Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

Published: February 29, 2024

Abstract

Source: Tram, K., et al. Colorimetric Detection of Bacteria Using Litmus Test. J. Vis. Exp. (2016).

This video demonstrates a colorimetric E. coli detection method, involving an E. coli biomarker activating a DNAzyme, leading to cleavage of RNA linkage and detachment of a DNAzyme-urease conjugate. A subsequent colorimetric assay detects the E. coli biomarker through ammonia-induced pH changes.

Protocol

1. Preparing E. coli Cells for Testing

  1. For a desired cell suspension, transfer 1 ml of cultured stock to a 1.5 ml microfuge tube.
  2. Centrifuge the cells at 6,000 x g for 10 min at 4 °C. Carefully remove the supernatant without disturbing the cell pellet.
  3. Add 10 µl of reaction buffer to the cell pellet and resuspend the cells. Sonicate the cell suspension for 5 min. Transfer the cell suspension to an ice box for 5 min.
  4. Sonicate the cell suspension for another 5 min.
  5. Centrifuge the cell suspension at 13,000 x g for 10 min at 4 °C. Use the supernatant for testing (10 µl).

2. Litmus Test

  1. In a 1.5 ml microfuge tube, prewash the tube by adding and vortexing 100 µl of reaction buffer (RB) in the microfuge tube and discarding the buffer.
  2. Transfer 15 µl of assembled EC1 to the washed microfuge tube.
  3. Wash the magnetic beads by placing the microfuge tube on a magnetic rack. Remove the supernatant by pipetting. Remove the microfuge tube from the rack, add 100 µl of RB, and carefully resuspend the magnetic beads.
  4. Wash the MB two more times by repeating step 2.3.
  5. Place the microfuge tube back on the magnetic rack, remove the supernatant, and add the 10 µl E. coli sample prepared from step 1.3.
  6. Mix the sample and magnetic beads carefully by gently tapping on the microfuge tube.
  7. Incubate the reaction at room temperature for 1 hr.
  8. To the reaction, add 90 µl of ddH2O and place the microfuge tube onto a magnetic rack.
  9. After approximately 3 min of magnetic separation, carefully transfer 85 µl of the supernatant to a 0.5 ml microfuge tube. Withdraw the supernatant slowly to avoid collecting any magnetic beads.
  10. To the above microfuge tube add 15 µl of 0.04% phenol red and 100 µl of substrate solution.
  11. Take a photograph at specific time intervals to record color change.
    NOTE: The change in pH can also be monitored using a pH meter with a microelectrode. The starting pH should be approximately 5.2-5.5 (the solution is yellow). If not, the solution can be adjusted by the addition of 1 mM Acetate Buffer pH 5.0).

Declarações

The authors have nothing to disclose.

Materials

Ethylenediaminetetraacetic acid (EDTA) VWR AMRESCO 105
Sodium Hydroxide (NaOH) pellets BIO BASIC CANADA INC SB6789
Tris-base VWR AMRESCO 497
Urea VWR AMRESCO M123
Xylenecyanol FF SIGMA-ALDRICH X-4126
0.04% Phenol red SIGMA-ALDRICH P3532
10x T4 polynucleotide kinase reaction buffer Lucigen 30061-1
E. coli K12 (MG1655) ATCC ATCC700926
Magnetic Bead (BioMag) Bangs Laboratories Inc
Magnetic Seperation Rack New England BioLabs S1506S
Microfuge tubes Sarstedt 72.69
Cell culture incubator Eppendorf Scientific M13520000
Branson Ultrasonic cleaner Branson N/A

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Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test. J. Vis. Exp. (Pending Publication), e22001, doi: (2024).

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