A non-amplification time-resolved fluorescence lateral chromatographic detection method for detecting salmonella and drug-resistant bacteria

Abstract

This invention discloses an amplification-free time-resolved fluorescence lateral chromatography method for detecting Salmonella and drug-resistant strains, belonging to the field of rapid detection technology. Based on CRISPR / Casl2a technology, this invention designs crRNAs targeting Salmonella and drug-resistant Salmonella strains. The crRNAs shown in SEQ ID NO. 1–SEQ ID NO. 8 can specifically detect Salmonella and drug-resistant Salmonella strains, while the crRNAs shown in SEQ ID NO. 9–SEQ ID NO. 14 can specifically detect drug-resistant Salmonella strains, effectively distinguishing between Salmonella and drug-resistant Salmonella strains. The time-resolved fluorescence kit for drug-resistant Salmonella provided by this invention achieves a detection limit (LOD) of 1.8 × 10⁻⁶. 2 With a concentration of CFU / mL, and high precision and accuracy, this technology enables effective and rapid screening and detection.

Description

Technical Field

[0001] This invention relates to an amplification-free time-resolved fluorescence lateral chromatography method for detecting Salmonella and drug-resistant bacteria, belonging to the field of rapid detection technology. Background Technology

[0002] Salmonella (S. aureus) is considered one of the most serious infectious foodborne pathogens. In international food safety incidents (EFSA. 2021), it consistently ranked first or second among public safety issues due to its invasiveness, virulence, and antibiotic resistance, leading to severe food poisoning. Furthermore, with the overuse of antibiotics in medicine and agriculture, bacterial resistance is becoming increasingly serious. The World Health Organization (WHO, 2020) lists antibiotic resistance as one of the current threats to global health, food security, and development. Therefore, a rapid, inexpensive, and sensitive detection method for Salmonella and its drug-resistant strains is crucial for ensuring food safety and human health.

[0003] To achieve simple and rapid detection of pathogens, the CRISPR / Cas12a system has gained favor among researchers due to its ability to recognize double-stranded DNA (dsDNA) and activate its cis and trans-cleavage activities. For example, Chen et al. developed a four-tag-based CRISPR / Cas12a system for detecting Yersinia pestis based on fluorescence signals. Li et al. developed a CRISPR / Cas12a-based fluorescent and electrochemical biosensor for detecting Listeria monocytogenes. These CRISPR / Cas biosensors have demonstrated excellent performance in standardized laboratory tests. However, these methods rely primarily on fluorescence and electrochemical signals for readings, heavily depending on specialized operation and sophisticated instruments (such as fluorescence readers and electrochemical workstations), which inconveniences practical detection and reduces the performance of grassroots monitoring. Therefore, the development of a low-cost, simple, and accurate CRISPR / Cas12a detection platform is urgently needed.

[0004] Lateral flow assay (LFB) is a promising point-of-care testing (POCT) platform that has been widely used in nucleic acid detection. However, traditional molecular-based LFB typically uses amplicon obtained by loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) as the ligation medium, which is prone to false positives caused by primers and primer dimers.

[0005] Combining CRISPR / Cas systems with LFBs can overcome current limitations. For example, Mukama et al. developed an LFB (CIALFB) platform based on CRISPR / Cas isothermal amplification, enabling rapid and sensitive detection of human papillomavirus (HPV) and Pseudomonas aeruginosa.

[0006] However, most CRISPR / Cas-based LFBs still use gold nanoparticle (AuNP)-labeled antibodies as both signal and internal control probes. This results in high detection costs, weak signals, and difficulty in achieving precise quantification, hindering their application in point-of-care testing (POCT). There is an urgent need to develop a low-cost, simple, strong-signal, and high-performance CRISPR / Cas12a-based LFB platform. To address these issues, CRISPR / Cas12a-based LFB platforms can be improved based on the following key factors: 1) selecting high-intensity and stable signal output materials for LFBs; 2) selecting capture and reporter probes; and 3) improving the methods for modifying probes on the LFB. Compared to gold nanoparticles or fluorescent dyes, time-resolved fluorescent microsphere (TRFM)-labeled LFBs exhibit higher sensitivity and lower background interference due to their longer Stokes shift.

[0007] As a common molecular probe, single-stranded DNA (ssDNA) meets the needs of foodborne pathogen detection (LFB) due to its simplicity, low cost, and high specificity. A detection method based on a CRISPR / Cas12a-based LFB platform, combined with QD and ssDNA probes, may achieve significantly enhanced performance. This detection method can successfully detect foodborne pathogens, offering low cost, speed, sensitivity, and specificity; its applications can be extended to clinical diagnosis and environmental monitoring of point-of-care testing (POCT). Summary of the Invention

[0008] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a method for rapid and sensitive detection of drug-resistant Salmonella under amplification-free conditions by combining CRISPR / Casl2a technology and crRNA.

[0009] Another objective of this invention is to provide a time-resolved fluorescence test strip method based on CRISPR / Casl2a technology. This method is a method for detecting Salmonella and drug-resistant bacteria using the AsCas12a(cpf1)-NLS enzyme / combined crRNA system.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] The first objective of this invention is to provide a kit for rapid detection of Salmonella and drug-resistant Salmonella based on CRISPR / Cas12a technology, comprising Cas12a nuclease, crRNA, and fluorescein-labeled reporter single-stranded DNA molecules.

[0012] In one embodiment of the present invention, the sequence of the crRNA for detecting Salmonella is shown as any one of SEQ ID NO.1 to SEQ ID NO.8.

[0013] In one embodiment of the present invention, the crRNA sequences for detecting Salmonella and drug-resistant Salmonella are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0014] In one embodiment of the present invention, the sequence of crRNA for detecting drug-resistant Salmonella is shown in any one of SEQ ID NO.9 to SEQ ID NO.14.

