Nh4 2so4

To evaluate the LAMP primer set designed in S. haematobium DNA amplification, we set up the reaction mixture using Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) combined with different betaine (Sigma, USA) and MgSO₄ (New England Biolabs, UK) concentrations. Thus, LAMP reactions mixtures (25 μL) contained 1.6 μM of each FIP and BIP primers, 0.2 μM of each F3 and B3 primers, 0.4 μM of each LB and LF primers, 1.4 mM of each dNTP (Bioron), 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Tween20- (New England Biolabs, UK), betaine (ranging 0.8, 1 or 1.2 M), supplementary MgSO₄ (ranging 4, 6 or 8 mM) and 8 U of Bst 2.0 WarmStart DNA polymerase with 2 μL of template DNA. To establish the standard protocol for LAMP reactions mixtures assayed, a range of temperatures (61, 63 and 65°C) was tested in a heating block for 30, 50 or 60 min and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction. Then, both optimal temperature and incubation time were determined and used in the following tests. Positive (S. haematobium DNA) and negative (no DNA template) controls were always included in each LAMP assay.

Oligonucleotides were purchased from Beijing Genomics Institution (Beijing, China), Tsingke Biotechnology Co. Ltd (Beijing,China), and Sangon Biotech. Co. Ltd (Shanghai, China). Lba Cas12a (Cpf1, 20 μM) and rCutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin (BSA), pH 7.9)) were ordered from MAGIGEN (Guangzhou, China) and New England Biolabs (Beijing, China). Large Fragment Bst DNA polymerase, 10× Bst Reaction Bufffer (200 mM Tris-HCl, 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% Triton X-100) and 100 mM MgSO₄ were bought from New England Biolabs (Beijing, China). dATP (100 mM), dTTP (100 mM) and dCTP (100 mM) were bought from Sangon Biotech. Co. Ltd (Shanghai, China). Dithiothreitol (DTT) was ordered from Sangon Biotech. Co. Ltd (Shanghai, China). N,N,N′,N′-Tetramethylethylenediamine (TEMED) and 30% acrylamide/bis solution were provided by Sigma-Aldrich (St. Louis, MO, USA). DNA loading buffer (6×) and Gel Red nucleic acid dye were ordered from TaKaRa Biotech (Dalian, China). All chemical reagents were of analytical grade, and RNase-free water was used throughout this study.

The LAMP primer sets designed were evaluated by using a reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol each of F3 and B3 primers, 1.4 mM each of dNTP (Intron), 1x Isothermal Amplification Buffer-20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Tween20 (New England Biolabs, UK)-betaine (1 M) (Sigma, USA), supplementary MgSO₄ (4 mM) (New England Biolabs, UK), and 8 U of Bst polymerase 2.0 WarmStart (New England Biolabs, UK) with 2 μL (1 ng) of template DNA. LAMP reactions were performed in 0.2 mL tubes that were incubated in a dry bath heat block at 63°C-65°C for 60 min and then heated at 80°C for 5-10 min to stop the reaction.
Schistosome DNA samples mentioned above were used to evaluate the specificity of the LAMP assay; the lower detection limit of the LAMP assay was established by using 10-fold serial dilutions prepared as previously described. Positive controls (DNA from all species tested) and negative controls (ultrapure water instead of DNA) were included in all LAMP reactions.

The outer primer to inner primer ratio was optimised and the concentration of primers were optimal with 2 μM of each forward inner primer (Eh-FIP-SER) and backward inner primer (Eh-BIP-SER), 0.167 μM of each forward outer primer (Eh-F3-SER) and backward outer primer (Eh-B3-SER), and 0.333 μM of backward loop primer (Eh-LB-SER). The concentrations of LAMP components such as dNTPs mix, betaine, MgSO4, and Bst DNA polymerase were optimised and determined empirically. The reaction was carried out with a final volume of 30 μL reaction mixture containing 1 × isothermal amplification buffer [20 mM of Tris-HCl (pH 8.8), 50 mM of KCl, 10 mM of (NH4)₂SO₄, 2 mM of MgSO₄, 0.1% of Tween 20] (New England Biolabs, Massachusetts, USA), 0.6 mM of dNTP mix (Thermo Fisher Scientific, USA), 0.8 M of betaine (Sigma, Missouri, USA), supplementary 6 mM of MgSO₄ (New England Biolabs, Massachusetts, USA), 16 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Massachusetts, USA) and 2 μL of DNA template. The reaction was carried out at 63 °C for 60 min followed by termination at 80 °C for 5 min. The LAMP product was subjected to agarose gel electrophoresis, LFD and calcein-manganese dye for post-LAMP analysis.