Method for rapidly detecting viable salmonella and application

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a method for rapidly detecting viable salmonella and application. The method comprises the following steps: mixing a sample to be detected with Felix O-1 bacteriophage, incubating at 37 DEG C to enable the Felix O-1 bacteriophage to infect and proliferate in the sample to be detected, and performing splitting decomposition treatment after infection proliferation is finished to obtain proliferated Felix O-1 bacteriophage DNA (Deoxyribose Nucleic Acid); felix O-1 bacteriophage DNA is used as a template for real-time fluorescence RPA amplification, and whether viable salmonella exists or not is judged through fluorescence signal intensity. According to the method provided by the invention, the defects that the traditional bacterial culture and biochemical confirmation method is long in time consumption and the original molecular biological method cannot conveniently distinguish dead and live are overcome. The method is short in detection time, the RPA reaction is carried out under a constant-temperature condition, equipment requirements are simple, and the method is suitable for field detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganism detection, and particularly relates to a method for rapidly detecting Salmonella live bacteria and application thereof. BACKGROUND

[0002] Salmonella is a foodborne pathogenic bacterium widely distributed in nature. The diversity of Salmonella serotypes and the lack of specific molecular targets pose significant challenges to rapid and accurate detection. Globalization and the complexity of global food supply chains make it easy for bacteria to contaminate production, processing, and circulation, leading to an increasing risk of bacterial food poisoning. Investigations in specific regions (such as Jiangxi Province) show that raw poultry meat and raw livestock meat are the main carriers of Salmonella contamination, and Chinese salad and cooked meat products are also contaminated to varying degrees, posing a significant risk of food poisoning. Salmonellosis refers to a foodborne disease caused by Salmonella infection. The pathogenicity of Salmonella directly depends on the presence of live bacteria. However, current mainstream rapid detection techniques (such as conventional PCR and immunoassay) generally cannot effectively distinguish between dead and live bacteria. This technical limitation may lead to overestimation of the actual risk: for example, in effectively sterilized foods or environments, residual dead bacterial DNA or antigens may be misjudged as active contamination, thereby triggering unnecessary product recalls and other issues. Therefore, developing specific detection techniques for Salmonella live bacteria is not only a key scientific requirement to overcome the limitations of existing methods, but also an urgent requirement to achieve precise prevention and control of foodborne diseases, ensure food safety, economic stability, and social public health safety.

[0003] Currently, nucleic acid detection techniques are one of the commonly used methods for detecting Salmonella, such as polymerase chain reaction (PCR) technology, which has the advantages of high sensitivity and strong specificity. However, traditional nucleic acid detection techniques have a significant drawback: they cannot distinguish between live and dead bacteria in samples. The nucleic acid residues of dead bacteria can lead to false positive results, affecting the accuracy and reliability of the detection results. In practical applications, determining whether Salmonella live bacteria are present in a sample is crucial for risk assessment and the implementation of targeted prevention and control measures. Therefore, developing a method that can accurately detect Salmonella live bacteria has become a pressing problem in this field. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art. The present application uses RPA to amplify phage DNA to achieve convenient and rapid detection of Salmonella live bacteria in original samples. The present application provides a method for rapidly detecting Salmonella live bacteria and application thereof. The following technical solutions are used: In a first aspect, the present application provides a method for rapidly detecting Salmonella live bacteria for non-diagnostic purposes, comprising the following steps: The sample to be tested is mixed with Felix O-1 bacteriophage, and then incubated at 37 DEG C to allow the Felix O-1 bacteriophage to proliferate in the sample to be tested; after the proliferation, lysis treatment is performed to obtain the proliferated Felix O-1 bacteriophage DNA. The real-time fluorescent RPA amplification is performed by using the Felix O-1 bacteriophage DNA as a template, and the presence of the living Salmonella is determined by the fluorescent signal intensity.

[0005] The present application uses the characteristics that the Felix O-1 bacteriophage only proliferates in living bacteria, extracts the proliferated bacteriophage DNA after 3 hours of culture, and uses a specific fluorescent RPA system to detect the bacteriophage nucleic acid, and the process does not require a thermal cycler, solves the false positive problem caused by the residual nucleic acid of dead bacteria, and is suitable for on-site rapid detection of food, environmental and other samples.