[0015] In one embodiment of the present invention, the sequence of crRNA for detecting drug-resistant Salmonella is shown in SEQ ID NO.9 and SEQ ID NO.12.

[0016] In one embodiment of the present invention, the sequence of the reporter single-stranded DNA molecule is 5'-TTTTTTT-3'.

[0017] In one embodiment of the present invention, the 5' end of the reporter single-linked DNA molecule is labeled with FAM and the 3' end is labeled with BHQ1.

[0018] In one embodiment of the present invention, the drug-resistant Salmonella is a quinolone-resistant Salmonella mutant strain.

[0019] In one embodiment of the present invention, the Cas12a nuclease includes As Cas12a(cpf1)-NLS nuclease.

[0020] A second objective of this invention is to provide a method for detecting Salmonella and drug-resistant Salmonella using the above-mentioned kit, wherein the method involves reacting As Cas12a(cpf1)-NLS enzyme, crRNA, reporter single-stranded DNA molecule and DNA of the sample to be tested in a buffer solution and measuring the fluorescence value.

[0021] In one embodiment, the working concentration of the crRNA is 80–120 nM.

[0022] In one embodiment, the working concentration of the As Cas12a(cpf1)-NLS enzyme is 120–180 nM.

[0023] In one embodiment of the present invention, the buffer solution comprises 40–60 mM NaCl, 5–15 mM Tris-HCl, 5–15 mM MgCl2 and 80–120 μg / mL BSA, with a pH of 7.9.

[0024] The third objective of this invention is to provide an amplification-free time-resolved chromatography quantitative kit for detecting Salmonella and drug-resistant Salmonella. The kit includes a lateral chromatography strip, streptavidin-modified time-resolved fluorescent microspheres, a 5' biotin-modified nucleic acid probe, Cas12a nuclease, and crRNA. The lateral chromatography strip includes a capture probe.

[0025] In one embodiment of the present invention, the sequence of the crRNA for detecting Salmonella is shown as any one of SEQ ID NO.1 to SEQ ID NO.8.

[0026] In one embodiment of the present invention, the crRNA sequences for detecting Salmonella and drug-resistant Salmonella are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0027] In one embodiment of the present invention, the sequence of crRNA for detecting drug-resistant Salmonella is shown in any one of SEQ ID NO.9 to SEQ ID NO.14.

[0028] In one embodiment of the present invention, the sequence of crRNA for detecting drug-resistant Salmonella is shown in SEQ ID NO.9 and SEQ ID NO.12.

[0029] In one embodiment of the present invention, the sequence of the nucleic acid probe is 5'-TTTTTTTTATT-3'.

[0030] In one embodiment of the present invention, the Cas12a nuclease includes As Cas12a(cpf1)-NLS nuclease.

[0031] In one embodiment of the present invention, the lateral chromatography test strip is provided with a backing plate, a glass fiber sample pad, a nitrocellulose membrane (NC membrane), and an absorbent pad; the sample pad, the NC membrane, and the absorbent paper are provided with a detection line and a control line on the NC membrane, the detection line is coated with a capture probe, and the control line is coated with bovine serum albumin-conjugated biotin.

[0032] In one embodiment of the present invention, the sequence of the capture probe is shown in SEQ ID NO.15.

[0033] In one embodiment of the present invention, the working concentration of the capture probe is 20-50 nM, and the working concentration of the bovine serum albumin-conjugated biotin is 0.1-0.5 mg / mL.

[0034] In one embodiment of the present invention, the capture probe uses a PBS solution containing 0.6–1.0 M KCl as a dilution buffer.

[0035] In one embodiment of the present invention, the spraying amount of both the capture probe and the biotin conjugated with bovine serum albumin is 1 μL / cm.

[0036] In one embodiment of the present invention, the detection line and the control line are 4-6 mm apart; the width of the test strip is 3-5 mm.

[0037] In one embodiment of the present invention, the glass fiber sample pad is prepared by immersing a glass fiber sample pad with a width of 18-22 mm in a buffer solution for 4.5-5.5 minutes until saturation, and then drying it at 45-55°C for 20-30 hours.

[0038] In one embodiment of the present invention, the buffer solution is 0.05M Tris-HCl, 2% (w / v) PEG8000 and 1.5% (v / v) Tween-20, pH=8.0.

[0039] In one embodiment of the present invention, the length of the overlap between the sample pad and the NC membrane is 2-4 mm, and the sample pad is placed on top of the NC membrane; the length of the overlap between the absorbent paper and the NC membrane is 2-4 mm, and the absorbent paper is placed on top of the NC membrane.

[0040] In one embodiment of the present invention, the detection line is 4-6 mm away from the sample pad, and the quality control line is 4-6 mm away from the absorbent paper.

[0041] In one embodiment of the present invention, the kit further contains a positive standard and a reaction buffer.

[0042] In one embodiment of the present invention, the reaction buffer is 0.02 mM EDTA, 0.4 mM Tris-HCl and 1 mM MgCl2.

[0043] The fourth objective of this invention is to provide a method for detecting Salmonella and drug-resistant Salmonella based on time-resolved fluorescent microspheres. The method involves using the aforementioned amplification-free time-resolved chromatography quantitative kit, and the specific steps are as follows:

[0044] (1) A fluorescent probe was obtained by coupling streptavidin-modified time-resolved fluorescent microspheres with a 5' end biotin-modified nucleic acid probe.

[0045] (2) Extract DNA from the sample to be tested;

[0046] (3) The fluorescent probe from step (1), the DNA from step (2), the Cas12a nuclease and the crRNA were left to stand at room temperature for 10 to 30 minutes to obtain a mixture.

[0047] (4) Add the mixture from step (3) to the sample pad, incubate, and quantify using an immunoassay analyzer and in conjunction with a standard curve.

[0048] In one embodiment of the present invention, in step (1), the coupling condition is coupling at 20-27°C for 10-20 minutes.

