2.4. Optical analysis and isothermal amplification
For the absorbance analysis of crystal violet, 40 µL of the finally
diluted crystal violet solutions were collected from the product
chambers and mixed with 120 µL of DW, which was then analyzed by
measuring the absorbance at 588 nm with a microplate reader (Infinite
M200 Pro, Tecan Group Ltd., Maennedorf, Switzerland).
For the real-time isothermal amplification, 50 µL of serially diluted
each bacterial sample was thermally lysed at 100 °C for 10 min using a
heat block (MS-100, Hangzhou Allsheng Instruments Co., Ltd., Hangzhou,
China). Ten µL of the lysed bacterial sample was then mixed with 10 µL
of isothermal target and probe amplification (iTPA) [24, 25] reagent
solution to prepare the final reaction solution (20 µL) containing inner
primers (1 µM each), outer primers (0.1 µM each), dNTPs (1.6 mM each),
10 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 10 mM
MgSO4, 0.8 mM spermine, 6 mM dithiothreitol, 5 ng/µL
bovine serum albumin, Bst polymerase large fragment (0.15 U/µL),
RNase inhibitor (0.3 U/µL), thermostable RNase H (0.3 U/µL), and a 50 nM
CPT probe. The reaction solution was subjected to the iTPA reaction at
65°C for 90 min using a CFX Connect™ Real-Time System (Bio-Rad
Laboratories, Inc., Richmond, CA, USA). A detail description of the iTPA
reaction is provided in Figure S3, or our previous reports [24, 25].