[0006] As a further preferred embodiment, if the fluorescent signal intensity is greater than 400000 RFU, it is determined that living Salmonella exists, otherwise, it is determined that living Salmonella does not exist.

[0007] As a further preferred embodiment, the concentration of the Felix O-1 bacteriophage is 3×10 3 PFU / mL - 3×10 7 PFU / mL; the infection and proliferation time is 1 h-4 h.

[0008] As a further preferred embodiment, the concentration of the Felix O-1 bacteriophage is 3×10 5 PFU / mL; the infection and proliferation time is 3 h.

[0009] As a further preferred embodiment, the proliferated Felix O-1 bacteriophage DNA is obtained by using a magnetic bead nucleic acid extraction kit and an automatic nucleic acid extractor.

[0010] As a further preferred embodiment, the reaction system when the real-time fluorescent RPA amplification is performed includes the following: 29.4 μL buffer A, 10 μM of 0.5 μL-8 μL of the upstream primer and the downstream primer, 0.6 μL probe, 2 μL Felix O-1 bacteriophage DNA template, 1 μL-3 μL buffer B, and the rest is made up to 50 μL with double distilled water.

[0011] As a further preferred embodiment, the probe is an EXO probe.

[0012] As a further preferred embodiment, the EXO probe sequence is 5'-TATCCAGTAGGTACTATCCATCTCTCTATGAA [FAM-dT][THF]C[BHQ-dT]GCAAACCCTTCTAC-C3-Spacer-3'.

[0013] As a further preferred embodiment, the nucleic acid sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0014] In a second aspect, the application provides application of the above method for rapid detection of live Salmonella in food or environmental samples.

[0015] The application has the following beneficial effects: (1) The application uses Felix O-1 bacteriophage to specifically infect live bacteria in the sample to be tested, effectively avoiding false positive interference caused by residual nucleic acid of dead Salmonella in traditional molecular detection, and achieving high-sensitivity and high-specificity detection of live Salmonella.

[0016] (2) The detection limit of the method provided by the application is low, and the detection limit of live Salmonella is 1.58 x 10 2 CFU / mL, and the detection limit of bacteriophage nucleic acid is 0.146 fg / reaction, which can detect low-concentration live Salmonella.

[0017] (3) The method provided by the application has a short detection time, and the detection can be completed within a few hours, including bacteriophage infection and proliferation and RPA reaction, meeting the requirement of rapid detection.

[0018] (4) The application does not require a thermal cycler, and the RPA reaction is carried out under constant temperature conditions, which is simple in equipment requirement and suitable for on-site detection, especially in environments lacking laboratory infrastructure.

[0019] (5) According to the data results of the embodiments of the application, the detection method provided by the application has a positive detection rate of 100% for 37 strains of different serotypes of Salmonella and a negative detection rate of 100% for 17 strains of non-Salmonella, which has broad intra-genus inclusivity and good inter-genus specificity. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0021] Figure 1 The results of different probe concentrations and different primer additions of RPA reaction components are shown in the graph; A is the EXO probe concentration; B is the primer addition amount.

[0022] Figure 2 The results of different input amounts of Felix O-1 bacteriophage are shown in the graph.

[0023] Figure 3 The results of different infection times of Felix O-1 bacteriophage are shown in the graph.

[0024] Figure 4 The detection limit of Felix O-1 bacteriophage nucleic acid is shown.

[0025] Figure 5 The detection limit of live Salmonella is shown.

[0026] Figure 6 The results of the method are shown in the graph; A is the fluorescence RPA method based on Felix O-1 bacteriophage; B is the traditional plaque observation method.

[0027] Figure 7 The results of the method are shown in the graph; A is the fluorescence RPA method based on Felix O-1 bacteriophage; B is the traditional plaque observation method.

[0028] Figure 8 The enrichment time and detection results of the method in the actual spiked sample are shown. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] Embodiment 1 A method for rapid detection of live Salmonella based on Felix O-1 bacteriophage infection and real-time fluorescent recombinase polymerase amplification (RPA) technology, the specific process is as follows: (1) Felix O-1 phage infection of Salmonella and proliferation: Felix O-1 phage (100 μL) was incubated with 900 μL of the sample, live Salmonella, inactivated Salmonella and an equal volume of nutrient broth (NB) at 37°C for 10 minutes. Then, the mixture was incubated in a constant temperature shaker at 37°C and 180 rpm for a period of time to obtain a suspension rich in phage; the Felix O-1 phage infection of live Salmonella was the positive group, the Felix O-1 phage infection of inactivated Salmonella was the negative group, and the Felix O-1 phage infection of NB was the blank control group.