[0049] In one embodiment of the present invention, in step (1), the final concentration of the streptavidin-modified time-resolved fluorescent microspheres is 1-10 ng / mL, and the final concentration of the 5' end biotin-modified nucleic acid probe is 10-80 nM.

[0050] In one embodiment of the present invention, in step (3), 20 μL of DNA, 50 nM of Cas12a nuclease, 100 nM of crRNA and 50 μL of fluorescent probe are mixed.

[0051] In one embodiment of the present invention, in step (4), the incubation conditions are 35-38°C chromatography for 10-20 min.

[0052] In one embodiment of the present invention, the method is carried out under conditions of pH 6.5 to 7.5.

[0053] In one embodiment of the present invention, the standard curve is prepared by extracting DNA from a negative solution containing different concentrations of Salmonella or drug-resistant strains, mixing streptavidin-modified time-resolved fluorescent microspheres with a 5' end biotin-modified nucleic acid probe to obtain a fluorescent probe, and then adding the fluorescent probe, DNA, Cas12a nuclease and crRNA to a sample pad after standing at room temperature for 10-30 min, incubating, performing quantitative analysis using an immunoassay analyzer, and fitting the data to obtain a standard curve.

[0054] When the target DNA is present, the trans-cleavage activity of the Cas12a enzyme is activated, indiscriminately degrading the biotin-ssDNA probe, leading to the failure of the capture probe on the T line; therefore, no fluorescence signal is generated. After the reaction solution reaches the C line, the bovine serum albumin-conjugated biotin can directly capture the free streptavidin-modified time-resolved fluorescent microspheres (TRFM-SA), forming a significant fluorescence signal. Conversely, in the absence of target DNA in the sample, the trans-cleavage activity of the Cas12a enzyme is not activated; the capture probe on the T line complements the intact bio-ssDNA probe to capture TRFM-SA, thus generating a significant fluorescence signal. The fluorescence intensity values ​​of the standards at the test and control lines were then measured to obtain the T and C values, respectively; a negative control was set up, i.e., the sample did not contain the test sample, and the fluorescence intensity T0 value was measured; using T / T0 as a parameter, a linear model was established with the logarithm of the concentration to obtain the corresponding standard curves for Salmonella and its quinolone-resistant mutant strains.

[0055] Beneficial effects:

[0056] 1. This invention designs crRNAs targeting Salmonella and drug-resistant Salmonella strains (quinolone-resistant Salmonella mutants) based on CRISPR / Casl2a technology. Among them, the crRNAs shown in SEQ ID NO.1 to SEQ ID NO.8 can be specifically used to detect Salmonella and drug-resistant Salmonella strains, and the crRNAs shown in SEQ ID NO.9 to SEQ ID NO.14 can be specifically used to detect drug-resistant Salmonella strains, effectively distinguishing between Salmonella and drug-resistant Salmonella strains.

[0057] 2. The crRNA combinations crRNA5+6-S and crRNA1+4-Q provided by this invention can effectively improve the detection sensitivity. It was found that the average slope of the crRNA5+6-S combination increased by 89.5% and 123.7% compared with the independent crRNA5-S and crRNA6-S, respectively; the average slope of the crRNA1+4-Q combination increased by 85.1% and 119.5% compared with crRNA1-Q and crRNA4-Q, respectively.

[0058] 3. This invention improves the reaction sensitivity of the time-resolved fluorescence kit by optimizing the reaction pH of the time-resolved fluorescent microspheres, the dilution buffer for the capture probe ssDNA, and the amount of TRFM-SA and bio-ssDNA probes added. Combined with the crRNA1+4-Q combination, the detection limit (LOD) for drug-resistant Salmonella strains can reach 1.8 × 10⁻⁶. 2 The time-resolved fluorescence reagent kit provided by this invention has high precision and accuracy, enabling effective and rapid screening and detection. Attached Figure Description

[0059] Figure 1 : The nucleotide sequence and position of the crRNA used.

[0060] Figure 2 For the screening of crRNA sequences and the construction of liquid environment detection methods (vertical axis is fluorescence intensity, horizontal axis is incubation time).

[0061] Figure 3 For independent crRNA fluorescence detection, A: fluorescence recovery graph of crRNA at different concentrations over time, B: slope (enzyme activity) comparison graph at different concentrations.

[0062] Figure 4 This is a comparison of the fluorescence recovery capabilities of combined crRNA and independent crRNA.

[0063] Figure 5 The diagram shows the specificity analysis of this detection method. A: Specificity analysis of the crRNA5+6-S combination; B: Specificity analysis of the crRNA1+4-Q combination.

[0064] Figure 6 To optimize the time-resolved fluorescent microsphere test strip, the following optimizations were made: A) pH optimization of the T-line of the time-resolved fluorescent test strip for Salmonella resistant to amplification; B) buffer optimization of the T-line of the time-resolved fluorescent test strip for Salmonella resistant to amplification; C) binding rate optimization of the time-resolved fluorescent microspheres in the time-resolved fluorescent test strip for Salmonella resistant to amplification; and D) optimization of the amount of bio-ssDNA added to the time-resolved fluorescent test strip for Salmonella resistant to amplification.

[0065] Figure 7 The standard curve for time-resolved fluorescent test strips for non-amplified drug-resistant Salmonella. Detailed Implementation

[0066] Example 1: Construction of a method for screening crRNA and detecting it in a liquid environment

[0067] In this embodiment of the invention, a rapid detection method for Salmonella and its quinolone-resistant mutant strains based on CRISPR / Casl2a technology is used for non-diagnostic or therapeutic purposes and includes the following steps:

[0068] Conserved and specific nucleic acid sequences were screened using Vmatch and BLAST alignment. These sequences were targeted at the Salmonella standard strain and the Salmonella qnrs gene, respectively, as target sequences for recognition by Cas12a nuclease (AsCas12a(cpf1)-NLS enzyme) and crRNA. The target sequences contained the “TTTN” PAM recognition site.

