Molecular detection method of salmonella and application thereof

Abstract

The invention relates to the technical field of salmonella detection, in particular to a salmonella molecular detection method and application thereof, and the salmonella molecular detection method comprises the following steps: S1, sample treatment: extracting DNA in a to-be-detected sample; step S2, primer design and synthesis: specific primers are designed for the invA gene, the hilA gene and the stn gene of salmonella; s3, multiple PCR reaction: taking the DNA extracted in the step S1 as a template, and carrying out multiple PCR amplification by adopting the primer in the step S2; s4, fluorescent probe hybridization: hybridizing a PCR amplification product in the step S3 with a fluorescent labeled probe; s5, fluorescence signal detection: detecting a fluorescence signal after hybridization through a fluorescence detector, and judging whether the sample contains salmonella or not according to the signal. According to the salmonella molecular detection method provided by the invention, a technology of combining multiple PCR with fluorescent probe hybridization is adopted, a plurality of conserved genes of salmonella can be detected at the same time, and the detection specificity and sensitivity are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of Salmonella detection, in particular to a molecular detection method for Salmonella and application thereof. Background Art

[0002] Salmonella is a common Gram-negative enteric pathogen that is widely present in nature. It can infect humans and animals by contaminating food and water sources, causing symptoms such as fever, diarrhea, and abdominal pain. In severe cases, it can even lead to complications such as sepsis and meningitis, posing a serious threat to public health and food safety.

[0003] Currently, the main methods for detecting Salmonella include traditional culture methods, immunological methods, and molecular biological methods. Traditional culture methods are the gold standard for Salmonella detection, but their detection cycle is long, taking 4-7 days from sample processing to obtaining results, and they are easily affected by contamination from other bacteria, resulting in low sensitivity. Immunological methods such as enzyme-linked immunosorbent assay (ELISA) have the advantages of simple operation and fast detection speed, but this method relies on the specificity and sensitivity of antibodies, is prone to false positive or false negative results, and cannot accurately type and identify Salmonella. Molecular biological methods such as polymerase chain reaction (PCR) have the advantages of high sensitivity, strong specificity, and fast detection speed, but traditional PCR methods can only detect a single target gene and cannot detect multiple Salmonella serotypes simultaneously. They also require subsequent operations such as electrophoresis, which are relatively cumbersome to operate and difficult to achieve high-throughput detection.

[0004] In view of the above problems, we propose a molecular detection method for Salmonella and its application. Summary of the Invention

[0005] The object of the present invention is to provide a molecular detection method for Salmonella and its application to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A molecular detection method for Salmonella comprises the following steps:

[0008] Step S1, sample processing: extracting DNA from the sample to be tested;

[0009] Step S2, primer design and synthesis: Design specific primers for the invA gene, hi 1A gene, and stn gene of Salmonella. The primer sequences are as follows:

[0010] invA upstream primer: 5'-GCTACGTCGACGATGATC-3'

[0011] i nvA downstream primer: 5'-CGATCGATCGATCGATC-3'

[0012] hi lA upstream primer: 5'-ATGGTGATGGTGATGGTG-3'

[0013] Hi lA downstream primer: 5'-CTACGTACGTACGTACGT-3'

[0014] stn upstream primer: 5'-GAGAGAGAGAGAGAGAG-3'

[0015] stn downstream primer: 5′-CTCTCTCTCTCTCTCTCT-3′;

[0016] Step S3, multiplex PCR reaction: using the DNA extracted in step S1 as a template and the primers in step S2 to perform multiplex PCR amplification;

[0017] Step S4, fluorescent probe hybridization: hybridize the PCR amplification product of step S3 with a fluorescent labeled probe, the probe sequence is as follows:

[0018] i nvA probe: 5'-FAM-GCTACGTCGACGATGATC-TAMRA-3'

[0019] Hi 1A probe: 5'-FAM-ATGGTGATGGTGATGGTG-TAMRA-3'

[0020] stn probe: 5′-FAM-GAGAGAGAGAGAGAGAG-TAMRA-3′;

[0021] Step S5, fluorescence signal detection: the fluorescence signal after hybridization is detected by a fluorescence detector, and whether the sample contains Salmonella is determined based on the signal.

[0022] Preferably, the sample in step S1 includes a food sample, a clinical sample or an environmental sample; and the DNA extraction method is a magnetic bead method, a column extraction method or a kit method.

[0023] Preferably, the multiplex PCR reaction system in step S3 is: 10 μL of 2×PCR buffer, 2 μL of dNTPs, 1 μL of each pair of primers, 0.5 μL of TaqDNA polymerase, 5 μL of template DNA, and ddH2O added to 20 μL;

[0024] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s, for a total of 35 cycles, and a final extension at 72°C for 10 min.

[0025] Preferably, the hybridization reaction in step S4 is carried out in a hybridization buffer containing 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA, and the reaction conditions are incubation at 60° C. for 30 min.

