleotide probe labeled with fluorescent dye is hybridized to a specific target of the LAMP product. After amplification, low-molecular-weight PEI is added to form an insoluble DNA–PEI complex. The precipitate is observed on an illuminator in which the fluorescence of the labeled probe bound to specific LAMP targets can be visualized [45]. The probe that remains unbound to nonspecific targets is not visualized because it is not hybridized with the LAMP–PEI complex. PEI only forms insoluble complexes with high-molecular-weight DNA, and thus cannot form precipitates of complexes with fluorescently labeled probes. Thus, the unbound probe remains in the supernatant and cannot be visualized in the precipitate. The precipitate emits colored fluorescence based on the type of labeled probe used. Specifically, the precipitate emits green fluorescence if a fluorescein isothiocyanate (FITC)-labeled probe is used [45], and red fluorescence with a 6-carboxyl-X-rhodamine (ROX)-labeled probe [45, 46], as shown in Figure 4. LAMP reaction tubes can be visualized under a UV illuminator or UV-LED. This method allows multiplex detection owing to differential labeling of probes that emit different fluorescent colors, according to a study by Mori, Hirano [45]. However, a major limitation is that PEI needs to be added after the LAMP reaction because it strongly inhibits this process, and may thus cause carryover contamination. However, as suggested above, the risk of contamination can be reduced by opening post-LAMP reaction tubes in separate rooms using discrete equipment.

Figure 4. Tube 1: Loop-mediated isothermal amplification (LAMP) negative reaction, Tubes 2 and 3: FITC-labeled probe, Tubes 4 and 5: ROX-labeled probe. Figure adapted from Mori, Hirano [45].


A biosensor is a device that combines a biological recognition element and a transducer that converts the recognition event into a measurable electrical signal. Such sensors have several advantages, including low cost, high sensitivity, ease of operation, portability, and robustness [47-49]. Biosensors have a significant impact on molecular diagnostics at point-of-care testing locations without laboratory equipment. They have many uses including as autoimmune disease markers, infectious disease markers, cancer biomarkers, and cardiac markers [50].

Several nucleic acid analyses employ the miniaturizing system, which includes fewer manual steps, prevents contamination, and limits cost by reducing the volume of reagents used [51]. In 2004, Hataoka et al. performed LAMP on a microchip. After amplification, products were examined using the developed integrated microchip-based electrophoresis. Through this system, amplification and analysis of the reaction was successfully achieved within a rapid time-frame of 15 min with high specificity and reproducibility [7]. The LAMP products were additionally detected in a miniature reactor employing the turbidity change principle caused by the pyrophosphate byproduct [52]. A few years ago, microLAMP, a microfluidic device that can run several samples on a single chip, was developed [53].

The chip provides a quantitative readout of the amplified product via optic sensor measurement or the naked eye. The high sensitivity, specificity, and rapidity of evaluation as well as portability of this device makes it ideal for on-site analysis.

Nucleic acid lateral flow immunoassay (NALIFA)

The nucleic acid lateral flow immunoassay, also known as lateral flow dipstick (LFD), is a detection method that provides rapid and accurate evaluation of the presence or absence of one or more target nucleic acids in a sample. Various recent studies have focused on the application of nucleic acid lateral flow [54, 55]. The sample flows laterally through a microporous membrane by capillary action from the application region to a membrane region where a specific capture reagent is present. NALIFA is applicable to the detection of both non-amplified and amplified target nucleic acids. Examples of a combined amplification platform include PCR [56, 57], nucleic acid sequence-based amplification (NASBA) [58, 59], and strand displacement amplification (SDA) [60]. The targeted analyte is visualized as a visible colored line at the capture reagent line.

The integration of LAMP and nucleic acid lateral flow produces an ideal method that facilitates rapid infectious disease diagnosis. The method requires a single reaction temperature and utilizes simplified instrumentation. Numerous studies on combinations of LAMP and chromatographic lateral flow have been conducted successfully [61-66]. The user only needs to prepare the sample and immerse the lateral flow device in an appropriate buffer. NALIFA detects labeled amplicons that have been hybridized with a FITC-labeled DNA amplicon complexed with a gold-labeled anti-FITC antibody. Amplicons can be labeled with either biotin or digoxigenin. DNA probe design is carried out manually based on the target sequence. The probe is usually designed from the sequence between the B1c and B2c regions of the LAMP amplicon and labeled with FITC at the 5ʹ end. Theoretically, the resulting DNA duplex is trapped at the test line and forms a reddish-purple color [66]. The non-hybridized biotin-labeled primer probe also binds the test line but has no color, owing to absence of the FITC-labeled amplicon. Subsequently, the solution moves up through the strip, and the gold-labeled anti-FITC antibody is trapped and forms a reddish-purple color at the control line.

A fully integrated, sample-in-to-answer-out diagnosis at the point-of-care in a low-resource setting using a compact and low-cost device was developed using a combination of LAMP and NALIFA [9]. This reported cartridge and portable, low-power heater has a fully enclosed combined format, which reduces the risk of amplicon carryover contamination. One limitation of this nucleic acid analysis is the need for a clean environment to eliminate contamination, which may generate a false-positive result and limit its application outside the laboratory. Thus, the use of micro and nanofabrication allows amplification reactions to be fully enclosed microsystems, which reduces the risk of contamination.

Comparative advantages/disadvantages of the different detection methods of LAMP assay is shown in Table 1

Table 1. Comparative advantages and disadvantages of different detection methods of loop-mediated isothermal amplification (LAMP) assay.

Future prospect 

The loop-mediated isothermal amplification (LAMP) assay will have a significant impact on molecular biology. Several studies have focused on developing a novel and robust method for nucleic acid detection. Future advances should aim at creating integrated platforms for diagnostics without loss of sensitivity, and should provide fast, specific, and low-cost analysis in point-of-care settings. Assays that are less time-consuming and can be used to simultaneously test multiple samples will be useful. The LAMP assay is an economic alternative because it is inexpensive and simple, and is therefore predicated to be the first choice for amplification in developing countries.


The development of molecular diagnostic tools for use in point-of-care settings is a topic of significant research interest in various fields. Diagnostic methods that are simple to use, do not require special equipment, display superior sensitivity and speed, have low contamination risk, and are suitable for high-throughput DNA and RNA detection are critical, especially in developing countries. In view of its rapidity, sensitivity, and specificity, LAMP has great potential for nucleic acid amplification in molecular diagnostics.


This research was supported by the Malaysian Ministry of Education through Prototype Development Research Grant Scheme 203/PPSP/6740018 and the MyBrain15 Fellowship scheme awarded to the first and second authors. The financial support provided by the Universiti Sains Malaysia (USM) through Research University Grant 1001/PPSP/813045 is also gratefully acknowledged.


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