2.1. Reagents and materials
Table 1 presents all of the strains and their respective sources. The E. coli O157:H7 strains were purchased from Beijing Beinachuang Biotechnology Research Institute (China). Escherichia coli (ATCC8739 and ATCC8099), Staphylococcus mimicus, and Staphylococcus xylosus strains were purchased from Baosai Biotechnology (China). Escherichia coli (ATCC25922), Vibrio parahaemolyticus (ATCC17802), and Staphylococcus aureus were purchased from the Guangdong Microbial Culture Collection Center (China). Vibrio parahaemolyticus (ATCC33847), Vibrio vulnificus, and Vibrio alginolyticus were purchased from the Beijing Beinachuanglian Biotechnology Research Institute. Listeria monocytogenes was purchased from the China Centre for the Management of Industrial Microbial Strain Preservation. The natural samples were purchased from Huacao Market in Shanghai. The primers, CrRNA, and reporter probes (ssDNA) were synthesized by Sangon (Shanghai, China). The water DNA extraction kit model DZ310 (FINDROP, Guangzhou, China) was used to extract DNA from strains. TwistAmp® basic kits, RNase inhibitor, and LbCpf1 nuclease were purchased from Morey Biosciences (Shanghai, China), BBI (Shanghai, China), and Tolo Biotech (Shanghai, China), respectively. Additionally, NanoDrop 2000c (Thermo Scientific, Shanghai, China) was used in the analysis.
Table 1
Strains used in this experiment, including their types and sources
No. | Bacterial species | Strains | Source |
1 | Escherichia coli O157:H7 | ATCC43888 | a |
2 | Escherichia coli O157:H7 | ATCC43895 | a |
3 | Escherichia coli | ATCC8099 | b |
4 | Escherichia coli | ATCC25922 | c |
5 | Escherichia coli | ATCC8739 | b |
6 | Vibrio parahaemolyticus | ATCC17802 | c |
7 | Vibrio parahaemolyticus | ATCC33847 | d |
8 | Vibrio vulnificus | ATCC27562 | d |
9 | Vibrio alginolyticus | ATCC33787 | d |
10 | Listeria monocytogenes | CICC21633 | e |
11 | Listeria monocytogenes | CICC21540 | e |
12 | Staphylococcus aureus | ATCC 6538 | c |
13 | Staphylococcus aureus | ATCC 43300 | c |
14 | Staphylococcus mimicus | ATCC27851 | b |
15 | Staphylococcus xylosus | ATCC29971 | b |
*a, Beijing Beinachuang Biotechnology Research Institute; b, Baosai Biotechnology; c, Guangdong Microbial Culture Collection Center; d, Beijing Beinachuanglian Biotechnology Research Institute; and e, China Centre for the Management of Industrial Microbial Strain Preservation.
2.2 Culture of bacterial and genome extraction
The E. coli O157:H7 strains were cultivated in a Luria–Bertani medium at 37°C for 12 h (1). After the colony-forming units were counted via the plate colony counting method, the bacteria solutions were diluted in a gradient to 2.4 × 106, 2.4 × 105, 2.4 × 104, 2.4 × 103, 2.4 × 102, 2.4 × 101, and 2.4 × 100CFU/mL. Then, 1 mL of the 2.4 × 106 CFU/mL pure culture suspension was collected for genomic DNA extraction, and NanoDrop was used to determine the concentration of genomic DNA. After testing, the concentration of genome DNA was 1.8 × 106 fg/µL, and the extracted genome was diluted to various concentrations from 1.8 × 106 to 1.8 × 100 fg/µL. The strains and DNA genomes were stored at − 20°C for subsequent experiments.
2.3 Design of primers, ssDNA reporters, and CrRNAs
The whole genome sequence of E. coli O157:H7 was retrieved from the National Center for Biotechnology Information to mine specific targets of E. coli O157:H7. Through local Basic Local Alignment Search Tool for Nucleotide sequences comparison, a specific gene (the rfbE gene) to detect E. coli O157:H7 was obtained. Primers, ssDNA reporters, and CrRNAs were synthesized and purified by Sangon Biotech (Shanghai, China). Three variations of CrRNAs were developed to optimize the CRISPR/Cas cleavage system. Additionally, two single-stranded reporters were employed for fluorescence-based detection and lateral flow detection within our detection platform. Reporter 1 (5'-BHQ1-ssDNA-HEX-3') was designed according to the carrier and structure of the fluorescence detection, and reporter 2 (5'-biotin-ssDNA-FITC-3') was used to match the lateral flow detection. The sequences of reporters, primers, and CrRNAs are shown in Table S1.
2.4 Method construction
As depicted in Fig. 1A, the detection method involved two key steps: RPA for the preamplification of DNA fragments and Cas12a for the trans-cleavage of ssDNA reporters. In the fluorescence-based detection, reporter 1 (BHQ1-ssDNA-HEX) was introduced into the cleavage reaction system. The whole reaction system was observed under ultraviolet (UV) light (λ = 480 nm). The presence of the target gene in the sample would result in the solution emitting yellow–green fluorescence visible to the naked eye. Conversely, the solution would not exhibit any fluorescence in the absence of the target gene (Fig. 1B). Fluorescence signal intensity was analyzed using a multipurpose microplate reader. The resulting data were used to plot a bar graph to analyze significant differences and thus indirectly determine the efficiency of the cleavage reaction.
