Loopamp real time turbidimeter la 320c

LAMP reactions were performed with the Loopamp Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) in a 25-µL reaction containing 1.6 mM of each of the FIP (Forward Inner Primer) and BIP (Backward Inner Primer) primers, 0.8 mM of the LF (Loop Forward) and LB (Loop Backward) primers, 0.2 mM of the F3 and B3 primers, 12.5 µL 2× reaction mix, 1 µL of Bst DNA polymerase (8 U), 1 µL Loopamp Fluorescent Detection Reagent (FD), and 1 µL DNA template. The mixtures were incubated in a Loopamp Real-time Turbidimeter LA-320C (Eiken Chemical Co., Ltd.) at 63 °C for 60 min and then at 80 °C for 5 min to stop the reaction. Mixtures lacking DNA template were used as negative controls. After amplification, the positive LAMP products could be observed directly by the color change of the FD. Moreover, LAMP products were examined by 2.5% agarose gel electrophoresis. To determine the optimum reaction temperature, the LAMP amplification was carried out for 60 min at constant temperatures from 60 to 67 °C in 1 °C intervals, with final incubation for 5 min at 85 °C to terminate the reaction.

To test the usability of two sets of LAMP primers, the LAMP approach either for Shigella strains or Salmonella strains was performed as the following description. In brief, the reaction was conducted with the Loopamp Kit in a final volume of 25 μl containing 1.6 μM each FIP and BIP primers, 0.8 μM each LF and LB primers, 0.4 μM each F3 and B3 primers, 12.5 μl 2 × reaction mix, 1 μl of Bst DNA polymerase (8 U), 1 μl FD, and 1 μl DNA template.
The reaction mixtures of normal LAMP were heated for 1 h at 63°C and then at 85°C for 5 min to stop the reaction. Three mainstream techniques were application to monitor the normal LAMP amplification. The positive amplifications could be directly observed color change by FD reagent, and the products were also detected by electrophoresis on 2% agarose gels with ethidium bromide staining. Moreover, real-time monitoring of normal LAMP reactions was conducted by recording the optical density (OD) at 650 nm every 6 s using the Loopamp Real-time Turbidimeter LA-320C (Eiken Chemical Co., Ltd, Japan). A positive result was defined as a threshold value of >0.1 within 60 min and analysis of each sample (dilution) was determined at least two times.

Amplification reactions mixtures of LAMP were performed with the Loopamp Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) in a final volume of 25 µl containing 1.6 mM FIP and BIP primers (each), 0.8 mM LF and LB primers (each), 0.2 mM F3 and B3 primers (each), 12.5 µl 2× reaction mix, 1 µl of Bst DNA polymerase (8 U), 1 µl Loopamp Fluorescent Detection Reagent (FD) and 1 µl DNA template. The mixture was incubated at 64°C for 1 h and then at 80°C for 5 min to stop the reaction. Mixture without DNA template was used as a negative control.
A total of 3 methods were used to confirm LAMP DNA amplification. The color change of positive amplification can be directly observed by FD and the positive products were detected by 2% agarose gels electrophoresis with ethidium bromide staining. Moreover, the LAMP products can be monitored by measuring the increased turbidity in real-time using Loopamp Real-time Turbidimeter LA-320C (Eiken Chemical Co., Ltd, Japan).

The LAMP reactions were carried out in 25 μl reaction mixture (DNA Amplification Kit; EIKEN CHEMICAL CO., LTD, Tochigi, Japan) containing the following reagents (final concentration):20 mM Tris-HCl (pH = 8.8), 10 mM KCl , 10 mM (NH4)₂SO4, 0.1 % Tween20, 0.8 M betaine, 8 mM MgSO4, 1.4 mM dNTP each and 8U Bst DNA polymerase. The amount of primer needed for one reaction was 40 pmol for FIP and BIP, and 5 pmol for F3 and B3. Finally, an appropriate amount of template genomic DNA was added to the reaction tube. The reaction was carried out and monitored at 63 °C in a Loopamp real-time turbidimeter(LA320-C, Eiken Chemical Co., Ltd., Tokyo, Japan).

The TgsGP primers as previously described were used [14 (link)]. The reaction mixture of 25 μL consisted of 40 pmol of the inner primers, 5 pmol of the outer primers, 20 pmol of the loop primers (Inqaba biotec, SA), 0.8M betaine (Sigma-Aldrich, St. Louis, MO, USA), 2.8 mM dNTPs mix, 1x Thermopol buffer (20 mM Tris-HCl pH 8.8, 10 mM KCl, 2 mM MgSO₄, 10 mM (NH₄)₂SO₄, 0.1% Triton X-100) (New England Biolabs, UK), and additional 4 mM MgSO₄, 8-unit Bst DNA polymerase (New England Biolabs, UK), double distilled water, and 2 μL of the template DNA. The positive and negative controls were similar to those used in the PCR reaction. The reactions were carried out in triplicate for 80 minutes in a Loopamp real-time turbidimeter LA320C (Eiken Chemical Co., Japan). Increase in turbidity indicates DNA amplification. After the reaction 1/20 dilution of SYBR green I dye (Sigma-Aldrich, St. Louis, MO, USA) was added to confirm the amplification.

The LAMP products were detected using two methods. First, the direct visual inspection was based on the change of the fluorescence of the LAMP reaction containing 1 µL of calcein [75 ,76 (link)]. A positive reaction was indicated by the color change from orange to green in the reaction, while a negative reaction was indicated by the failure of color change from orange to green. The advantage of the fluorescence detection was that the color change of the LAMP reactions was observed directly either by the naked eye under natural light or the UV light. Second, in the detection of turbidity method [22 (link)], the amplification products of the LAMP assay were examined using spectrophotometry. Specifically, the value of the optical density at 650 nm (OD₆₅₀) of the magnesium pyrophosphate (Mg₂P₂O₇) produced in the LAMP reactions was measured every 6 s using a Loopamp real-time turbidimeter (LA-320C, Eiken Chemical Co., Ltd., Tokyo, Japan) and was plotted against reaction time to generate the curve to determine the positive or negative reactions.

The main reagents used in this study include the reaction buffer and Bst enzyme (Isothermal Amplification Kit, Haitaizhengyuan, Beijing), the Biotin-11-dUTP (Thermo Scientific, Shanghai), the colorimetric indicators (Haitaizhengyuan, Beijing), the nanoparticle LFB, the LFB running buffer, the Nano drop ND-1000 (Calibre, Beijing, China) and a Loopamp Realtime Turbidimeter LA-320C (Eiken Chemical Co., Ltd., Japan.
The LFB was constructed according to the instructions by Wang et al.’s report^{14 (link)}. Briefly, the streptavidin-coated nanoparticles were adhered onto the conjugate pad. On the nitrocellulose membrane pad, there were three lines, including two test lines (conjugated with rabbit anti-fluorescein antibody and sheep anti-digoxigenin antibody, respectively) and one control line (conjugated with biotinylated bovine serum albumin). Finally, the assembled sample pad, conjugate pad, nitrocellulose membrane and the absorbent pad were cut (4 mm in width) and packaged in the plastic shells. The packaged biosensors were stored in dry environment at room temperature. The running buffer was the phosphate buffered saline (PBS) with the pH of 7.4.