Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

Abstract

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.

Keywords: biotechnology; detection; diagnosis; diseases; rapid methods.

Figure 1

Schematic representation of LAMP reaction and its principle. LAMP reaction involved reaction mixture consists of dNTPs mix, Bst polymerase, fluorescence dye, primers and DNA template. Primer design: Design of primer for LAMP reaction and is characterized by the use of four different primers specially designed to recognize six distinct regions of the target DNA. Forward Inner Primer (FIP) consists of a F2 region at the 3'‐end and an F1c region at the 5'‐end; Forward Outer Primer (F3 Primer) consists of a F3 region which is complementary to the F3c region of the template sequence; Backward Inner Primer (BIP) consists of a B2 region at the 3'‐end and a B1c region at the 5'‐end. Backward Outer Primer (B3 Primer) consists of a B3 region which is complementary to the B3c region of the template sequence. LAMP reaction: The amplification starts when F2 region of FIP hybridizes to F2c region of the target DNA and initiates complementary strand synthesis, follow by F3 primer that hybridizes to the F3c region of the target DNA and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5'‐end. This single‐stranded DNA with a loop at the 5'‐end then serves as a template for BIP. B2 hybridizes to B2c region of the template DNA. DNA synthesis is initiated leading to the formation of a complementary strand and opening of the 5'‐end loop. Subsequently, B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand. This results in the formation of a dumbbell‐shaped DNA. The nucleotides are added to the 3'‐end of F1 by Bst DNA polymerase, which extends and opens up the loop at the 5'‐end. The dumbbell‐shaped DNA now gets converted to a stem‐loop structure (refer a and b). This structure serves as an initiator for LAMP cycling, which is the second stage of the LAMP reaction. Loop primers can be added as well for exponential amplification of LAMP. The final products obtained are a mixture of stem‐loop DNA with various stem lengths and various cauliflower‐like structures with multiple loops. Fig. 1 was modified from Notomi et al. (2000). [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

A summary of the commonly used LAMP detection method. (a) LAMP endpoint detection with turbidity, (b) LAMP calorimetric detection using calcein dye, (c) LAMP calorimetric detection using SYBR Green I dye, (d) LAMP calorimteric detection using HNB dye, and (e) analysis of LAMP product using gel electrophoresis. [Colour figure can be viewed at http://wileyonlinelibrary.com]