Duplex fluorescent pcr detection primer probe group for duck salmonella enteritidis and salmonella typhimurium, kit, and use thereof
Patent Information
- Application Number
- GB2025000087
- Authority / Receiving Office
- GB · GB
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-01-05
- Filing Date
- 2024-01-05
- Publication Date
- 2025-02-19
AI Technical Summary
Existing multiplex PCR detection methods are prone to false positive and false negative problems when detecting Salmonella enteritidis and Salmonella typhimurium, and the detection effect is poor when the content of pathogenic bacteria in the sample is low, affecting the accuracy of the results.
A dual fluorescent PCR detection primer and probe set was designed. By screening Salmonella Enteritidis lygD gene and Salmonella Typhimurium-specific gene STM4497 as targets, specific primers and probes were designed, combined with fluorescent labeling and quenching group modification, to optimize PCR. Reaction conditions to improve detection sensitivity and specificity.
It achieves fast, sensitive and specific detection results, reduces false positives and false negatives, and improves the accuracy of detection results. The sensitivity is 100 to 1000 times higher than the traditional PCR method, and is suitable for rapid identification of Salmonella in duck farms.
Abstract
Description
Primer probe set, kit and application of dual fluorescence PCR detection of Salmonella enteritidis and Salmonella typhimurium Technical Field
[0001] The present application specifically relates to a dual fluorescence PCR detection primer probe set for Salmonella enteritidis and Salmonella typhimurium, a kit and applications thereof, and belongs to the field of animal quarantine technology. Background Art
[0002] Duck salmonellosis is a general term for diseases caused by Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella anatidae. Ducks of all ages can be infected, primarily in ducklings, and can cause mass mortality. Shandong Province is a major duck-raising province. With the increasing intensification and stocking density of large-scale duck farms, Salmonella infections in ducks are steadily increasing. An outbreak in ducks can spread rapidly, posing a significant threat to the duck industry. Therefore, Salmonella contamination in ducks warrants close attention.
[0003] Salmonella Enteritidis and Salmonella Typhimurium are the most common serotypes isolated from duck farms. Different Salmonella serotypes vary in pathogenicity, host range, and target populations. Therefore, the identification of Salmonella serotypes by laboratories and clinics is crucial for disease treatment. Multiplex PCR methods offer the advantages of both rapidity and sensitivity, as well as high throughput. Its fundamental principle is to incorporate multiple pairs of amplification primers into a single reaction system to simultaneously amplify multiple pairs of specific genes. This allows for the simultaneous detection of multiple pathogenic genes, saving time and reducing costs. However, current multiplex PCR assays often encounter incompatible amplification conditions between multiple targets, and the optimal PCR conditions for each primer pair vary. Consequently, the amplification efficiency and number of amplified fragments are often unsatisfactory, leading to false positives and false negatives when the pathogenic bacteria content in the sample is low, thus affecting the final test results.
[0004] Summary of the Invention
[0005] In view of this, the present application provides a dual fluorescence PCR detection primer probe set that can simultaneously detect Salmonella enteritidis and Salmonella typhimurium. The multiplex fluorescence PCR detection technology established in this application not only inherits the advantages of multiplex PCR, but also further improves the sensitivity of detection. It can avoid false positives caused by a large amount of impurities in the sample and false negatives caused when the pathogenic bacteria content in the sample is low, further ensuring the accuracy of the test results.
[0006] The present application also provides a kit containing the above primer probe set, which provides a simple method for rapid identification of Salmonella Enteritidis and Salmonella Typhimurium. The kit of the present application has the advantages of strong specificity and high sensitivity.
[0007] Another object of the present application is to provide an application of the above primer probe set or kit.
[0008] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:
[0009] The present application provides a dual fluorescence PCR detection primer probe set for Salmonella enteritidis and Salmonella typhimurium. The nucleotide sequence of the detection primer pair for Salmonella enteritidis is: upstream primer: 5'-CTTTTGTGCAGCGAGCAT-3' (SEQ ID NO.1), downstream primer: 5'-ACAACAGGCTGATTTACTAAACC-3' (SEQ ID NO.2); the nucleotide sequence of the detection probe for Salmonella enteritidis is 5'-ACCTCTAAGCCGGTCAATGAGTT-3' (SEQ ID NO.3); the nucleotide sequence of the detection primer pair for Salmonella typhimurium is: upstream primer: 5'-ACCTGAAGTATCTGTTGCG-3' (SEQ ID NO.4); downstream primer: 5'-CAAATAACCCACGTTCAGT-3' (SEQ ID NO.5). NO.5); the nucleotide sequence of the detection probe for Salmonella typhimurium is 5'-CTTTCAGATCATCGTCGACATGC-3' (SEQ ID NO.6).
