Loop-mediated isothermal amplification (LAMP)

Since the development of the LAMP technology by the working group of Notomi et al. (2000), the technology has been used in over 500 studies in the field of research and diagnostics (Schaudies 2014). Reasons for that are mainly the user-friendly software’s often freely available for the design of LAMP primer sets, the availability of the reagents, the simple operation of that technology without the need of a thermocycler and the great combinability with easy result-visualization techniques (Mori et al. 2001; Goto et al. 2009; Schaudies 2014). The outcome of a LAMP reaction are long, stacked stem-loop or also called dumpbell- structures of DNA with numerous inverted repeats of the target, that were described by Gill et al. (2011) as cauliflower-like structures with multiple loops. The yield of a LAMP reaction with a volume of 25 µL achieves about 10–30 μg that is often over 20-fold higher than the yield of a PCR reaction containing the same reaction volume (Mori et al. 2001; Schaudies 2014). During DNA amplification, pyrophosphate ions are produced as by-products. In LAMP reaction high amounts of pyrophosphate ions are produced and form a white precipitate of magnesium pyrophosphate. The turbidity of this insoluble magnesium pyrophosphate-complex is a feature of the LAMP technology that enabled the design of various “indirect” or “unspecific” result documentation techniques in the last two decades. The result-visualization can be performed either by using the same devices as for PCR, including portable devices, or by using the already mentioned “indirect” visualization techniques, where the by-product magnesium pyrophosphate is displayed in an intensified manner (naked eye detection).

To realize an easy naked eye result-monitoring after LAMP reaction, fluorescent dsDNA intercalating dyes (ethidum bromide, Moradi et al. 2012; propidium iodide and SYBR® _Green,

Hill et al. 2008) could be added to the reaction mix. Nie (2005) was the first, who published RT-LAMP application for potato virus detection. He was working with PVY detection and the article of 2005 demonstrated the visualization of high accumulation of pyrophosphate during DNA amplification in RT-LAMP via turbidity observation by the naked eye and pyrophosphate absorbance measurement at 405 nm and 600 nm wavelength. After Goto et al. (2009), it was possible to support the turbidity observation of pyrophosphate by adding the metal ion indicator

HNB indirectly senses the presence of pyrophosphate and thus occurred amplification events due to the high amplification rate of LAMP.

Some intercalating dyes or other detection chemicals such as cationic polymers can inhibit the LAMP reaction (Schaudies 2014). Therefore, for result imagining it was common to add the detection reagent after amplification, like in the application of agarose gel electrophoresis. However, all detection systems requiring open tubes after the amplification process do increase the chance of cross-contamination. Cross-contamination is a drawback in the highly reactive LAMP assay, which happens via LAMP-products arising as aerosols (Tao et al. 2011; Schaudies 2014; Siemonsmeier 2019). Therefore, many research efforts have been made to find detection chemicals that can be add prior to the LAMP reaction. Detection agents not affecting the LAMP process, like metal indicators (hydroxynaphthol blue: Goto et al. 2009; Calcein: Tomita et al. 2008 and Eriochrome black T: Oh et al. 2016) have been found to accelerate the “indirect” detection of amplification processes. In cases where LAMP primer sets are capable of strongly enhancing the target sequence with strong magnesium pyrophosphate formation and turbidity, it was easier to directly monitor the result with the naked eye (Mori et al. 2001) that makes tube opening after LAMP-reaction unnecessary. In that respect, Real-Time LAMP or quantitative LAMP (qLAMP) was tested and applied. This was done by fluorescence measurement (Parida et al. 2011) or again in an “indirect” manner by turbidity measurement of magnesium-pyrophosphate precipitate using a turbidimeter that was established by Mori et al. (2004). Further detection chemicals used for LAMP product monitoring are listed in Hadersdorfer (2013) and Schaudies (2014).

The rate-limiting step of LAMP and SMAP 2 technologies is the creation of stem-loop or dumpbell-structures of DNA with numerous inverted repeats. After performing this step, the remaining amplification is less prone to the purity of the target input. This might be the reason for the widely used claim that the LAMP assay is very robust and not demanding in terms of sample input and PCR inhibitors (Kaneko et al. 2007; Francois et al. 2011; Yang et al. 2014b). Consequently, Hadersdorfer et al. 2011 was the first skipping RNA purification for plant samples using the robust Bst DNA polymerase with strain displacement activity. In a LAMP assay, they reliably detected Plum pox virus in crude plant sap extracts of Prunus leaves (Hadersdorfer et al. 2011), young shoots and fruits (Hadersdorfer et al. personnel communication).

Introduction

1.3.3.1.1 Principle LAMP The nucleic acid strand displacement activity of Bst DNA polymerase in combination with the usage of a target specifically designed set of at least four primers (F3, B3, FIP and BIP), enable amplification of a target sequence at isothermal conditions, often at 60-65°C. F3 and B3 primers confine the target sequence in forward and backward direction, like PCR primers do, by binding to the target regions F3c and B3c (Figure 9 A). Forward inner primer (FIP) and backward inner primer (BIP) are hybrid primers holding at the 3′-end a

complement to the target strand region (F2c and B2c), called F2 and B2. In addition, the hybrid primers contain at the 5′-end homologous sequences called F1c and B1c (Figure 9 A) that are located downstream to the F2c and B2c sequences on the target strand or its complement, respectively. FIP and BIP bind within their F2 and B2 sequence to the confined region of F3 and B3 primers, and thus modify the amplifiable target sequence by their attachment sequence, F1c and B1c, directed for infinite annealing of FIP and BIP primers. The DNA polymerase displaces the evolved FIP- and BIP-originated nucleic acid strands based on amplification starting from F3 or B3 primers (Figure 9 B). This results in stem-loop structures or also called dumbbell-like structures (Figure 9 E). The dumbbell-like structures serve as starting molecules for annealing of again FIP and BIP primers leading to consecutive amplification and displacement processes (Figure 9 F-I). The following website helps to understand the sophisticated principle of LAMP: http://loopamp.eiken.co.jp/e/lamp/anim.html.

Figure 9: Schema of the LAMP reaction.

For initiation of the reaction B3 and the complementary part of BIP hybridize to the target strand (B). After formation of the complement and strand- displacement action of the DNA polymerase, a loop is formed owing to the newly formed self- complementary sequence-ends (C). The same occurs for the counterpart (D). This results in a stem-loop or dumbbell-like DNA structure (E) that is used as an initial structure for further amplification. During further amplification, various frag- ments of differing size (F-I) arise. That is because the DNA polymerase elongates the newly formed 3′-end from the loop plus the 3’-end formed by annealing of BIP to the same loop (F+G). The same happens for the resultant sequence, but FIP anneals instead of BIP (H). Afterwards, the resulting DNA sequences are again ready for further elongation and strand- displacement that leads to long fragments with varying size and shape (I). The displayed figure is according to Notomi et al. (2000) and Hadersdorfer (2013).