[0069] First, target-specific crRNAs were designed for the target sequences. Four low-repetition sequences, crRNA-Salmonella1 (crRNA1-S) to crRNA-Salmonella4 (crRNA4-S), and four high-repetition sequences, crRNA5-S to crRNA8-S, were designed and synthesized for standard Salmonella strains. Sequences targeting the QnrS gene of drug-resistant Salmonella were designed and synthesized for crRNA-QnrS1 (crRNA1-Q) to crRNA-QnrS6 (crRNA6-Q). Figure 1 Then, transcription and purification are performed. The synthesized Cas12a crRNA consists of two parts: a 20bp repeat sequence at the 5' end, which can bind to the AsCas12a(cpf1)-NLS enzyme, and the repeat sequence is 5'-GGAUUUAGACUAAACUAAGAU-3'; the second part is a 21bp spacer sequence, which can pair complementaryly with the target gene; the PAM sequence of Cas12a is located at the 5' end of the non-binding strand of the target DNA, between the repeat sequence and the spacer sequence.

[0070] Design of a single-stranded DNA molecule (ssDNA-FQ): A 7-T single-stranded DNA molecule with FAM and BHQ1 groups at both ends. 5'-fam-ttttttt-BHQ1-3'.

[0071] Table 1. Oligonucleotide sequences used in CRISPR / Cas12a detection

[0072]

[0073]

[0074] Before the assay, Salmonella standard strains and drug-resistant Salmonella were incubated overnight in nutrient broth at 37°C to obtain pure bacterial cultures. The concentration of each bacterial species was then determined by standard plate counting.

[0075] DNA was extracted using a thermal lysis method, also known as water extraction. 1 mL of bacterial solution was resuspended in 200 μL Tris-EDTA (TE) buffer and lysed at 100 °C for 8 min. The lysed bacterial solution was centrifuged at 12000 × g for 2 min, and the supernatant was used as the nucleic acid sample for subsequent experiments. DNA concentration was measured using a microspectrophotometer (Kaiao Technology Development Co., Ltd., Beijing, China) before assay.

[0076] Fluorescence detection reaction system: The Cas12a lysis assay consisted of 2.1 μL of 1×NEB buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 ug / mL BSA, pH 7.9, at 25 °C), 150 nM AsCas12a(cpf1)-NLS enzyme, 100 nM crRNA, 300 nM ssDNA-FQ reporter probe, and 4 μL of nucleic acid sample. Finally, nuclease-free water was added to a final volume of 25 μL.

[0077] Fluorescence detection reaction conditions: The fluorescence detection reaction system was placed at 37℃ for 30 min before the fluorescence intensity was detected.

[0078] The reaction product was excited by excitation light at a wavelength of 492 nm in an ELISA reader, and the fluorescence intensity was detected at a wavelength of 525 nm to obtain the detection results.

[0079] pass Figure 2 It is known that the crRNA sequences crRNA5-S and crRNA6-S designed for Salmonella showed the best fluorescence recovery, while the crRNA sequences crRNA1-Q and crRNA4-Q designed for the QnrS gene of drug-resistant Salmonella showed the best fluorescence recovery and can be used for subsequent detection.

[0080] Example 2: Combining crRNA to improve the sensitivity of CRISPR-Cas12a detection

[0081] Bacterial DNA nucleic acid extraction kit was used to extract bacterial concentrations of 10. 1 ~10 4 DNA of Salmonella and drug-resistant Salmonella was collected at CFU / mL. The target DNA was then subjected to fluorescence detection using the method described in Example 1 to independently determine the detection limits of crRNA5-S, crRNA6-S, crRNA1-Q, and crRNA4-Q screened in Example 1. Results were obtained from... Figure 3 As shown in Figure A, the fluorescence recovery signal generated by Cas12a nuclease digestion is directly proportional to the concentration of target DNA in the assay. For groups containing either crRNA5-S or crRNA6-S, targeting Salmonella standard strains, when the target bacterial activity was 10... 2 It can be detected at CFU / ml, but the fluorescence recovery is not obvious; adding crRNA1-Q or crRNA4-Q respectively, targeting drug-resistant Salmonella strains, only when the bacterial activity reaches 10 3 It can only be detected when the concentration of CFU / ml is high.

[0082] Next, we will combine two crRNA sequences designed for Salmonella (crRNA5+6-S) and two crRNA sequences designed for the QnrS gene of drug-resistant Salmonella (crRNA-Q1 and crRNA-Q4) (crRNA1+4-Q). We will then determine whether combining crRNAs in the same reaction to form two different CRISPR-Cas12a systems can enhance the activation of overall cleavage and thus improve the sensitivity of detection.

[0083] The total volume of the fluorescence detection reaction system was 25 μL, containing 150 nM Cas12a nuclease, 100 nM crRNA combination (crRNA5+6-S combination or crRNA1+4-Q combination), 2 nM reporter single-stranded DNA molecule, 10 U RNase inhibitor (TaKaRa), 5 μL of target DNA corresponding to different bacterial activities, and 2.1 μL of 1×NEB buffer. The reaction system was incubated at 37 °C for 30 minutes.

[0084] When the bacterial activity is 1.4 × 10 4 At (CFU / mL), binding to crRNA significantly increased the slope and sensitivity of the detection reaction. Results are as follows... Figure 4 As shown, the fluorescence recovery of the combined crRNA (crRNA5+6-S) targeting Salmonella and the combined crRNA (crRNA1+4-Q) targeting drug-resistant Salmonella was significantly higher than that of single crRNAs, especially at low concentrations, showing a significant increase compared to background fluorescence, thus significantly reducing the limit of detection (LOD). We performed linear regression on the fluorescence recovery data and present the results as slope ± 95% confidence intervals. Analysis of covariance (ANCOVA) was used to compare the slope with the individual RNP control: ****p<0.0001, ***p<0.001, ns = not significantly higher than the RNP control. The mean slope of the crRNA5+6-S combination increased by 89.5% and 123.7% compared to independent crRNA5-S and crRNA6-S, respectively; the mean slope of the crRNA1+4-Q combination increased by 85.1% and 119.5% compared to crRNA1-Q and crRNA4-Q, respectively, indicating a significant increase in detection sensitivity.