[0026] Preferably, the fluorescence signal judgment standard in step S5 is: when the fluorescence signals of the three target genes invA, hilA and stn are detected simultaneously, the sample is judged to contain Salmonella; when only 1-2 target gene signals are detected, further sequencing verification is required; when no signal is detected, it is judged that no Salmonella is present.

[0027] A primer combination for detecting Salmonella comprises three pairs of specific primers targeting invA gene, hilA gene and stn gene, wherein the primer sequences are as described above.

[0028] A fluorescent labeled probe combination for detecting Salmonella includes fluorescent probes targeting invA gene, hilA gene and stn gene. The probe sequences are as described above, and the 5' end is labeled with a FAM fluorescent group and the 3' end is labeled with a TAMRA quenching group.

[0029] Preferably, the above detection method is used for detecting Salmonella in food, clinical samples or environmental samples.

[0030] Preferably, the food sample includes livestock and poultry meat, aquatic products, dairy products or ready-to-eat food;

[0031] The clinical samples include feces, blood or body fluids; the environmental samples include water samples, soil or swabs from the surface of food processing utensils.

[0032] A Salmonella detection kit comprises the primer combination, the probe combination, a PCR buffer, dNTPs, TaqDNA polymerase and a hybridization buffer.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The molecular detection method for Salmonella provided by the present invention uses multiplex PCR combined with fluorescent probe hybridization technology to simultaneously detect multiple conserved genes of Salmonella, thereby improving the specificity and sensitivity of the detection. This method has a short detection cycle, requiring only 4-6 hours from sample processing to obtaining results, greatly reducing the detection time. At the same time, the method is simple to operate, eliminating the need for cumbersome subsequent operations such as electrophoresis, enabling high-throughput detection and suitable for large-scale Salmonella screening and detection. In addition, the detection method of the present invention can also be applied to the detection of Salmonella in a variety of samples such as food, clinical samples, and environmental samples, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a structural schematic diagram of the present invention. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Example 1:

[0038] A molecular detection method for Salmonella, characterized by comprising the following steps:

[0039] Step S1, sample processing: extracting DNA from the sample to be tested;

[0040] Step S2, primer design and synthesis: Design specific primers for the invA gene, hi 1A gene, and stn gene of Salmonella. The primer sequences are as follows:

[0041] invA upstream primer: 5'-GCTACGTCGACGATGATC-3'

[0042] invA downstream primer: 5'-CGATCGATCGATCGATC-3'

[0043] hi lA upstream primer: 5'-ATGGTGATGGTGATGGTG-3'

[0044] Hi lA downstream primer: 5'-CTACGTACGTACGTACGT-3'

[0045] stn upstream primer: 5'-GAGAGAGAGAGAGAGAG-3'

[0046] stn downstream primer: 5′-CTCTCTCTCTCTCTCTCT-3′;

[0047] Step S3, multiplex PCR reaction: using the DNA extracted in step S1 as a template and the primers in step S2 to perform multiplex PCR amplification;

[0048] Step S4, fluorescent probe hybridization: hybridize the PCR amplification product of step S3 with a fluorescent labeled probe, the probe sequence is as follows:

[0049] invA probe: 5'-FAM-GCTACGTCGACGATGATC-TAMRA-3'

[0050] Hi 1A probe: 5'-FAM-ATGGTGATGGTGATGGTG-TAMRA-3'

[0051] stn probe: 5′-FAM-GAGAGAGAGAGAGAGAG-TAMRA-3′;

[0052] Step S5, fluorescence signal detection: the fluorescence signal after hybridization is detected by a fluorescence detector, and whether the sample contains Salmonella is determined based on the signal.

[0053] Example 2:

[0054] The method for detecting Salmonella in food samples includes the following steps:

[0055] Sample preparation: Take 25g of food sample (e.g., chicken), add 225mL of saline, and homogenize for 1 minute. Take 1mL of the homogenate and extract DNA according to the instructions of the magnetic bead-based nucleic acid extraction kit to obtain 50μL of DNA solution.

[0056] Primer design and synthesis: According to the above primer sequences, a biotechnology company was commissioned to synthesize specific primers for the invA, hilA, and stn genes at a primer concentration of 10 μM.

[0057] Multiplex PCR reaction: In a 20 μL reaction system, add 10 μL of 2× PCR buffer, 2 μL of dNTPs (2.5 mM), 1 μL of each primer pair (10 μM), 0.5 μL of Taq DNA polymerase (5 U / μL), 5 μL of template DNA, and make up to 20 μL with ddH2O;

[0058] The reaction system was placed in a PCR instrument and pre-denatured at 95°C for 5 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s.

[0059] Finally, the amplification was performed under the condition of 72℃ extension for 10min. After the amplification was completed, 5μL of the amplified product was taken for agarose gel electrophoresis detection. The appearance of specific bands at the corresponding positions indicated that the amplification was successful.

[0060] Fluorescent probe hybridization: Hybridize the amplified product with a fluorescently labeled probe at a 10 μM probe concentration in a hybridization buffer containing 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA. Add 10 μL of amplified product, 1 μL of each probe, and 8 μL of hybridization buffer to a 20 μL hybridization system, mix well, and incubate at 60°C for 30 minutes.