[0031] (2) Extraction of progeny phage DNA: The phage DNA was extracted using a magnetic bead nucleic acid extraction kit and an automatic nucleic acid extractor. 200 μL of the sample to be tested was added to the 2nd and 8th columns of the pre-dispensed reagent. The 1T pre-dispensed reagent was placed on the base bracket, and then the magnetic rod sleeve was inserted into the extraction instrument magnetic rod sleeve frame slot. The nucleic acid extraction process was carried out according to the following steps (as shown in Table 1). The extracted genomic DNA was stored at -20°C to avoid repeated freeze-thawing.

[0032] Table 1 Nucleic acid sample extraction procedure (3) Design and screening of RPA primers and probes: The gene sequence of the phage was derived from the NCBI GenBank database (GenBank accession number: NP944921.1), and the conserved region of the gene was determined by comparing the RPA primer and probe design software (Primer and Probe Design Tool for RPA / RAA, https: / / ezassay.com / primer). The RPA primers of the phage were designed according to the gene sequence of the conserved region, and were synthesized by Shanghai Shengong Biotechnology Co., Ltd. The upstream primer sequence was (SEQ ID NO. 1) 5'-CAGCAAGAGCAGTAAATAACTTGTACAAGGC-3', the downstream primer sequence was (SEQ ID NO. 2) 5'-GTGCTCTTCCTCTTGAAACTAACTCCCAAGT-3', and the EXO probe sequence was 5'-TATCCAGTAGGTACTATCCATCTCTCTATGAA [FAM-dT] [THF] C [BHQ-dT] GCAAACCCTTCTAC-C3-Spacer-3' (in the sequence table, SEQ ID NO. 3[FAM-dT][THF] C [BHQ-dT] SEQ ID NO. 4-C3-Spacer), wherein [FAM-dT] represents a fluorescein-modified thymine deoxyribonucleotide, [THF] represents tetrahydrofuran, [BHQ-dT] represents a (black hole quencher) modified thymine deoxyribonucleotide, and C3-Spacer represents a 3-carbon spacer.

[0033] (4) Establishment of fluorescent RPA method: The extracted phage DNA was detected by the fluorescent RPA method using the EXO probe, and the component optimization was performed according to the instructions of the Amply Future DNA isothermal rapid amplification kit (fluorescent type); the RPA reaction system (50 μL) included: 29.4 μL of buffer A, 10 μM of upstream primer and downstream primer, 4 μL of each, 0.6 μL of 10 μM probe, 2 μL of DNA template, 1.5 μL of buffer B, and double distilled water was added to 50 μL. The reaction tube was mixed and placed in the fluorescence amplification platform for amplification and light reading. The amplification program was: 37 °C for 25 minutes, and the fluorescence signal was collected every 30 seconds, and the fluorescence curve was automatically drawn. In order to objectively judge the real-time fluorescence RPA amplification curve results, the threshold was set as follows: running 12 independent no-template control experiments, establishing a negative control data set, obtaining the end-point fluorescence intensity data; calculating the mean = 130000 RFU, the standard deviation = 27000 RFU, and the threshold = RFU (this setting covers 99.99% confidence interval, ensuring that the false positive rate is <0.01%); if the end-point fluorescence intensity >400000 RFU, the result is positive, otherwise it is negative.