[0085] The nucleic acid samples to be tested were serially diluted, and the detection results of two sets of crRNA combinations, crRNA5-S combined with crRNA6-S or crRNA1-Q combined with crRNA4-Q, were compared with the detection results of crRNA alone. It was found that the combined crRNAs could increase the detection sensitivity of Salmonella standard strains from 10 to 10. 3 CFU / ml decreased to 10 2The CFU / ml concentration significantly improves detection sensitivity without changing the detection time or other conditions.

[0086] Example 3: Determination of the specificity of combined crRNA detection

[0087] The fluorescence detection reaction system consisted of a total of 25 μL, 50 nM Cas12a nuclease, 100 nM crRNA combination (crRNA5+6-S combination or crRNA1+4-Q combination), 2 nM reporter single-stranded DNA molecule, 10 U RNase inhibitor (TaKaRa), 5 μL nucleic acid sample, and 2.1 μL 1×NEB buffer. The reaction system was incubated at 37 °C for 30 minutes.

[0088] DNA was extracted from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, standard strains of Salmonella, and drug-resistant Salmonella as nucleic acid samples, and all were diluted to 10⁻⁶. 3 After CFU / mL, fluorescence detection reactions were performed, such as... Figure 5 As shown, Figure 5 A represents the fluorescence recovery after adding the crRNA5+6-S combination to the sample. Figure 5 B represents the fluorescence recovery in different samples after adding the crRNA1+4-Q combination. The sample numbers are as follows: 1. *Escherichia coli*, 2. *Staphylococcus aureus*, 3. *Pseudomonas aeruginosa*, 4. *Aeromonas hydrophila*, 5. *Vibrio parahaemolyticus*, 6. *Salmonella* standard strain, 7. Drug-resistant *Salmonella*, 8. *Escherichia coli* + *Staphylococcus aureus* + drug-resistant *Salmonella*, 9. *Vibrio parahaemolyticus* + *Escherichia coli* + *Pseudomonas aeruginosa* + *Staphylococcus aureus* + *Aeromonas hydrophila*, 10. *Escherichia coli* + *Pseudomonas aeruginosa* + *Salmonella* standard strain + *Staphylococcus aureus*; 11. *Escherichia coli* + *Staphylococcus aureus* + *Pseudomonas aeruginosa* + *Vibrio parahaemolyticus* + *Aeromonas hydrophila* + drug-resistant *Salmonella*; 12. *Escherichia coli* + *Staphylococcus aureus* + *Pseudomonas aeruginosa* + *Aeromonas hydrophila* + drug-resistant *Salmonella* standard strain; 13. *Salmonella* standard strain + drug-resistant *Salmonella*.

[0089] It can be seen that when using the crRNA5+6-S combination, only samples containing the standard Salmonella strain and drug-resistant Salmonella showed fluorescence recovery. When using the crRNA1+4-Q combination, only samples containing drug-resistant Salmonella showed significant fluorescence recovery, demonstrating good specificity. It can specifically detect Salmonella and distinguish between sensitive strains and drug-resistant bacteria.

[0090] Example 4: Preparation of streptavidin (SA) modified time-resolved fluorescent microspheres (TRFM-SA) probes

[0091] Preparation of relevant solutions:

[0092] Activation buffer: 0.05M 2-(N-morpholino)ethanesulfonic acid (MES, C6H) at pH 4.5-6.5. 13 NO4S·H2O) solution;

[0093] Coupling buffer: 0.01M phosphate-buffered saline (PBS) pH 7.0-8 (avoid solvents containing free amines);

[0094] Activator: 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C8H) 17 N3·HCl) solution and 10 mg / mL N-hydroxysuccinimide (NHS, C4H5NO3) solution;

[0095] Blocking buffer: Phosphate-buffered saline (PBS) at pH 6.0-7.00 M containing 1% BSA and 0.05% Tween-20;

[0096] Washing solution: 0.1% Tween-20, pH 6.0-7.0, 0.05M Tris-HCl;

[0097] Streptavidin-modified time-resolved fluorescent microspheres (TRFM-SA) complex solution / reaction sustained-release solution: containing 5% sucrose, 1% bovine serum albumin, and 1% Tween-20 in 0.05M Tris-HCl at pH 6.0-7.0.

[0098] The specific preparation method of TRFM-SA is as follows:

[0099] (1) Take 50 μL (1% solids content) of time-resolved fluorescent microspheres (TRFM) containing Eu3+ (purchased from Xiamen Dibai Biotechnology Co., Ltd., particle size 300nm) placed at 4℃, disperse by sonication, add 800-1000 μL of activation buffer, and centrifuge at 4℃ for 10-15 min at 15000 rpm;

[0100] (2) Discard the supernatant, add 500-800 μL of activation buffer, sonicate to resuspend, and repeat centrifugation and washing 2-3 times;

[0101] (3) Discard the supernatant, add 200 μL of activation buffer and sonicate to resuspend, add 50 μL of 10 mg / mL EDC solution and 50 μL of 10 mg / mL NHS solution as activators, and activate at room temperature by shaking at 500 rpm in the dark for 40 min.

