Loop-mediated isothermal amplification in the field: reality beyond the potentiality

* Correspondence: donatoantonio.raele@izspb.it To the Editor, Loop-mediated isothermal amplification (LAMP) is a very promising technique for DNA amplification, first described by Notomi et al. [1] and summarized by Raele et al. [2]. It mainly relies on the action of a DNA polymerase that, beyond the common 5’-3’ DNA synthesis, has strand displacement activity. The final product of LAMP is a series of concatemers, whose repeating units are the replicates of the selected DNA target. The specificity is granted by the six different sites to be recognized on the template to prime the reaction, six different sites have to be recognized by the primers on the template [3], and, from a more practical perspective, LAMP has benefits of simplicity and rapidity, as the reaction may be carried out at a constant temperature, without need of thermal cycles (and thermal cycler as well), and it takes about 40–50 min. Because of those features, LAMP has been suggested by many authors as suitable for use in the field and on the spot. Among them, Centeno-Cuadros et al. set up a LAMP protocol [4] suitable for the sex determination of three raptor species in the field, and more recently Lee [5] pointed out the suitability of the LAMP approach for a wider range of in-field applications, including the detection of pathogens and parasites in the environment for monitoring purposes. Despite its potential, the actual application of LAMP in the field has been hampered by some technical challenges. Among them, there is the need to expose the reaction to UV rays to detect positivity after addition of fluorescent intercalating dyes. Another problem is the unavailability of handily portable thermo-blocks. Only recently, new products have been devised to make it possible to carry out LAMP outside of laboratories. In particular, portable instruments for the real-time detection of fluorescence have been introduced, and this may overcome the difficulties linked to the fluorescence detection by UV lamp, which needs a dark environment, often not possible to be achieved in the field. To verify the actual feasibility of such a system in the field, we undertook a 1-day campaign aimed to apply LAMP to detect the presence of Borrelia burgdorferi s.l. , or Lyme borrelia (LB), in ixodid ticks immediately after collection in the National Park of Gargano (Apulia, Italy). Such a group of pathogens was chosen because of its clinical, epidemiological, and bacteriological relevance, and for being typical vector-borne pathogens, mostly transmitted to humans through the bite of the sheep tick Ixodes ricinus [6]. The location (the National Park of Gargano) was selected because of previous laboratory detection of B. afzelii from some pools of I. ricinus collected there (unpublished data). The campaign was carried out by two investigators on a sunny day of June 2017, in order to find better conditions for the collection of ticks. One of the investigators carried a light portable fridge, the other a small outdoor folding table; they both were also equipped with a backpack. Backpacks contained: gloves, a 1-m2 fabric made of white cotton flannel (for tick collection, TC), a 1.5-m wooden dowel (for TC), sterile forceps (for TC), sterile petri dishes (for TC) sterile plastic tubes filled with 70% ethanol (for tick storage), a portable stereomicroscope (for tick identification), a pipette set (P10, P200, P1000) with respective sterile filter tips, sterile 1.5-mL microcentrifuge tubes (for DNA extraction, DE), sterile pestles and mortars (for DE), Plant Material DNA extraction kit (Optigene, Horsham, UK) (for DE), a portable Genie III instrument (Optigene) (for LAMP reaction, LR), Received: 07.08. 2018 Accepted/Published Online: 25.01. 2019 Final Version: 12.02. 2019 Letter to the Editor