[0034] Example 2 A rapid detection method for Felix O-1 phage infecting Salmonella live bacteria based on real-time fluorescent recombinase polymerase amplification (RPA) technology and optimization of Felix O-1 phage infection and real-time fluorescent recombinase polymerase amplification (RPA) technology An equal volume (100 μL) of different concentrations (3×10 3 PFU / mL, 3×10 4 PFU / mL, 3×10 5 PFU / mL, 3×10 6 PFU / mL, 3×10 7PFU / mL) O-1 phage to 900 μL of live and inactivated Salmonella and equal volume of NB, respectively, and incubated at 37 °C for 10 min, then incubated in a constant temperature incubator at 37 °C and 180 rpm for 0 h, 1 h, 2 h, 3 h, and 4 h. After magnetic separation, the supernatant was taken and the DNA of the phage was extracted according to the method described in Example 1. The extracted DNA was added to the RPA reaction system according to the steps described in Example 1. The Ct values of the RPA fluorescence curves of O-1 phage with different input amounts and different infection times infecting Salmonella strains (live and inactivated) of different states (live and inactivated) were observed. The difference between the Ct values of the RPA fluorescence curves of the samples and NB was obtained to obtain the ΔCt value. The smaller the Ct value, the faster the amplification speed, and the more the target DNA content in the sample under the same conditions. The effects of the changes of each variable on the RPA fluorescence curve ΔCt value of O-1 phage infecting Salmonella strains (live and inactivated) of different states (live and inactivated) were compared to explore the ability of each variable to detect live Salmonella.

[0035] The results are shown in Figures 2-3 Felix O-1 phage added at a concentration of 3×10 5 PFU / mL (100 μL in volume) and for 3 h, live Salmonella can be effectively detected in a shorter infection time, and false positives of the negative control can be avoided.

[0036] Example 3 A detection limit verification of a method for rapidly detecting live Salmonella based on Felix O-1 phage infection and real-time fluorescent recombinase polymerase amplification (RPA) technology is as follows: Phage nucleic acid detection limit: 3×10 10 PFU / mL of Felix O-1 phage stock solution (250 μL) was used to extract phage DNA according to the method described in Example 1. After quantification and analysis of the nucleic acid, 10-fold serial dilutions (7.3×10 5 fg / mL, 7.3×10 4 fg / mL, 7.3×10 3 fg / mL, 7.3×10 2 fg / mL, 7.3×10 1 fg / mL, 7.3×10 0 fg / mL, 7.3×10 -1 fg / mL, 7.3×10 -2 fg / mL) were prepared. 2 μL of each dilution of nucleic acid was used as a template for real-time fluorescent RPA detection. By analyzing the amplification curve characteristics, the lowest detection limit (Limit of Detection, LOD) of the phage nucleic acid was determined.

[0037] Limit of detection for live Salmonella: (1.58 × 10⁻⁶) 5 CFU / mL, 1.58×10 4 CFU / mL, 1.58×10 3 CFU / mL, 1.58×10 2 CFU / mL, 1.58×10 1 Live Salmonella culture (900 μL, CFU / mL) was mixed with FelixO-1 bacteriophage (100 μL, 3×10⁻⁶ CFU / mL) separately. 5 A mixture of PFU / mL was used as the live bacteria group. The negative control group used an equal volume of heat-inactivated Salmonella culture (95 °C, 30 min) instead of the live bacteria culture, while the blank control group used an equal volume of NB instead of the live bacteria culture. All mixtures were incubated at 37 °C for 10 minutes, then transferred to a 37 °C shaker (180 rpm) for 180 minutes. After incubation, the supernatant was used to extract phage DNA as described above, and real-time fluorescence RPA detection was performed according to the aforementioned method. By analyzing the fluorescence amplification curves of samples with different live bacteria concentrations, combined with the background signal of the negative / blank controls, the LOD value of this method for live Salmonella was determined.

[0038] The result is as follows Figures 4-5 As shown, it is composed of Figure 4 The results of the phage nucleic acid detection limit show that when the nucleic acid concentration is ≥ 73 fg / mL, all repeated experiments produce amplification curves (endpoint fluorescence value > 400000). The nucleic acid amount corresponding to this concentration is 0.146 fg / reaction, that is, the LOD of RPA for phage nucleic acid is 73 fg / mL.

[0039] Depend on Figure 5 The detection limit for live Salmonella is 1.58 × 10⁻⁶. 2 At a concentration of CFU / mL live Salmonella, significant positive amplification was observed (endpoint fluorescence value > 400,000), while no amplification signal was found in the inactivated bacteria group and the NB control group. Based on this, the LOD of this method for live Salmonella was determined to be 1.58 × 10⁻⁶. 2 CFU / mL.