[0102] (4) After activation, centrifuge, discard the supernatant, and wash 2-3 times with PBS buffer;

[0103] (5) Discard the supernatant, add 400 μL of coupling buffer and sonicate to resuspend, then add 30 μg of streptavidin and label with shaking at room temperature in the dark for 2 h;

[0104] (6) After labeling, add 10% (v / v) blocking buffer and shake at room temperature in the dark for 40 min;

[0105] (7) After sealing, centrifuge and discard the supernatant, then wash 2-3 times with 800-1000 μL of labeled washing solution;

[0106] (8) Discard the supernatant, add 200 μL of fluorescent microsphere reconstitution solution to obtain streptavidin-labeled time-resolved fluorescent microspheres (TRFM-SA), and store at 4℃ for later use.

[0107] Example 5: Preparation of time-resolved fluorescent microsphere test strips

[0108] (1) Synthetic labeling probe: Synthetic biotin probe (bio-ssDNA) probe (see Table 1) is obtained by coupling bio-ssDNA probe with TRFM-SA synthesized in Example 4 at 25°C for 10-20 min. The labeling probe is prepared and used immediately when the test strip is used.

[0109] (2) Sample pad preparation: The glass fiber sample pad (20 mm wide) was immersed in buffer (0.05 M Tris-HCl, 2% (w / v) PEG8000 and 1.5% (v / v) Tween-20, pH=8.0) for 5 min to complete saturation, and then dried at 50 °C for 24 h.

[0110] (3) NC membrane coating: The capture probe (50 nM) and bovine serum albumin-conjugated biotin (0.25 mg / mL) as shown in Table 1 were sprayed onto the NC membrane as the detection line (T line) and control line (C line), respectively. The spraying amount was 1 μL / cm. The width of the T line and the C line depended on the diameter of the spraying instrument tubing, which was about 2 mm. The T line was about 5 mm away from the sample pad, and the C line was about 5 mm away from the absorbent paper. The two lines were about 4-6 mm apart. The membrane was dried at 37℃ for 2-3 h.

[0111] (4) Assembly of test strips: The sample pad, NC membrane and absorbent paper are glued to the PVC base plate to assemble the test strip. The key to the assembly of the test strip is to ensure that the components have consistent transferability. The sample pad is stacked on the NC membrane, with an overlap of about 3 mm. Similarly, the absorbent paper is stacked on the NC membrane, with an overlap of about 3 mm. The glued plate is cut into test strips about 4 mm wide using a strip cutter. The strips are assembled with plastic base and clips and sealed at 4°C for later use.

[0112] Example 6: Optimization of a Time-Resolved Fluorescence Reagent Kit for Drug-Resistant Salmonella

[0113] (1) As Figure 6 As shown in Figure A, Eu3+ fluorescent microspheres are unstable in aqueous solution. Therefore, high concentrations in the reaction system, the concentration of charged groups on the particle surface, and electrolyte concentrations can all lead to particle aggregation in aqueous solution. The pH of the solution significantly affects the sensitivity, fluorescence index (FI), and stability of TRFM-SA. The fluorescence value of the T-line on the test strip and the FI... t / FI C The fluorescence value increases with increasing pH of the Tris-HCl buffer. It reaches its maximum at pH 7, after which the fluorescence value begins to decrease. Between pH 6.5 and 7.0, the fluorescence value of the test strip reaches 40 w (au), and the FI... t / FI C A ratio close to 1 meets the detection requirements.

[0114] (2) Figure 6 As shown in Figure B, although the capture probe ssDNA is low-cost and simple to design, its instability when modified on the NC membrane cannot be ignored. Therefore, we screened capture probe ligation agents: six ligation reagents were prepared, including C2H5OH, C3H8O3, Tween 20, PBS, KCl, KCl+Tween 20, and KCl+PBS. We found that the three ligation reagents containing KCl could effectively anchor the DNA probe and produce a brighter band in the T-line, while the other ligation reagents showed significant diffusion. Figure 6 As shown in Figure B, the mixed ligand of KCl and PBS produces a stronger fluorescence peak and the largest FI. T / FI C Ratio. A mixture of KCl (0.8M) and PBS was chosen as the T-line buffer and binding agent.

[0115] (3) Figure 6 As shown in Figure C, different amounts of TRFM-SA significantly affect detection sensitivity. Specifically, small amounts reduce sample detection, while large amounts lead to poor system dispersion. The effects of different TRFM-SA dosages were tested. Based on previous literature, we adjusted FI... t / FI C The ratio was defined as the optimal condition closest to 1, and a TRFM-SA concentration of 3 μg / mL was found to meet the requirements with appropriate FI. t / FI C The ratio and the brightness band between comparable T and C lines. Therefore, a concentration of 3 μg / mL was selected as the optimal concentration.

[0116] (4) Figure 6As shown in D, the concentration of the bio-ssDNA probe: the concentration of the reporter probe determines the sensitivity of the CFA-LFB detection platform. Tests were conducted at different concentrations (0, 10, 15, 20, 25, 30, 50, and 80 nM). The results showed that the FIT / FIC ratio gradually increased with increasing bio-ssDNA probe dosage, and this ratio approached 1 at 20 nM. Finally, 20 nM of bio-ssDNA probe was selected as the optimal concentration.

[0117] Example 7: Plotting the Standard Curve for the Time-Resolved Fluorescence Reagent Kit for Drug-Resistant Salmonella

[0118] The method for plotting a standard curve is as follows:

