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LAMP-based methods

Apart from enzyme-free self-templated amplification systems currently under development (Dong et al., 2012; Michaelis et al., 2014; Jung and Ellington, 2014), a variety of different methods for isothermal enzymatic in vitro DNA amplification have been developed over the past 20 years (see reviews by Gill and Ghaemi, 2008; Fakruddin et al., 2013; Yan et al., 2014; Li and Macdonald, 2015). Most isothermal amplification systems have the advantage of being easily operated with simple equipment since no thermal cycling is necessary as in PCR. Moreover, they are useful tools in point-of- care (POC) applications in clinical settings making them highly attractive for the diagnostic industry. Loop-mediated isothermal amplification (LAMP) is an approach to nucleic acid amplification that is especially suitable due to its high specificity, rapidness, user friendliness and low price. Niessen (2015) reviewed its application for the diagnosis of filamentous fungi and yeasts. The method has been applied for the species-specific diagnosis of Fusarium graminearum (Niessen and Vogel, 2010) and F. tricinctum (Niessen et al., 2012) in wheat and barley as well as for the group-specific detection of gushing-inducing Fusarium spp. (Denschlag et al., 2012, 2013) and producers of trichothecene mycotoxins (Denschlag et al., 2014) in cereals and malt. Moreover, is has been applied to the detection and identification of Saccharomyces brewing yeasts and wild yeasts in beer and other sources (Hayashi et al., 2007, 2009).

The method relies on auto-cycling strand displacement DNA synthesis performed by thermophilic DNA polymerases under isothermal conditions with a set of four specifically designed primers. These hybridize to six different parts of the target DNA sequence (Notomi et al., 2000). A comprehensive explanation of the reaction mechanisms involved can be found in the literature (Notomi et al., 2000; Tomita et al., 2008; Niessen, 2015). Fig. 8.2 shows a schematic representation of the different reaction steps leading to DNA synthesis during LAMP. The method basically makes use of the large fragment of the Bst DNA polymerase from Geobacillus stearothermophilus. The large fragment of the enzyme contains the 5' * 3' polymerase activity but lacks 5'-* 3' exonuclease activity. Similar enzymes (Bsm, GspM, GsM 2.0, GspSSP) from other bacterial hosts as well as optimized versions of the original Bst polymerase (Bst 2.0, Bst warmstart) are now commercially available (Chander et al., 2014; Wozniakowski and Samorek-Salamonowicz, 2014; Kang et al., 2014). The Bst DNA polymerase large fragment displaces third-strand DNA with high efficiency during primer-initiated polymerization of new DNA, leaving a double-stranded product and a single-stranded DNA strand, which can act as the matrix for further primer annealing and DNA polymerization.

Since Bst DNA polymerase has a very high activity, vast amounts of high-molecular-weight DNA are produced within a short time. The exceptionally high specificity of LAMP is because a set of four primers with six binding sites must hybridize correctly to their target sequence before DNA biosynthesis occurs. A third pair of primers (loop primers) can be added optionally to the reaction in order to further amplify the amount of DNA produced during LAMP (Nagamine et al., 2002). One of the primer pairs is constructed in such a way that the reverse complement of a binding site downstream of the F2c/B2c binding site (F1c/ B1c) is attached to the 5'-end of a primer binding to that site. These composite primers are essential for the specificity of the amplification reaction and thus have to be chosen very carefully. Both parts of each FIP/BIP primer should be checked for cross-reactivity by in silico analysis (e.g. BLAST) prior to application in LAMP reactions. A pair of outer primers (F3/B3) anneals upstream of the F2c/B2c binding site to displace the initial LAMP product strand from the DNA matrix. Specificity of outer primers can be regarded as being of lower importance since they are not involved in any of the following amplification reactions and a low number of base mismatches will not prevent amplification. The process is initiated by attachment of primers to the DNA target. Primers are elongated and the second matrix strand is displaced from the target DNA. The newly synthesized product itself is displaced from the matrix strand by the F3/B3 product strand. Primers F3 and B3 have no further function once the amplification process has been initiated. As the final product of the amplification initiation step, a dumbbell-structured, singlestranded DNA is formed by hybridization of both

Schematic representation of the LAMP reaction

Figure 8.2 Schematic representation of the LAMP reaction. A, Primers, binding sites, and reaction conditions. B, Initiation of the LAMP reaction resulting in production of a double-loop stem structure (dumbbell structure). C, Autocycling enzymatic DNA amplification during LAMP resulting in multimers of different size of the monomeric double-loop stem structure. Redrawn from Niessen (2015), with kind permission of Springer-Verlag, Heidelberg, Germany.

ends of the molecule to complementary downstream sequences, forming two loops. Starting from this structure, primers FIP and BIP continuously hybridize to newly generated binding sites and are elongated, displaced and refolded while forming ever-longer multimers of the basic dumbbell structure. Loop primers are designed to hybridize to the single-stranded loop structures present in the dumbbell structures as well as in the multimeric DNA formed during autocycling DNA amplification. They prime the production of novel template DNA to which FIP/BIP primers can bind to initiate synthesis of even higher concentrations of DNA. Addition of loop primers therefore does not increase the sensitivity of amplification but rather enables earlier detection of a LAMP signal as compared with a reaction run without loop primers.

Direct detection of amplification in LAMP can be done by addition of DNA intercalating dyes (SYTO 9, SYBR Green 1, ethidium bromide) or fluorescent hybridization probes. Indirect detection is accomplished via Mg-pyrophosphate turbidity or calcein fluorescence (see reviews by Niessen etal., 2013; Niessen, 2015). Quantification of template DNA concentrations is possible but not very accurate due to the autocycling nature of the amplification reaction (Denschlag et al., 2013; Niessen, 2015). Beside its speed and ease of use, robustness is another major advantage of LAMP assays over PCR. It has been demonstrated that the reaction is quite insensitive against inhibitors from the sample matrix (Kaneko et al., 2007; Francois et al., 2011). Simple procedures for sample preparation are therefore sufficient in many cases to obtain a signal after addition of mycelia or fungal spores just washed off cereal or malt grains directly to the LAMP reaction mix (Luo et al., 2012, 2014; Denschlag et al., 2014). Application of the LAMP method for the analysis of brewing cereals and malt was demonstrated by Denschlag et al. (2012, 2013) who designed primers that detected the hyd5 gene coding for the class 2 hydrophobin Hyd5p in Fusar- ium spp., which have been associated with gushing in beer. The assay detected F. cerealis, F. culmorum, and F. graminearum and had a detection limit of three Fusarium-contaminated grains in 200 g. Analysis of gushing-positive and gushing-negative malts revealed good correlation with gushing-test results (modified Carlsberg's test) and showed that the latter test seems to overestimate gushing potential.

The same authors developed another LAMP-based assay detecting tox5 and tox6, two genes involved in the production of trichothecene mycotoxins in Fusarium spp. (Denschlag et al., 2014). The assay was applied to the analysis of wheat. LAMP results corresponded well with the presence of DON in respective samples at threshold values of 163 ppb and 1000 ppb when DNA extraction or a simple lavage of the samples was used for sample preparation, respectively. Niessen et al. (2012) demonstrated the usefulness of LAMP for the detection of F. tricinctum in barley samples and in single barley grains by immersing single seeds into the LAMP master mix prior to the reaction.

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