[0040] Example 4 A rapid detection method for Salmonella live bacteria based on Felix O-1 phage infection and real-time fluorescent recombinase polymerase amplification (RPA) technology, verifying intrageneric inclusion and intergeneric specificity, is described below: 37 Salmonella strains (10) were selected 6 The CFU / mL assay method was used to explore the inclusivity of the genus; these 37 Salmonella strains belonged to 17 serotypes, namely Salmonella Londonii (CFU / mL). Salmonella London Salmonella enteritidis ()Salmonella Enteritidis ), Salmonella Thompson ( Salmonella Thompson Salmonella Lyseneca ( Salmonella Rissen Salmonella Stanley ( Salmonella Stanley Salmonella S. Paulo ( Salmonella Saintpaul Salmonella typhimurium ( Salmonella Typhimurium Salmonella Potsdam Salmonella Potsdam ), Ohio Salmonella ( Salmonella Ohio Salmonella Newport ( Salmonella Newport Salmonella Münsterii Salmonella Münster ), Salmonella Munich Salmonella München ), Kentucky Salmonella ( Salmonella Kentucky ), Salmonella Mbandaka Salmonella Mbandaka Salmonella Livingstonis Salmonella Livingstone Salmonella infantis ( Salmonella Infantis Salmonella Indiana ( Salmonella Indiana The method provided in this invention was used for detection, while a traditional plate-based method was used for comparison. 1 mL of different serotypes of Salmonella was evenly spread onto NA plates. After the bacterial suspension stopped flowing on the plates, bacteriophage suspension was added to each plate. The plates were then incubated at 37 °C for 14 h, and the presence or absence of phage plaques was observed as a verification method. Intragenous inclusion = (Number of detected positive strains ÷ Total number of positive strains) × 100% Seventeen non-Salmonella strains were selected to investigate the intergeneric specificity of the method, including Staphylococcus aureus. Staphylococcus aureus Listeria monocytogenes ( ) Listeria monocytogenes ), Bacillus cereus ( Bacillus cereus Staphylococcus epidermidis ( Staphylococcus epidermidis Candida albicans ( Candida albicans ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Bacillus thuringiensis ( Bacillus thuringiensis Staphylococcus xylose ( Staphylococcus xylosus Shiga toxin-producing Escherichia coli (STEC) BNCC 186739 Escherichia coli BNCC 186739), Shiga toxin-producing Escherichia coli (STEC) FC 7856 ( Escherichia coli FC 7856), Shiga toxin-producing Escherichia coli (STEC) FC 7662 ( Escherichia coli FC 7662), Escherichia coli ATCC 25922 ( Escherichia coli ATCC 25922), Escherichia coli CMCC 44484 ( Escherichia coliCMCC 44484), Cronobacter sakazakii ( Cronobacter sakazakii Shigella ( Shigella spp), Listeria innocense ( Listeria innocua ) and Yersinia enterocolitica ( Yersinia enterocolitica 17 non-Salmonella strains, including 10 6 The concentration of CFU / mL was detected using the method provided in this invention, with live Salmonella bacteria serving as a positive control. Intergeneric specificity = (number of detected non-positive strains ÷ total number of non-positive strains) × 100%.

[0041] The result is as follows Figures 6-7 As shown, and by Figure 6 The results of the genus inclusion test showed that all 37 different serotypes of Salmonella were positive for RPA, and the method provided by this invention has an genus inclusion rate of up to 100% (37 / 37).

[0042] Depend on Figure 6 As shown in the result B, Salmonella strains with serotypes such as Salmonella São Paulo (SM-2024-33D) and Salmonella Kentucky (SM-2024-289D) tested positive for RPA using the method of this invention. In contrast, no visible plaques were found on traditional plaque assay plates. This indicates that the method provided by this invention has higher sensitivity and can detect Salmonella more comprehensively compared to traditional methods.

[0043] Depend on Figure 7 Results of intergeneric specificity showed that 17 non-Salmonella strains (10) were selected. 6 The CFU / mL result and the RPA and plaque results for the strain were both negative, indicating that the intergeneric specificity of this detection method is as high as 100% (17 / 17).

