Simplified closed tube loop mediated isothermal amplification (LAMP) assay for visual diagnosis of Leishmania infection
Introduction
Leishmaniasis is a vector-borne disease caused by flagellate protozoa of the genus Leishmania affecting millions of people worldwide and remains a global health problem. Infection with Leishmania could cause atypical type of manifestations depending on infected species ranging from self-healing – cutaneous leishmaniasis (CL) to potentially fatal outcome – visceral leishmaniasis (VL) (Adams et al., 2018). Since the emergence of CL and VL caused by L. martiniquensis and L. siamensis has been reported in northern and southern Thailand, the number of infected cases has increased continuously among patients with HIV/AIDS (Leelayoova et al., 2017; Manomat et al., 2017). Notably, underlying HIV influences the transmission of leishmaniasis, probably due to a decrease in host cell immunity.
Currently, polymerase chain reaction (PCR) is considered the primary diagnostic test, especially in cases of HIV co-infection because the technique is comparatively harmless with trustworthy sensitivity and specificity (Georgiadou et al., 2015; Manomat et al., 2017). However, PCR requires costly instruments requiring 5 to 24 hours to detect specific amplicons making it inappropriate for fieldwork (Rajapaksha et al., 2019). In contrast, loop mediated isothermal amplification (LAMP) assay uses a simple approach based on single temperature amplification and a set of four to six primers that are used to amplify a mixture of various lengths of stem-loop DNA under isothermal conditions (60 to 65°C) by strand displacement activity of Bst polymerase (Fallahi et al., 2020; Ghodsian et al., 2019; Notomi et al., 2015). Therefore, LAMP can synthesize large amounts of DNA in a short period in terms of simplicity, sensitivity and specificity. The technique overcomes the need of an expensive thermal cycler; furthermore, the technique is robust, reliable, cost effective and field-friendly (Yano et al., 2007).
To visualize and measure LAMP products, several assays including turbidity, fluorescence and color have been developed (Gadkar et al., 2018; Sriworarat et al., 2015). Malachite green (MG) as a colorimetric assay was developed and validated to detect Leishmania spp. in pre-reaction preparations (Nzelu et al., 2016; Nzelu et al., 2019; Sriworarat et al., 2015; Tiwananthagorn et al., 2017). However, ambiguous reading interpretations sometimes occurred between a very pale blue and transparency for the MG-LAMP assay (Lucchi et al., 2016; Thita et al., 2019), whereas, SYBR green I, a fluorescent dye requiring post-amplification preparation, has been widely used for LAMP assay due to clear interpretation under both ultraviolet (UV) and visible lights (Fischbach et al., 2015; Karthik et al., 2014; Nzelu et al., 2019; Singpanomchai et al., 2018; Tao et al., 2011). Alternatively, fluorescent detection reagent (FDR) is a good choice of fluorescent dye because the dye can be used in a pre-reaction to avoid the post-amplification preparation step, a common contamination prone step in the LAMP assay (Adams et al., 2018; Adams et al., 2010; Nzelu et al., 2019). Recently, SYBRTM Safe, a low cost nonmutagenicity fluorescent dye, was shown to eliminate ambiguous evaluation due to clear reaction in detecting DNA of Leishmania spp. without the need of post-amplification preparation. However, the technique additionally required a UV or blue light transilluminator to detect LAMP amplicons (Thita et al., 2019). Thus, SYBR green I-LAMP seems to be a good choice of colorimetric and fluorescent assay in the field environment, especially where a transilluminator is unavailable.
Several studies developed pre-reaction preparations of SYBR green I based on two main techniques. The first involves SYBR green I pre-spotting inward to the tube lid or on the folded tinfoil where in a brief spin down after inactivation allowed amplicons staining (Dixit et al., 2018; Hong et al., 2012; Zhou et al., 2014). Second, dye capsules such as microcrystalline wax or agar encapsulating SYBR green I are used once the reaction was terminated, and the melting dye capsules released the dye to stain the DNA products (Karthik et al., 2014; Tao et al., 2011). Thus, unambiguous interpretation of LAMP products can be clearly observed by the naked eye, directly, under visible light where a positive result turns green and negative remains orange (Dixit et al., 2018; Dragan et al., 2012; Fallahi et al., 2015; Fallahi et al., 2014; Hong et al., 2012; Karthik et al., 2014; Tao et al., 2011; Zhou et al., 2014). Otherwise, dye capsules need the dye encapsulating step that is inconvenient to prepare during fieldwork, whereas pre-spotting dye might be unsuitable for a large population study when is associated with mass preparation of reaction mixtures. The non-protective dye could accidentally spill down into and inhibit the reaction mixture during amplification resulting in false negative interpretation.
In the present study, we partially secured SYBR green I using parafilm that formed a semi-layer blocking between the dye and LAMP reaction mixture to ensure and improve simplicity of the pre-reaction preparation. This simplified technique needed no post-amplification preparation with high sensitivity and specificity as well as prevented accidental spilling of the dye during LAMP amplification. Thus, the dye is comparatively cheaper than other LAMP commercial dyes and easily accessible making this approach both affordable and suitable to detect leishmaniasis DNA for fieldwork as well as all levels of hospitals including health services, particularly in low income countries.
Section snippets
Ethics statement
All participants who were >18 years old were informed and enrolled in the study. Written informed consent was obtained from all participants before sample collection and anonymous analysis. This study was approved by the Ethics Committee of the Royal Thai Army Medical Department (IRBRTA 952/2562).
DNA preparation for clinical samples and Leishmania parasites
Eight milliliters of EDTA anti-coagulated blood samples were collected from eligible participants >18 years old visiting the HIV Clinic at Satun Hospital, Satun Province every 6 months for follow-up
Results
The use of semi-layer parafilm to partially secure SYBR green I from spilling in closed tube LAMP assay successfully amplified the 18s rRNA gene of Leishmania DNA that, under visible light, the presence of LAMP amplicons was indicated by green color while the remaining orange color represented the absence of amplification after the dye spinning down (Fig. 1C). In addition, glowing fluorescence was associated with the presence of LAMP products, whereas failure of amplification was represented by
Discussion
In this present study, parafilm was introduced and additionally applied to a conventional closed tube LAMP assay that relied on a nonprotective prespotting dye at the inward lid (Dixit et al., 2018; Zhou et al., 2014) to prevent spilling of the prespotted SYBR green I. The semi-layer modification of the parafilm showed no obstruction of reaction mixture staining after spinning down of the tube. Furthermore, a clear interpretation was observed, similar to that of the conventional closed tube
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Worarachanee Imjaijitt, Office of Research and Development, Phramongkutklao College of Medicine & Phramongkutklao Hospital (ORD, PCM & PMK), for her assistance and useful comments on statistical analysis. We also thank Asst. Prof. Tawin Inpankaew for providing Trypanosoma evansi DNA. This work was partially supported by Phramongkutklao College of Medicine and the Thailand Research Fund (grant no. MRG6180152). Toon Ruang-areerate received supplementary financial support from the
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