Reporter 2 (biotin-ssDNA-FITC) was used as an indicator for the lateral flow detection. According to Fig. 1C, the test strip comprised a backing card with fixed components: nitrocellulose (NC) membrane, sample pad, conjugate pad, and absorbent pad. The sample pad and the conjugate pad were pretreated with different treatment buffers and then dried at 37°C. The conjugate pad was coated with gold particle-labeled anti-FITC-rabbit antibodies (2 µg). The NC membrane was coated with streptavidin (2 mg/mL) and goat anti-rabbit IgG antibody (1 mg/mL) to form the control line (C-line) and the test line (T-line), respectively. In addition, 1% bovine serum albumin was added to the goat anti-rabbit IgG antibody solution to eliminate the false positive signals. The NC membrane, conjugate pad, absorbent pad, and sample pad were sequentially fixed onto the backing card with an overlap of 1–2 mm and then cut into 3 mm-wide strips.
2.5 RPA and CRISPR/Cas12a cleavage reactions and lateral flow assay
The RPA reaction was performed using the TwistAmp® basic kit (Morey Biosciences, Shanghai, China), and the amplification system was optimized according to the conditions previously optimized by our team (21). The whole system was incubated at a constant temperature for 12 min, and the amplification of the target was confirmed via agarose gel electrophoresis.
The volume of the CRISPR/Cas12a cleavage system was 20 µL. The system consisted of 10 × Cpf1 reaction buffer, 50 nM Cpf1 (Cas12a) nuclease, 0.5 µM CrRNA, 1 unit RNase inhibitor, 1 µL ssDNA reporter, and diethyl pyrocarbonate water. Similar to the case of RPA amplification, the cleavage system was incubated at a constant temperature for 20 min. Subsequently, 5 µL of the cleavage product was mixed with 100 µL running buffer for lateral flow immunochromatography testing. After 5 min of reaction, the test results were determined according to the color development of the T-line. The gray value of the T-line was assessed using ImageJ software, and the signal was normalized.
2.6 Optimization of reaction conditions for RPA and CRISPR/Cas12a cleavage reaction
The sequences of the primers and CrRNA and the incubation time and temperature directly affect the efficiency of the platform; therefore, these factors were optimized in this study to obtain the optimal reaction conditions. Four primers were designed for RPA, and the results were analyzed via agarose gel electrophoresis. Similarly, three CrRNAs were analyzed to identify the most effective one for the CRISPR/Cas12a cleavage reaction. The cleavage reaction temperatures were optimized under a temperature range of 36.2–47.0°C, and time optimization was conducted at 0–30 min. Finally, the concentrations of reporters used in the CRISPR/Cas12a system were varied from 0 to 1500 nM to determine the optimal concentration.
2.7 Sensitivity and specificity of the platform
Sensitivity and specificity are important indicators for evaluating the feasibility of the detection method; therefore, these two aspects were experimentally evaluated. Sensitivity analysis involved bacterial culture solutions at concentrations ranging from 2.4 × 106 to 2.4 × 100 CFU/mL and various genomic DNA concentrations from 1.8 × 106 to 1.8 × 100 fg/µL. In addition, the specificity of the detection method was validated using two E. coli O157:H7 strains, three Escherichia coli strains, and 10 other strains. Distilled water without bacteria and genomes was used as a blank template control.
2.8 Detection of artificially contaminated samples
The official food safety standard in China explicitly prohibits the presence of E. coli O157:H7 in any food (GB29921-2021). Therefore, different concentrations of bacterial culture solutions (2.4 × 106, 2.4 × 104, 2.4 × 102, and 2.4 × 100 CFU/mL) were used to infect romaine lettuce. Specifically, 5 g of samples was added to 10 mL of distilled water, and then the mixture was homogenized. Immediately, 0.1 mL of broth was mixed with 1 mL of bacterial culture solution, and then 1 mL of mixture was used for DNA extraction.
2.9 Detection of natural samples
The detection of natural samples is a significant part of evaluating the feasibility of the detection method. Fifteen food varieties were randomly purchased from the market for the analysis of natural samples. These included chicken, fish, eggs, shrimp, milk, romaine lettuce, congee, noodles, cheese, goose egg, beef, pork, tofu, cucumber, and salmon. Each sample, weighing 25 g, was transferred to 225 mL of a medium buffer and incubated at 37°C for 12 h. Subsequently, 1 mL of the cultured solution was extracted and used for the detection process.
2.10 Statistical analysis
To enhance the objectivity of sample testing, photographs of the test strips were taken, and ImageJ software (NIH, Bethesda, MD, USA) was utilized to scan the color areas on all strips, displaying the peak area for each. The efficiency of CRISPR/Cas12a cleavage was assessed using a multipurpose microplate reader (Tecan, Mannedorf, Switzerland). Histograms were generated using Origin 2023b software (Origin Lab, Northampton, MA, USA). Average values and standard errors were calculated with SPSS 26.0 software (IBM Corp., Armonk, NY, USA).