Eriochrome black T, Indicator

Eriochrome black T, Indicator is a complexing agent for metal ions (e.g., Ca²⁺, Mg²⁺) and is used as an indicator in complexometric titrations. Eriochrome black T, Indicator forms colored complexes with metal ions through covalent coordination bonds, and indicates the endpoint of the titration by color change. Eriochrome black T, Indicator can be used as an anionic azo dye in photocatalytic degradation studies to evaluate the performance of photocatalysts. The reaction solution of Eriochrome black T, Indicator combined with Mg²⁺ is initially purple. During loop-mediated isothermal amplification (LAMP), the color changes from purple to sky blue due to the consumption of Mg²⁺ by the formation of magnesium pyrophosphate, indicating a positive reaction. The optimal concentration of Eriochrome black T, Indicator in LAMP is 60 μM, and the detection limit for Mycobacterium tuberculosis is 1 pg DNA/reaction^[1][2]. In Vitro:Eriochrome black T, Indicator can be used to detect Mycobacterium tuberculosis by loop-mediated isothermal amplification (LAMP) reaction. It indirectly indicates the presence of bacterial DNA through the consumption of magnesium ions in the LAMP reaction. Eriochrome black T, Indicator can bind to Mg²⁺ to form a purple complex. In the initial stage of the LAMP reaction, the concentration of Mg²⁺ in the solution is sufficient; and Eriochrome black T, Indicator appears purple. When the byproduct magnesium pyrophosphate (Mg₂P₂O₇) precipitates, Eriochrome black T, Indicator turns sky blue^[2].

1.?DNA extraction
(1) Biological specimen processing:
Sputum or bronchoalveolar lavage fluid was treated by the Petroff method and DNA was extracted by boiling.
The biological specimen was washed twice with TE buffer, the precipitate was boiled for 5-10 minutes, and the supernatant was taken as template DNA after centrifugation.
(2) Preparation of purified DNA:
Genomic DNA was extracted from Mycobacterium tuberculosis H37Rv strain for reaction optimization and detection limit determination: the bacterial liquid was suspended in TE buffer, inactivated at 80°C for 1 hour, lysed with lysozyme and proteinase K at 37°C, and purified DNA was obtained by chloroform-isoamyl alcohol extraction and isopropanol precipitation.

2. LAMP reaction system (taking 25 μL system as an example)
Reaction components:
Inner primers (FIP, BIP) 1.6 μM each, outer primers (F3, B3) 0.2 μM each, loop primers (FLP, BLP) 0.8 μM each;
0.8 M betaine, 1X ThermoPol buffer, 8 U Bst DNA polymerase, 1 ng template DNA;
3.5 mM MgSO₄, 1.4 mM dNTPs, 60 μM EBT indicator.
Incubation conditions: 64°C constant temperature incubation for 30 minutes, no denaturation or annealing steps required.

3. Result observation
Positive judgment: The color of the reaction solution changes from purple to sky blue, indicating the presence of target DNA.
Negative control: The reaction solution without template DNA should remain purple.