Endonuclease 4

Alternatively, after capture, the beads were washed five times with 500 µl of Buffer I, once with 500 µl of 1× NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT) and resuspended in a 20 µl reaction system including 20 U Endonuclease IV (New England BioLabs, M0304S) and 1× NEBuffer 3 followed by 2 h incubation at 37 °C.

Each test reaction was prepared using our developed primers and probe combined with either the TwistAmp™ nfo kit (TwistDX, Cambridge, United Kingdom) or the TwistAmp™ exo kit (TwistDX, Cambridge, United Kingdom), with final reaction conditions of 1× rehydration buffer and 1/5 rehydrated lyophilized pellet, forward primer (420 nM), reverse primer (420 nM), probe (120 nM), Ribolock (10 U), and Moloney Murine Leukemia virus reverse transcriptase (mMLV, 40 U) including 1 μL template (from section “2.4.2 Sample processing”) and magnesium acetate (14 mM) to a final reaction volume of 10 μL. Reactions using the TwistAmp™ exo kit also contained Endonuclease IV (2 U; New England Biolabs, Victoria, Australia). Reactions were incubated at 39°C for 20 min before lateral flow detection.

The DNA oligonucleotides were synthesized and purified through HPLC by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The sequences of the miRNA and DNA are given in Table S1. The Endonuclease IV and 10×NEBuffer 3 (1000 mM NaCl, 500 mM Tris-HCl (pH 7.9), 100 mM MgCl₂ and 10 mM DTT) were obtained from New England Biolabs (Ipswich, MA, USA). DSN and 10 × DSN master buffer (500 mM Tris-HCl, 50 mM MgCl₂, 10 mM D, L-dithiothreitol (DTT), pH 8.0) were purchased from Evrogen (Moscow, Russia). The fetal bovine serum (FBS) was gained from Gibco (Carlsbad, CA, USA). The RNase inhibitor, DEPC-treated water, 10 × TBE buffer (225 mM Tris-Boric Acid, 50 mM EDTA, pH 8.0), 10 × phosphate-buffered saline (PBS), 4S green plus nucleic acid stain, and 10 × RNA glycerol gel loading buffer were obtained from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Furthermore, 30% Acrylamide/bis-acrylamide, 29:1 (3.3% crosslinker), Ammonium Persulfate (APS), and N, N, N′, N′-Tetramethylethylenediamine (TEMED) were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Other chemicals were of analytical grade and were obtained from Sinopharm Chemical Reagents (Shanghai, China).

For each condition, a plug slice of 4 × 2 mm was equilibrated with 150 μL of the 1x endonuclease buffer, provided by the manufacturer, at 37°C for 30 min. To detect apurinic/apyrimidinic sites and pyrimidine dimers, DNA was digested respectively with 5 U of endonuclease IV or T4 endonuclease V (New England Biolabs, France), in 150 μL of fresh buffer for 1 h at 37°C.

The CTX-M-1/15 LEC-LAMP assay contained 1× Isothermal Amplification Buffer (New England Biolabs, Hitchin, UK), 6 mM MgSO₄ (Roche Diagnostics), 1.8 mM deoxynucleotide triphosphate set (New England Biolabs), CTX-M-1/15 LEC-LAMP oligonucleotides (1.6 μM FIP, 1.6 μM BIP, 0.2 μM F3, 0.2 μM B3, 0.4 μM LF, 0.4 μM LB–CTX-M-1, 0.4 μM LB–CTX-M-15), IAC LEC-LAMP oligonucleotides (1.6 μM FIP, 1.6 μM BIP, 0.2 μM F3, 0.2 μM B3, 0.4 μM LF, 0.4 μM LB–IAC), 8 U Bst 2.0 WarmStart DNA polymerase (New England Biolabs), 1 U Endonuclease IV (New England Biolabs), 500 copies IAC DNA gBlock Gene Fragment template, 2 μL bacterial DNA template for test reactions or 2 μL molecular grade water for no template control (NTC) reactions, and molecular grade water to give a final reaction volume of 25 μL. LEC-LAMP reactions were performed in duplicate at 63°C for 1 h in a LightCycler 480 instrument II (Roche Diagnostics). Fluorescence measurements were recorded every min using the FAM (495 to 520 nm), HEX (535 to 565 nm) and Cy5 (646 to 662 nm) detection channels producing amplification curves representing positive reactions as signal acquisition exceeding background fluorescence.