Mgso4

Target amplification, enzymatic digestion, pyrophosphorolysis and ligation, and rolling circle amplification were carried out essentially as described in Silva et al. with modifications described below:
The PCR mastermix from [1 (link)] was modified to perform RT-PCR in 6 µL of mixture per reaction by the addition of 1x Luna WarmStart reverse transcriptase (E3006, NEB) and the reaction performed in 1x Q5U buffer (M0515, NEB) with 2 mM MgSO₄ (NEB), and 6 µL of RNA target. The thermocycling conditions used were 37 ˚C 1 min; 55 ˚C 10 min; 98 ˚C 1 min; 50 cycles of 98 ˚C 10 s, 63 ˚C 15 s, 72 ˚C 15 s; and 72 ˚C 5 min. Proteinase K digestion and pyrophosphorolysis were performed essentially as described previously [1 (link)]. For the RCA, 10 µL of detection mixture per reaction was prepared by mixing 1x RCA buffer (51.8 mM Tris-HCl pH 8.8, 27.6 mM NH₄SO₄, 3.72 mM KCl, 3.49 mM MgSO₄, 0.0567% Tween-20), 300 U/mL Bst 3.0 WarmStart (M0374, NEB), 0.8 mM dNTPs with dUTP (Promega), 0.3% polydimethylsiloxane emulsifier (A5757, Sigma Aldrich), and 0.253 µM primer mix. 5 µL PPL reaction sample was added to 10 µL of detection mix in a 384-well plate. Samples were incubated at 57 °C for 200 min in a QuantStudio5 RealTime PCR System (ThermoFisher). The fluorescence read-out was taken every minute in the FAM, JUN, VIC and Cy5 channels.

The LAMP master mix formulation included FIP and BIP primers at concentrations of 1.6 μM each, F3 and B3 primers at 0.2 μM each, and both LF and LB primers at 0.4 μM each. The list of primers used to amplify the λ phage DNA target is provided in Supplementary Materials Additionally, the LAMP mix comprised of Bst DNA polymerase (New England Biolabs, Ipswich, MA, USA) at 8000 U/mL, 10× isothermal amplification buffer containing 2 mM of MgSO₄ (New England Biolabs), an added 6 mM of MgSO₄, dNTPs at 1.4 mM each, and the SYTO 9 dye at 50 μM. 25 µL of this formulated mix was utilized as the dispersed phase for the droplet microfluidic operations. For assays requiring positive controls, the master mix was supplemented with 1 μL of lambda DNA at specified concentrations. Conversely, the negative control was established by adding 1 μL of distilled water to the master mix.

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.

All primers and plasmids were synthesized by Sangon Biotech (Shanghai, China); (NH4)₂SO₄, KCl, KOH, Tween 20, and neutral red were purchased from Sigma-Aldrich (Shanghai, China). Deoxynucleotide triphosphates (dNTPs) and MgSO₄ were obtained from New England Biolabs Ltd. (Beijing, China). Glycerol-Free Bst polymerase (MDX018-08A) was purchased from Meridian Biotech Ltd. (Shenzhen, China); the DNA extraction kit (D7113) was purchased from Magen Biotech Ltd. (Guangzhou, China). DMF chips were provided by Digifluidic Ltd. (Zhuhai, China).

ALV-J (HN06), ALV-A (GD-13), ALV-B (CD08), ALV-K (GDFX0601), ALV-E (HN1301) and Salmonella enteritis (SE) were maintained in our laboratory. Escherichia coli ATCC15922 and Pseudomonas aeruginosa ATCC27853 were obtained from HuanKai Microbial (Guangzhou, China). Mycoplasma gallisepticum (MG) S6 was donated by Professor Ding (College of Veterinary Medicine, South China Agricultural University). Bst DNA polymerase, 10 × ThermoPol Reaction Buffer, dNTPs and MgSO₄ were products of New England Biolabs (Beverley, MA, USA). Betaine was acquired from Sigma (St. Louis, MO, USA). Disposable Nucleic Acid Detection Strip were purchased from Ustar (Hangzhou, China). The DNA Extraction Kit, Gel Extraction Kit, and Plasmid Mini Kit were produced from OMEGA (Norcross, GA, USA).

The LAMP reaction was carried as described elsewhere with some modifications [17 (link)]. In brief, a 20 μL reaction mixture was prepared that contained 1 μL of target genomic DNA along with 1.6 μM each of the primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM of LF and LB. 4U of the Bst 2.0 DNA polymerase (New England Biolabs, Ipswich, MA, USA), 8 mM of MgSO₄ (New England Biolabs), 1.5 mM of dNTPs (Thermo Fisher Scientific), 1× isothermal amplification buffer (New England Biolabs), and 1.25 M of N-methyl formamide (NMF) and isobutylamide (IBA). Furthermore, 2 μM of SYTO 9 (Thermo Fisher Scientific) was added to enhance fluorescence in the presence of DNA in the real-time assay. The amplification reaction was performed at 60℃ for 45 min and terminated by heating at 80℃ for 3 min using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The results were shown as a graph on the monitor of real-time analysis software (BioRad CFX Manager, Bio-Rad Laboratories). The fluorescence curve was captured from the BioRad CFX Manager graph.