Materials and Reagents

Materials

1.5 mL tube (Axygen, catalog number: MCT-150-C)
0.2 mL PCR tube (Axygen, catalog number: PCR-02-C-P)
10 and 100 μL pipettes (Eppendorf, catalog numbers: 3120000224 and 3120000828)
10 and 100 μL micropipettes (Axygen, catalog numbers: TGL-10FT-17-R and TF-100)
Petri dishes (Guangdong Huankai, catalog number: 20221700)
Inoculation loop (Guangdong Huankai, catalog number: 00416571)

Strains

Bacillus cereus ATCC 14579 (ATCC, catalog number: ATCC 14579)
Bacillus thuringiensis ATCC 10792 (ATCC, catalog number: ATCC 10792)
Bacillus subtilis ATCC 6633 (ATCC, catalog number: ATCC 6633)
Listeria monocytogenes ATCC 19115 (ATCC, catalog number: ATCC 19115)

Reagents

Nutrient agar plate (Guangdong Huankai, catalog number: 1024089)
Taq DNA polymerases (Thermo Fisher Scientific, catalog number: 10966018)
Marker D, 100–2,000 bp (Sangon Biotech, catalog number: B600335-0050)
GoldView^TM (Coolaber, catalog number: GD001-1mL*5)
50× TAE buffer (Sango Biotech, catalog number: B548101-0500)

Equipment

Incubator (SHANGHAI MINQUAN INSTRUMENT, model: LAB-0001-0004-SHMQ)
Benchtop centrifuge (Eppendorf, model: 5415D)
Ultrasonic cleaning machine (LANGEE, model: UC-9120)
Thermal cycler (Bio-Rad, model: C1000 Touch)
Electrophoresis imaging system (Bio-Rad, model: GelDoc XR Biorad)
Spectrophotometer (Thermo Fisher, model: NanoDrop^TM One Microvolume UV-Vis)
Autoclave (ZEALWAY, model: GR60DA)

Software

Image Lab (Bio-Rad)

Procedure

Cultivate the strains (listed in materials) conserved in glycerol at 37 °C, at 200 rpm overnight (for approximately 8–12 h).
Dip a small amount of the overnight culture using a 10 μL inoculation loop and streak lightly on the nutrient agar plate. Then, cultivate the plate at 37 °C overnight.
Suspend a single colony into 30 μL of ddH₂O in a 1.5 mL tube.
Note: The size of the colonies of B. cereus ATCC 14579, B. thuringiensis ATCC 10792, B. subtilis ATCC 6633, and L. monocytogenes ATCC 19115 is recommended in Figure 1 and Table 1, and the quality of DNA is listed in Table 2.

Figure 1. B. cereus (A), B. thuringiensis (B), B. subtilis (C), and L. monocytogenes (D) colonies formed on Petri dishes. Scale bars represent 10 mm.

Table 1. Colony size of B. cereus, B. thuringiensis, B. subtilis, and L. monocytogenes
Microorganism Colony size
B. cereus ATCC 14579 2–4 mm
B. thuringiensis ATCC 10792 1–2 mm
B. subtilis ATCC 6633 1–3 mm
L. monocytogenes ATCC 19115 0.2–0.5 mm

Treat the bacterial suspension by sonication at 40 kHz and 120 W at room temperature for 5 min and then centrifuge at 10,000 × g for 1 min at room temperature.
Note: No need to set pauses between the pulses.
Carefully transfer the upper aqueous phase to a new 1.5 mL tube without disturbing the pellet.

The DNA samples are tested using 1 μL of supernatant as DNA template by PCR. Primers for PCR are listed in Table 3. Taq DNA polymerases are added and parameters for PCR are as follows: initial denaturation: 95 °C, 3 min; denaturation: 95 °C, 30 s; annealing: 55 °C, 30 s; extension: 72 °C, 1 min; final extension: 72 °C, 10 min; number of cycles: 25×. After electrophoresis on 1.2% agarose gels (in 1× TAE diluent), all amplicons are visualized under UV light with GoldView^TM staining. The results are shown in Figure 2.

Note: The bands are brighter if you increase the PCR reaction cycles.

Figure 2. B. cereus, B. thuringiensis, B. subtilis, and L. monocytogenes DNA analyzed by electrophoresis. Lane M: 100–2,000 bp DNA ladder (Marker D). B. c: B. cereus; B. t: B. thuringiensis; B. s: B. subtilis; L. m: L. monocytogenes. DNA: DNA extracts by ultrasonic method for 5 min. Positive control: DNA extracted with a commercial kit (1 μL DNA template). CK: Check control (no template).

Table 2. The quality of DNA sample by spectrophotometry analysis

Microorganism	Nucleic Acid (ng/μL)	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀
B. cereus ATCC 14579	20.343	1.517	0.641
B. thuringiensis ATCC 10792	19.43	1.5	0.605
B. subtilis ATCC 6633	21.935	1.303	0.634
L. monocytogenes ATCC 19115	6.321	1.808	1.018

Table 3. Primer sequences used in B. cereus, B. thuringiensis, L. monocytogenes, and B. subtilis, respectively

Microorganism	Target gene	Primer sequence (5′ to 3′)
B. cereus ATCC 14579	gmk	F-TTAAGTGAGGAAGGGTAGG R-AATGTTCACCAACCACAA
B. thuringiensis ATCC 10792	pycA	F-GTGAAAGCAAGAACACAAGC R-ATAGTTTTTGTATCCAACTGCG
B. subtilis ATCC 6633	SigB	F-ATGACACAACCATCAAAAACTACGA R-TTACATTAACTCCATCGAGGGATCT
L. monocytogenes ATCC 19115	prfA	F-GTCAAAACATACGCTCTTATC R-ACATAATCAGTCCAAAGTAGATGC

Acknowledgments

This protocol was adapted from previous work (Chen et al., 2022; Li et al., 2022). This research was supported by Guangdong Major Project of Basic and Applied Basic Research (2020B0301030005), Guangdong Provincial Key Laboratory (2020B121201009), and Guangdong Province Academy of Sciences Special Project for Capacity Building of Innovation Driven Development (2020GDASYL-20200301002).

Competing interests

There are no conflicts of interest or competing interests.

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