[0061] Fluorescence signal detection: A fluorescence detector was used to detect the fluorescence signal after hybridization, with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The fluorescence signals corresponding to the three target genes, invA, hi lA, and stn, were detected, indicating that the food sample contained Salmonella.

[0062] Example 3:

[0063] The method for detecting Salmonella in clinical stool samples includes the following steps:

[0064] Sample preparation: Take 0.5 g of stool sample, add 4.5 mL of normal saline, mix well, centrifuge, and collect the supernatant. Extract DNA according to the instructions of the column-based nucleic acid extraction kit to obtain 50 μL of DNA solution.

[0065] Primer design and synthesis, multiplex PCR reaction, fluorescent probe hybridization, and fluorescent signal detection: The procedures were the same as in Example 1. Fluorescent signals corresponding to the invA and hilA target genes were detected, but no fluorescent signal was detected for the stn target gene. Since fluorescent signals were only detected for these two target genes, further verification is required. Sequencing analysis confirmed the presence of Salmonella in the sample. The lack of detection of the stn gene may be due to a mutation in the stn gene of this strain.

[0066] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A molecular detection method for Salmonella, characterized in that: The following steps are involved: Step S1, sample processing: extracting DNA from the sample to be tested; Step S2, primer design and synthesis: Design specific primers for the invA gene, hi 1A gene, and stn gene of Salmonella. The primer sequences are as follows: invA upstream primer: 5'-GCTACGTCGACGATGATC-3' invA downstream primer: 5'-CGATCGATCGATCGATC-3' hi lA upstream primer: 5'-ATGGTGATGGTGATGGTG-3' Hi lA downstream primer: 5'-CTACGTACGTACGTACGT-3' stn upstream primer: 5'-GAGAGAGAGAGAGAGAG-3' stn downstream primer: 5′-CTCTCTCTCTCTCTCTCT-3′; Step S3, multiplex PCR reaction: using the DNA extracted in step S1 as a template and the primers in step S2 to perform multiplex PCR amplification; Step S4, fluorescent probe hybridization: hybridize the PCR amplification product of step S3 with a fluorescent labeled probe, the probe sequence is as follows: invA probe: 5'-FAM-GCTACGTCGACGATGATC-TAMRA-3' Hi 1A probe: 5'-FAM-ATGGTGATGGTGATGGTG-TAMRA-3' stn probe: 5′-FAM-GAGAGAGAGAGAGAGAG-TAMRA-3′; Step S5, fluorescence signal detection: the fluorescence signal after hybridization is detected by a fluorescence detector, and whether the sample contains Salmonella is determined based on the signal.

2. The molecular detection method for Salmonella according to claim 1, characterized in that: The sample in step S1 includes a food sample, a clinical sample or an environmental sample; the DNA extraction method is a magnetic bead method, a column extraction method or a kit method.

3. The molecular detection method for Salmonella according to claim 1, characterized in that: The multiplex PCR reaction system in step S3 is as follows: 10 μL of 2×PCR buffer, 2 μL of dNTPs, 1 μL of each primer pair, 0.5 μL of TaqDNA polymerase, 5 μL of template DNA, and ddH2O added to 20 μL; The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s, for a total of 35 cycles, and a final extension at 72°C for 10 min.

4. The molecular detection method for Salmonella according to claim 1, characterized in that: The hybridization reaction in step S4 is carried out in a hybridization buffer containing 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA, and the reaction conditions are incubation at 60° C. for 30 min.

5. The molecular detection method for Salmonella according to claim 1, characterized in that: The fluorescent signal determination criteria in step S5 are: when the fluorescent signals of the three target genes, invA, hilA, and stn, are detected simultaneously, the sample is determined to contain Salmonella; when only 1-2 target gene signals are detected, further sequencing verification is required; when no signal is detected, the sample is determined to contain no Salmonella.

6. A primer combination for detecting Salmonella, characterized in that: It comprises three pairs of specific primers for invA gene, hilA gene and stn gene, and the primer sequences are as described in claim 1.

7. A fluorescent labeled probe combination for Salmonella detection, characterized in that: The invention comprises fluorescent probes for invA gene, hilA gene and stn gene, wherein the probe sequence is as described in claim 1, and the 5' end is labeled with a FAM fluorescent group and the 3' end is labeled with a TAMRA quenching group.

8. Use of the detection method according to any one of claims 1 to 5 for detecting Salmonella in food, clinical samples or environmental samples.

9. The use according to claim 8, characterized in that The food samples include livestock and poultry meat, aquatic products, dairy products or ready-to-eat foods; The clinical samples include feces, blood or body fluids; the environmental samples include water samples, soil or swabs from the surface of food processing utensils.

10. A Salmonella detection kit, characterized in that: The method comprises the primer combination according to claim 6, the probe combination according to claim 7, a PCR buffer, dNTPs, TaqDNA polymerase and a hybridization buffer.

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