Hydroxynaphthol blue hnb

LAMP reactions were performed in a final volume of 25 µL reaction buffer [10 mM Tris–HCl (pH 8.8), 50 mM KCl, 10 mM (NH₄)₂SO₄, 8 mM MgSO₄, and 0.1% Tween 20], 8 U Bst 2.0 DNA polymerase (New England Biolabs, Ipswich, MA, USA), (1.4 mM) of each deoxynucleoside triphosphate (dNTP), 1.6 mM of each FIP and BIP primer, 0.2 mM of each F3 and B3 primer, 0.4 mM of FLP and BLP, and 2 µL of target DNA. The mixture was incubated at 63°C for 60 min, then heated at 80°C for 2 min to terminate the reaction. Reactions were carried out using either a Loop Amp Realtime Turbidimeter (LA-320c, Eiken Chemical Co, Japan) or a 2720 Thermocycler (Applied Biosystems, USA) set at a constant temperature for colorimetric detection. A positive reaction was defined as a threshold value greater than 0.1. Turbidity data were analyzed using the LA-320c software package that reports when the change in turbidity over time (dT/dt) reaches a value of 0.1, which we then assigned to be the threshold time (Tt). For determination of amplification measured by color change (purple to sky blue), 0.15 µL of 120 µM hydroxy naphthol blue (HNB, Sigma-Aldrich Inc, St. Louis, MO, USA) was added to the reaction mixture. All experiments were performed in duplicate at least 3 times.

Fusarium graminearum isolate R9 (Table S2), which is resistant to MBC because of a point mutation at codon 167 (Phe to Tyr, F167Y) in the β₂-tubulin gene (Genbank accession no. FJ214663), and F. graminearum isolate 2021 (Table S2), which is sensitive to MBC, were used in this study. Other F. graminearum isolates were collected from diseased wheat spikelets from different geographical regions in China (Table 2, 3).
Carbendazim was provided by the Shenyang Academy of Chemistry and Industry, China. The fungicide was dissolved in 0.1 M hydrochloric acid (HCl) and adjusted to 1 mg mL⁻¹. Bst DNA polymerase was purchased from NEB. Betaine and hydroxynaphthol blue (HNB) were purchased from Sigma, and MgCl₂ and dNTPs were purchased from Takara. Double-distilled water (ddH₂O) was used in all experiments. All other reagents were analytical grade.

Anti-aflatoxin mAb 1C11 and anti-idiotypic nanobody phage V2–5 specific for mAb 1C11 were produced in our laboratory as previously described. Aflatoxin B₁, B₂, G₁, and G₂ standard solutions, polyethylene glycol 8000 (PEG8000), Tween-20, betaine, bovine serum albumin (BSA), and hydroxy naphthol blue (HNB) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The helper phage M13KO7, E. coli ER2738, and Bst DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). dNTPs were purchased from Takara (Kyoto, Japan). The water was produced by a Milli-Q purification system (Merck KGaA, Darmstadt, Germany). The iLAMP method was validated with an Agilent 1100 HPLC system (Santa Clara, CA, USA).