[0119] Drug-resistant strains were added to negative milk samples to prepare cultures with bacterial activities of 0 and 6.4 × 10⁻⁶, respectively. 1 5.4×10 2 2.7×10 3 1.4×10 4 6.7×10 4 3.3×10 5 The spiked sample solution at CFU / mL was used for detection on the fluorescent test strip (the diluent was 0.01M pH 7.4 PBS solution; TRFM-SA solution: 0.05μg / μL TRFM-SA, containing 1.5% bovine serum albumin, 1% Tween-20, pH 6.7 0.05M Tris-HCl solution). Nucleic acid was extracted from the standard solutions of different concentrations using a nucleic acid extraction kit to obtain the corresponding nucleic acid samples. The final concentrations of 4ng / mL TRFM-SA and 20nM TRFM-SA prepared in Example 4 were used. Bio-ssDNA was used as a fluorescent probe. 20 μL of nucleic acid sample, 50 nM Cas12a nuclease and 100 nM crRNA combination (crRNA1+4-Q) and 50 μL of fluorescent probe were mixed evenly and incubated at room temperature for 15 min. The mixture was then slowly added to the sample well of the test strip and chromatographically analyzed at 37 °C for 15 min. The T-line fluorescence value of the test strip was recorded using an HG-98 immunoassay analyzer. Six replicates were measured for each concentration. The T-line fluorescence value of the spiked sample solution with bacterial activity of 0 CFU / mL was set as T0, and the T-line fluorescence value of other spiked sample solutions was T. A standard curve was plotted with the logarithm of the bacterial activity of drug-resistant Salmonella as the x-axis and T / T0×100 (%) as the y-axis. The inhibition rate was set as (1-T / T0)×100%.

[0120] like Figure 7 It can be seen that as the concentration of drug-resistant Salmonella increases, the fluorescence on the T-band of the test strip becomes lighter and lighter, so the T / T0 ratio decreases. Figure 7The figure shows the curve of T / T0 as a function of the concentration of drug-resistant Salmonella. When the concentration of drug-resistant Salmonella is 5.4 × 10⁻⁶, the curve is displayed. 2 -3.3×10 5 At that time, the logarithm of the concentration of drug-resistant Salmonella showed a linear relationship with T / T0, with the linear equation being Y = -22.532X + 254.89, R 2 =0.9854, the detection limit (LOD) can reach 1.8×10 2 CFU / ml.

[0121] Example 8: Performance Testing of the Time-Resolved Fluorescence Reagent Kit for Drug-Resistant Salmonella

[0122] (1) Precision test, taking the detection of drug-resistant Salmonella as an example.

[0123] Eight time-resolved immunochromatographic assay kits were taken from the same batch and their fluorescence intensity (T0) at the test line was measured in the negative control group (pH=6.7 PBS) and the positive test group (drug-resistant Salmonella 10). 4 Intra-batch differences were analyzed by measuring the fluorescence intensity (T) at the T line of the CFU / / ml test kit. The fluorescence intensity (T0) at the test line of the negative control group (pH=6.7 PBS) and the positive test group (drug-resistant Salmonella 10) were measured using eight different batches of time-resolved immunochromatographic assay kits. 4 The fluorescence intensity value (T) at the T line (CFU / mL) was used to analyze inter-batch differences. Table 2 shows that, using the coefficient of variation calculation formula, the intra-batch coefficients of variation for T0, T, and T / T0 (%) were 3.36%, 5.74%, and 2.81%, respectively; the inter-batch coefficients of variation for T0, T, and T / T0 (%) were 4.26%, 5.80%, and 5.54%, respectively. This indicates that the kit has low intra-batch and inter-batch coefficients of variation, high precision, and good accuracy, basically meeting the requirements of a quantitative detection kit.

[0124] Table 2. Precision test results of time-resolved test strips for drug-resistant Salmonella.

[0125]

[0126] (2) Accuracy test

[0127] To verify the accuracy and sensitivity of the Salmonella resistant test strips, spiked recovery tests were conducted on several negative milk and dairy product samples (raw milk, pasteurized milk, yogurt, and milk powder). Three different spiked concentrations (high, medium, and low) were set up for each sample, and three parallel tests were conducted for each concentration gradient. The spiked recovery rate was used as the accuracy evaluation index, and the relative standard deviation (RSD%) of the detection results for a specific concentration of sample was used as the precision evaluation index. The formulas for calculating the spiked recovery rate and relative standard deviation are as follows:

[0128]

[0129]

[0130] Dairy product samples that tested negative were processed at a rate of 1×10⁻⁶. 2 1×10 3 1×10 4 Spiked recovery experiments of drug-resistant Salmonella were conducted using three concentration gradients of CFU / mL. Each concentration was repeated 10 times, and the average T / T0 was calculated. The results are shown in Table 1. The CV values ​​of all results were less than 5.7%, indicating that the kit has good accuracy within the linear range. PBS solution containing 0.05% Tween-20 was used as the sample diluent in this study to ensure the performance of the method.

[0131] Accurately weigh 5g of milk powder sample, add 40mL of ultrapure water at 60-80℃, and sonicate or shake until fully dissolved. Extract sample DNA using a heating lysis method or a water extraction method for immunochromatographic detection.

[0132] Accurately pipette 10 mL of pasteurized milk sample, centrifuge at 6000 rpm and 4℃ for 10 min, take the middle and lower layer liquid, dilute it 5 times with PBS containing 0.05% Tween-20, and extract the sample DNA using the heating lysis method for immunochromatographic detection.

[0133] Take an appropriate amount of raw milk sample, add it directly, and extract the sample DNA using the heating lysis method for immunochromatographic detection.

[0134] Table 3. Accuracy test results of the time-resolved immunochromatographic assay kit.

[0135]

[0136] As shown in Table 3, when the prepared time-resolved immunochromatographic kit was used to detect spiked milk samples, the final spike recovery rate of milk was between 79.69% and 128.74%, and the coefficient of variation was less than 5.7%, indicating that the prepared drug-resistant Salmonella immunofluorescence quantitative kit can be used for rapid on-site screening and detection.

