Introduction
In contrast to PCR, isothermal amplification or LAMP is carried out at constant temperature (60-65°C) avoiding the requirement of thermal cycling. “LAMP” stands for Loop-mediated Isothermal Amplification, a technique for rapid and specific amplification of DNA based on a sophisticated primer design. An optimized Bst polymerase with high strand displacement activity generates an amplification factor of up to 109 which is comparable to 30 cycles in a PCR assay. This allows the detection of a target gene within 10-30 minutes.
Advantage
LAMP has been observed to be more resistant to inhibitors in complex samples (e.g. blood, plant tissue) compared with PCR due to the use of a different polymerase (Bst vs. Taq). This allows the rapid detection of a target gene from minimally processed samples and makes LAMP the method of choice for a fast screening of individual DNA samples.
Limitations of LAMP may occur if precise target quantifications are requested or if amplification of several genes in a single tube (multiplexing) is essential.
Detection
Although some methods have been developed to visualize the DNA amplification by basic equipment or even the naked eye (increase of turbidity, color change of added dyes, hybridization to gold-bound ss-DNA) in general real-time detection of the DNA amplification by a fluorescent DNA-intercalator dye is recommended. Addition of SYBR Green or EvaGreen Fluorescent DNA Stain to the assay allows a sensitive measurement of the increasing amount of DNA without any influence on the reaction. So, LAMP combined with real-time detection has advantages over conventional PCR in terms of shorter assay time and simpler equipment.
LAMP Primer Design
Typically, 4 different primers are used to identify 6 distinct DNA regions allowing the specific amplification of the target gene. An additional pair of loop primers further accelerates the amplification allowing to cut down the total detection time to 10-20 min.
The manual design of primers may be challenging do to the complex LAMP reaction sequence. To simplify the design process and increase the likelihood of reaction success the use of a LAMP primer design software (i.g. Primer Explorer) is recommended.
As sensitivity and non-template amplification of in silico designed primers may vary, we recommend to evaluate 2–4 real primer sets for optimal performance before choosing a final set.
