Loop-mediated isothermal amplification (LAMP) is a relatively new DNA amplification technique that due to its simplicity, robustness, and low cost delivers considerable advantages over other amplification methods. LAMP utilizes autocycling and strand displacement to amplify DNA samples via the activity of the large fragment of Bst DNA polymerase. The isothermal nature of LAMP dispenses with the need for expensive thermocyclers used in conventional polymerase chain reactions (PCR). A number of diagnostic tools incorporating LAMP have been developed for clinical applications, including infectious disease diagnosis, as well as food quality control applications and environmental monitoring.
LAMP uses 2-3 primer pairs recognizing multiple distinct regions of target DNA for a highly specific amplification reaction (figure 1). The amplification products are generally quite long, and target amplification is so extensive that several different detection modes are possible. One of the most useful is the use of a pH indicator dye, such as phenol red, in the reaction mixture. The polymerization of deoxynucleic acids into DNA during LAMP reactions releases hydrogen ions as a biproduct, resulting in the acidification of the reaction mixture and the color of phenol red will turn from magenta to yellow when this occurs.
Figure 1. LAMP amplification schematic. Forward and reverse internal primers target and duplicate specific analyte DNA sequences, as well as create loop structures with primer extension. Subsequent amplification of the generated looped sequences is exponential.
While it is quite easy to manually assess this color change in tubes, microplate-based high throughput requires an automated means to assess the assay results. Automated LAMP assay assessment can be accomplished by monitoring the ratio of 420 nm and 560 nm absorbance (420:560) kinetically (figure 2). Only reactions that result in DNA amplification produce sufficient hydrogen ions to alter the pH of the reaction mixture and significantly and increase the 420:560 ratio. With good primer design only true positive samples will result in a marked increase in the 420:560 ratio, while samples that either lack target (NC) or have inappropriate primers (IC) will have little change in the ratio.
Figure 2. Kinetic measurement of the LAMP assay over 60 minutes. Data are displayed as a ratio of the 420 nm and 560 nm absorbance values (420:560). The positive reaction threshold ratio value is depicted as a dashed line. These data represent the mean and standard deviation of eight determinations.
In this TekTalk we feature two new application notes that demonstrate the use of the Agilent BioTek Synergy Neo2 hybrid multimode reader to monitor LAMP assays kinetically, as well as highlight the ability to improve assay performance with an increased instrument temperature range. Additionally, we provide tips for general LAMP assay optimization, including primer selection and design.
Characterization of LAMP Assays Using a Multimode Microplate Reader
Over the course of the last 20 years, isothermal nucleic acid amplification tests, such as loop-mediated isothermal amplification (LAMP) have emerged as an important diagnostic tool, not only for clinical applications, but also for food quality control and environmental monitoring. LAMP is an assay technology that has gained traction for its ability to detect nucleic acid sequences under a number of different conditions without specialized equipment. While typically run as an end point reaction in PCR tubes using manual observation for positive/negative determinations, the use of colorimetric pH change detection lends itself to automated kinetic monitoring. Here we describe the adaptation of a PCR tube-based assay to microplates and the subsequent use of the Agilent BioTek Synergy Neo2 multimode reader to run LAMP assays at low volumes in 384-well microplates.
Improved LAMP Assay Performance
with Increased Temperature
Loop-mediated isothermal amplification (LAMP) is an autocycling and strand
displacement DNA synthesis amplification method involving the use of the
large fragment of Bst DNA polymerase. While typically performed at a uniform temperature of 65 °C, there are certain advantages with the use of increased temperatures when running this assay technology. This application note describes the use of the Agilent BioTek Synergy H1 multimode reader with a reaction temperature of 69 °C to reduce time required to elicit positive LAMP assay reactions.
Synergy Neo2 multimode reader is designed for the screening laboratory, with speed and ultra-high performance. Multiple PMTs and laser TRF enable rapid measurements; the monochromator-based and filter-based optics enhance sensitivity and flexibility. Environmental controls optimize conditions for live cell assays, while Gen5 software offers complete reader control, powerful data analysis, and automation and LIMS integration.
Primer selection and design
The LAMP assay reaction is dependent on the faithful annealing of the designed primers to their complementary target sequences. Compared to other amplification technologies LAMP assay primer design is more difficult. Primer design that incorporates primer length, guanine-cytosine (GC) content and unique target sequences play a critical role in reaction fidelity. Individual primers are typically are 15-25 bases in length, with a GC content of 40-60%. Regarding primer sequences, if possible, it is best to avoid runs of 3 or more of one base or dinucleotide repeats (e.g., ACCC or ATATATAT). Primer pairs should have similar Tm values, with a maximum difference of 5 °C. The external primers, which initiate the reaction generally have a Tm of 55-63 °C, while the internal and loop primers, which drive exponential amplification have higher Tm value (60-68 °C). The use of 5’-tails as part of the internal primer design and primers in the formed loop also improve the fidelity of primer extension reactions. Fortunately, there are a are number of different LAMP primer designing software packages available to optimize sequence selection. PrimerExplorer from Eiken is one example, while GLAPD is an online genome-based LAMP primer design package developed at the Shanghai Center for Bioinformation. New England Biolabs also offers a user-friendly primer design tool on their website.
Preheating of the reader is critical for a successful assay run. The relatively high assay temperature compared to ambient room temperature requires a substantial input of heat energy to warm all the components in the read changer to equilibrium. While the LAMP assay technology is isothermic, and does not require rapid temperature changes, consistent results require that the temperature is stable. Initiating the assay run before temperature equilibration has occurred can result in positive reaction being delayed or inconsistent results across the microplate. We generally preheat for 1 hour when increasing reader temperature from ambient temperature to 65 °C.
Lamp Assay Resources
- Improved LAMP Assay Performance with Increased Temperature
Characterization of LAMP Assays Using a Multimode Microplate Reader
- Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride
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