Xylenol orange

This activity was determined according to a previously described method [13 (link)] with minor changes. An aliquot of 50 mM H₂O₂ and various concentrations (0–2 mg/ml) of samples were mixed (1:1 v/v) and incubated for 30 min at room temperature. After incubation, 90 μl of the H₂O₂-sample solution was mixed with 10 μl HPLC-grade methanol and 0.9 ml FOX reagent was added (prepared in advance by mixing 9 volumes of 4.4 mM BHT in HPLC-grade methanol with 1 volume of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfate in 0.25 M H₂SO₄). The reaction mixture was then vortexed and incubated at room temperature for 30 min. The absorbance of the ferric-xylenol orange complex was measured at 560 nm. All tests were carried out six times and sodium pyruvate was used as the reference compound [14 (link)].

The assay is conducted at 30 °C by adding Fe²⁺ to a mixture of protein/enzyme (0.8 uM), Tris-HCl buffer (50 mM, pH 7.0), and (NH₄)₂Fe(SO₄)₂ (40 µM). A 10-µl aliquot is taken at 5 min after the assay reaction started and mixed with a 100-µL solution of Xylenol Orange (XO) (125 µM) and H₂SO₄ (25 mM). After incubation at room temperature for 5 min, the concentration of ferric ions is determined by measuring the absorbance at 595 nm using a TECAN microplate reader (Infinite M200 Pro, TECAN, Zürich, Switzerland) with i-control v1.7 software. The auto-oxidation of the ferrous ions is measured under the same assay conditions but without adding protein/enzyme.

The FOX assay was carried out with some modifications of the previously described method (Gay and Gebicki 2002 (link); Pinto et al. 2007 (link)). The xylenol orange reagent containing 2.0 mM ferrous sulfate, 0.29 mM xylenol orange tetrasodium salt, and 440 mM perchloric acid in methanol/water (9:1) was freshly prepared. To find the optimum absorbance at which the concentration of the ferric-xylenol orange (Fe³⁺–XO) complex can be determined, 30 µL of cumene hydroperoxide (0–10.52 mM dissolved in water) and 150 µL xylenol orange reagent were mixed well. The mixtures were then incubated for 15 min at room temperature (~ 20 °C) and the absorption spectra of the mixtures were measured in the wavelength range of 400–650 nm using a Spectramax ID3 multi-detection microplate reader (Molecular Devices, LLC, San Jose, California, USA). When the absorbance of the samples exceeded 2.0, the samples were diluted in 75% methanol in water. The actual absorbance of the sample was then calculated, taking the dilution factor into account.

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.