[0044] Example 5 A rapid detection capability validation method for live Salmonella bacteria in actual spiked samples based on Felix O-1 phage infection and real-time fluorescent recombinase polymerase amplification (RPA) technology is described below: Salmonella was spiked onto 25 mL samples of commercially available milk to prepare low-concentration spiked samples with Salmonella concentrations of 4 CFU / 25 mL, 40 CFU / 25 mL, 75 CFU / 25 mL, and 750 CFU / 25 mL. Enrichment was performed in a shaker at 37±1 ℃ and 200 rpm. During enrichment, 2 mL of culture was aseptically collected at specified time points (1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). 900 μL of each collected culture was then added to 3×10⁻⁶ CFU / mL of [a solution / concentration]. 5Felix O-1 phage (100 μL) at 108PFU / ml, the mixture was incubated at 37 ℃ for 10 min, and then transferred to a shaking bed (37±1 ℃, 200 rpm) for 3 h. After the end of the infection, the phage DNA was extracted and detected by real-time fluorescent RPA. According to the fluorescence curve, the positive and negative samples under different enrichment times were determined, and the enrichment time was finally determined. In addition, 1 mL of culture was used for Salmonella chromogenic medium for Salmonella colony counting.

[0045] The experimental results are shown in Figure 8 Table 1, and the positive samples with Salmonella concentrations of 4 CFU / 25 mL, 40 CFU / 25 mL, and 75 CFU / 25 mL were detected after only 6 h of enrichment, and the positive sample with a Salmonella concentration of 750 CFU / 25 mL was detected after 5 h of enrichment. This indicates that the method has strong resistance to interference and a low detection limit for actual food samples. The method can detect 10 0 CFU / 25 mL of ultra-low amount of spiked food samples within a single day.

[0046] The embodiments of the present application are described above in combination with the drawings, and the principles and implementation modes of the present application are described herein. The above description of the embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above specific implementation modes. The above specific implementation modes are only illustrative and not restrictive. Those skilled in the art can make many forms under the inspiration of the present application without departing from the scope of the present application and the protection scope of the claims.

Claims

1. A method for rapid detection of live Salmonella bacteria for non-diagnostic purposes, characterized in that, Includes the following steps: The sample to be tested was mixed with Felix O-1 phage, and then incubated at 37°C to allow Felix O-1 phage to infect and proliferate in the sample. After the infection and proliferation were completed, the sample was lysed to obtain the proliferated Felix O-1 phage DNA. Real-time fluorescent RPA amplification was performed using Felix O-1 phage DNA as a template, and the presence of live Salmonella bacteria was determined by the intensity of the fluorescence signal.

2. The method according to claim 1, characterized in that, If the fluorescence signal intensity is greater than 400,000 RFU, then live Salmonella bacteria are considered to be present; otherwise, live Salmonella bacteria are not present.

3. The method according to claim 1, characterized in that, The concentration of the Felix O-1 bacteriophage was 3 × 10⁻⁶. 3 PFU / mL - 3×10 7 PFU / mL; infection and proliferation time is 1 h-4 h.

4. The method according to claim 3, characterized in that, The concentration of the Felix O-1 bacteriophage was 3 × 10⁻⁶. 5 PFU / mL; infection and proliferation time was 3 h.

5. The method according to claim 1, characterized in that, The DNA of the proliferated Felix O-1 phage was obtained using a magnetic bead nucleic acid extraction kit and an automated nucleic acid extractor.

6. The method according to claim 1, characterized in that, The reaction system for real-time fluorescence RPA amplification includes the following: 29.4 μL Buffer A, 0.5 μL-8 μL each of 10 μM upstream and downstream primers, 0.6 μL probe, 2 μL Felix O-1 phage DNA template, 1 μL-3 μL Buffer B, and bring the total volume to 50 μL with double-distilled water.

7. The method according to claim 6, characterized in that, The probe is an EXO probe.

8. The method according to claim 7, characterized in that, The EXO probe sequence is 5′-TATCCAGTAGGTACTATCCATCTCTCTATGAA [FAM-dT][THF]C[BHQ-dT]GCAAACCCTTCTAC-C3-Spacer-3′.

9. The method according to claim 6, characterized in that, The nucleic acid sequences of the upstream and downstream primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

10. The application of the method according to any one of claims 1-9 in the detection of live Salmonella in food or environmental samples.

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