[0137] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> A non-amplification time-resolved fluorescence lateral chromatography method for detecting Salmonella and drug-resistant bacteria <130> BAA220529A <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 46 <212> DNA <213> Artificial sequence <400> 1 ggatttagac taaacuaaga ttttggcgat agacgcttca gtgtca 46 <210> 2 <211> 46 <212> DNA <213> Artificial sequence <400> 2 ggatttagac taaacuaaga ttttcaccga ccacatggca taagta 46 <210> 3 <211> 46 <212> DNA <213> Artificial sequence <400> 3 ggatttagac taaacuaaga ttttaacaccc ccgaaatcat tataaa 46 <210> 4 <211> 46 <212> DNA <213> Artificial sequence <400> 4 ggatttagac taaacuaaga ttttaacaga taacgtatac cacact 46 <210> 5 <211> 46 <212> DNA <213> Artificial sequence <400> 5 ggatttagac taaacuaaga ttttagcgac gatctctacg gttata 46 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <400> 6 ggatttagac taaacuaaga ttttcataat atcctcacac gacact 46 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 ggatttagac taaacuaaga ttttagcgta catgcgtggg tagata 46 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <400> 8 ggatttagac taaacuaaga ttttacgtta acagtcggac aattta 46 <210> 9 <211> 47 <212> DNA <213> Artificial Sequence <400> 9 ggatttagac taaacuaaga ttttcacata aagacttaag tgatctc 47 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <400> 10 ggatttagac taaacuaaga ttttcgacgt gctaacttgc gtgata 46 <210> 11 <211> 46 <212> DNA <213> Artificial Sequence <400> 11 ggatttagac taaacuaaga ttttgccatc gcaagttggc attgtt 46 <210> 12 <211> 46 <212> DNA <213> Artificial sequence <400> 12 ggatttagac taaacuaaga ttttaaatca cacgcacgga actcta 46 <210> 13 <211> 46 <212> DNA <213> Artificial sequence <400> 13 ggatttagac taaacuaaga ttttcccgaa caaactttgc ccatca 46 <210> 14 <211> 46 <212> DNA <213> Artificial sequence <400> 14 ggatttagac taaacuaaga ttttctaaac aaaccctctc catatt 46 <210> 15 <211> 42 <212> DNA <213> Artificial sequence <400> 15 aataaaaaaa aaaaaaaaaa aaataaaaaa aaaaaaaaaa aa 42

Claims

1. A non-amplification time-resolved chromatographic quantitative kit for detecting Salmonella and quinolone-resistant Salmonella, characterized in that, The kit includes a lateral chromatography test strip, streptavidin-modified time-resolved fluorescent microspheres, a 5' biotin-modified nucleic acid probe, and Cas12a nuclease; the detection line of the lateral chromatography test strip is coated with a capture probe; the capture probe is diluted with PBS solution containing 0.6-1.0 M KCl as a dilution buffer; The sequence of crRNA for detecting Salmonella is shown in any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8; The sequence for detecting quinolone-resistant Salmonella crRNA is shown in any one of SEQ ID NO. 9, SEQ ID NO. 11 to SEQ ID NO. 14; The sequence of the nucleic acid probe is 5'-TTTTTTTTATT-3'; The sequence of the capture probe is shown in SEQ ID NO.

15.

2. The kit of claim 1, wherein The lateral chromatography test strip is provided with a backing plate, a glass fiber sample pad, a nitrocellulose membrane, and absorbent paper; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with a capture probe; the control line is coated with bovine serum albumin-conjugated biotin.

3. The kit according to claim 1 or 2, characterized in that, The working concentration of the capture probe is 20-50 nM; the working concentration of the bovine serum albumin-conjugated biotin is 0.1-0.5 mg / mL.

4. The reagent kit according to claim 2, characterized in that, The glass fiber sample pad is prepared by immersing a glass fiber sample pad with a width of 18-22 mm in a reaction buffer for 4.5-5.5 min to achieve saturation, and then drying it at 45-55℃ for 20-30 h.

5. The reagent kit according to claim 2, characterized in that, The sample pad overlaps with the nitrocellulose membrane for 2-4 mm, with the sample pad positioned above the nitrocellulose membrane; the absorbent paper overlaps with the nitrocellulose membrane for 2-4 mm, with the absorbent paper positioned above the nitrocellulose membrane.

6. The reagent kit according to claim 2, characterized in that, The detection line and the control line are 4-6 mm apart; the width of the test strip is 3-5 mm; the detection line is 4-6 mm away from the sample pad; and the control line is 4-6 mm away from the absorbent paper.

7. A method for detecting Salmonella and quinolone-resistant Salmonella based on time-resolved fluorescent microspheres, not for the purpose of disease diagnosis, characterized in that... The method involves detection using the amplification-free time-resolved chromatography quantitative kit described in any one of claims 1 to 6, and the specific steps are as follows: (1) A fluorescent probe was obtained by coupling streptavidin-modified time-resolved fluorescent microspheres with a 5' end biotin-modified nucleic acid probe; (2) Extract DNA from the sample to be tested; (3) The fluorescent probe from step (1), the DNA from step (2), the Cas12a nuclease and the crRNA were placed at room temperature for 10-30 min to obtain a mixture; (4) Add the mixture from step (3) to the sample pad, incubate, and quantify using an immunoassay analyzer and in conjunction with a standard curve.

8. The method according to claim 7, characterized in that, In step (1), the coupling conditions are 10-20 min at 20-27℃, the final concentration of the streptavidin-modified time-resolved fluorescent microspheres is 1-10 ng / mL, and the final concentration of the 5' end biotin-modified nucleic acid probe is 10-80 nM.

9. The method according to claim 7, characterized in that, The method is carried out under conditions of pH 6.5 to 7.

5.

10. The method according to claim 7, characterized in that, The standard curve is prepared by extracting DNA from negative solutions containing different concentrations of Salmonella or quinolone-resistant strains, mixing streptavidin-modified time-resolved fluorescent microspheres with a 5' end biotin-modified nucleic acid probe to obtain a fluorescent probe, and then adding the fluorescent probe, DNA, Cas12a nuclease, and crRNA to a sample pad after standing at room temperature for 10-30 min and incubating. Quantitative analysis is performed using an immunoassay analyzer, and the data are fitted to obtain the standard curve.

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