Optimized LAMP-XO Assay for V. parahaemolyticus

Optimization of LAMP-xylenol orange (LAMP-XO) assay was carried out using 4 sets of previously described primers targeting four different genes: Set 1 for rpoD gene for total V. parahaemolyticus (pathogenic and non-pathogenic strains) detection (Nemoto et al., 2011 (link); Lamalee et al., 2023 (link)) and sets 2–4 for tdh, trh1, and trh2 genes, respectively, for V. parahaemolyticus pathogenic strain detection (Table 1) (Nemoto et al., 2009 (link); Yamazaki et al., 2010 (link)). The optimization for each gene was done separately. For rpoD detection, in addition to the main primers utilized, we designed four additional primers (loop forward and loop backward 2; F1c2 and B1c2, and forward inner and backward inner 2; FIP2 and BIP2) (Table 1) to further improve the reaction kinetics (Jaroenram et al., 2022 (link)). The primers were examined for possible cross dimerization by basic local alignment search tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Briefly, the protocol (Table S1) was done in a 25-µL reaction mixture containing each target-specific primer set (forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer B3), forward loop primer (LF), and backward loop primer (LB)) at different amounts (Table 1), dNTP mix (New England Biolabs, Ipswich, MA, USA), betaine (Sigma-Aldrich, St.Louis, MO, USA) MgSO₄ (New England Biolabs, Ipswich, MA, USA), Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Ipswich, MA, USA), 2.5 µL of 1× low-buffer solution with pH 8.5 (100 mM (NH₄)₂SO₄, 500 mM KCl, 20 mM MgSO₄, and 1% Tween-20), and 1 µL of a gDNA template. The final volume was adjusted to 25 µL using UltraPure™ distilled water (DW) (Invitrogen, Grand Island, Germany). The negative control containing only DW (no gDNA templates) was included in each run. LAMP reaction was done in a Loopamp Realtime Turbidimeter LA-320C (Eiken Chemical Co Ltd, Tokyo, Japan) at a given condition (temperature and time) followed by DNA polymerase inactivation at 80 °C for 5 min. Following this initial protocol, the optimal incubation temperature was first determined, in that the LAMP reactions were carried out at various temperatures (60, 63, and 65 °C) for 75 min. The obtained optimal temperature was then subjected to optimizing six respective parameters: dNTP mix (1.2–1.8 mM), betaine (0.2–0.8 M), MgSO₄ (4–10 mM), Bst 2.0 WarmStart DNA polymerase (6–12 U), reaction time (30, 45, 60, and 75 min), and XO dye, (0.03–0.12 mM) (Table S2). The last parameter was performed in a heat block, and the result was inspected visually. Color of LAMP-XO was changed from purple to yellow in a positive test while color was still purple in a negative test. To confirm LAMP-XO results, LAMP amplicons were analyzed by 3% agarose gel electrophoresis (AGE) (Vivantis, Malaysia), stained with ethidium bromide (Invitrogen, Waltham, MA, USA) and visualized under UV illumination. Each parameter used 1 µL gDNA of 10⁶ copies/µL/reaction as a template except for the incubation temperature and time that used 1 µL aliquot of 10-fold serially diluted DNA (10⁴, 10³, 10², 10 copies/µL) instead. The DNA copy has been calculated using the formular “amount of DNA (ng) × 6.022 × 10²³/ length of a DNA template (bp) × 1  × 10⁹ × 650” (https://www.technologynetworks.com/tn/tools/copynumbercalculator). For each primer set/target gene, any given temperature, time, and components’ concentration that maximize DNA amplification based on signal intensities by the turbidimeter and the degree of color change from purple (negative) to yellow (postitive) was selected to establish the standard LAMP-XO protocol.