[0010] The present application screened out the enteritidis lyg D gene-specific fragment Sdf I and the typhimurium-specific gene STM4497 as identification targets by comparing and analyzing the whole genome sequences of Salmonella Enteritidis and Salmonella Typhimurium. The specific sequence of the coding region in the enteritidis-specific fragment lyg D was selected as the target region, and the enteritidis primer and probe design were selected based on the multiple sequence alignment; the specific sequence of the coding region of the typhimurium-specific gene STM4497 was selected as the target region, and the typhimurium primer and probe design were performed based on the multiple sequence alignment. The primer length is about 20 bases, the GC content is 50%-60%, there is no secondary structure and repeatability within the primer, there is no complementary sequence between primers and within the primer, and the melting temperature (Tm value) between primers differs by less than 2°C. The probe length is about 25 bases, and the Tm value is about 5°C higher than the primer Tm value.
[0011] Preferably, the detection probe is a probe modified with a fluorescent label and a quencher group. More preferably, the detection probe for duck enteritis Salmonella is modified with a FAM fluorescent label and a BHQ1 quencher group, the 5' end of the Salmonella enteritis probe sequence is labeled with a reporter fluorescent group FAM (6-carboxy-fluorescein), and the 3' end is labeled with a quencher fluorescent group BHQ1. The detection probe for duck enteritis Salmonella is 5'FAM-ACCTCTAAGCCGGTCAATGAGTT-BHQ13'; the detection probe for Salmonella typhimurium is modified with a HEX fluorescent label and a BHQ1 quencher group, the 5' end of the Salmonella typhimurium probe sequence is labeled with a reporter fluorescent group HEX (5-hexachloro-fluorescein), and the 3' end is labeled with a quencher group BHQ1. The detection probe for Salmonella typhimurium is 5'HEX-CTTTCAGATCATCGTCGACATGC-BHQ13'.
[0012] The present application also provides a use of the above primer probe set in preparing a product for detecting Salmonella enteritidis and / or Salmonella typhimurium.
[0013] In the present application, the types of products preferably include reagents or kits.
[0014] The present application also provides a kit for detecting Salmonella enteritidis and / or Salmonella typhimurium, wherein the kit comprises the above-mentioned primer probe set.
[0015] In the present application, the kit preferably also includes a fluorescent PCR reaction solution, Taq DNA polymerase, a positive control or a negative control. In the present application, the PCR reaction solution is preferably composed of 1.5 volumes of an upstream primer for Salmonella Enteritidis at a concentration of 10 μmol / L, 1.5 volumes of a downstream primer for Salmonella Enteritidis at a concentration of 10 μmol / L, and 0.5 volumes of a probe for Salmonella Enteritidis at a concentration of 10 μmol / L; 1 volume of an upstream primer for Salmonella Typhimurium at a concentration of 10 μmol / L, 1 volume of a downstream primer for Salmonella Typhimurium at a concentration of 10 μmol / L, and 0.3 volumes of a probe for Salmonella Typhimurium at a concentration of 10 μmol / L; 10 volumes of 2× buffer, 2 volumes of MgCl2 at a concentration of 25 mmol / L, 2 volumes of dNTPs at a concentration of 2.5 mmol / L, and 0.2 volumes of diethyl pyrocarbonate-treated water. The present application does not specifically limit the specific sources of the above reagents. In the kit described herein, the concentration of Taq DNA polymerase is preferably 5 U / μL, and the negative control is preferably autoclaved diethyl pyrocarbonate-treated water. The positive control is preferably a DNA fragment that identifies Salmonella Enteritidis and Salmonella Typhimurium. More preferably, the positive control identification target for Salmonella Enteritidis is the lygD gene-specific fragment SdfI, and the positive control identification target for Salmonella Typhimurium is the specific gene STM4497.
[0016] The fluorescent PCR buffer used in this application includes but is not limited to: dNTP, MgCl2, etc. The negative control used in this application: diethyl pyrocarbonate treated water: tap water is distilled twice, purified by a Millipore MILLI-Q PF PLUS water purifier, and water with a resistivity of ≥18.0 MΩ.cm is collected to obtain Millipore-Q purified water; DEPC is then added to the Millipore-Q purified water to a final concentration of 0.1%, stirred at 37°C for 12 hours, and sterilized with high-pressure steam at 15 lbf / in2 (1.034×105Pa) for 15 minutes. Preparation of positive control: The internal specific fragment SdfⅠ of the lygD gene of Salmonella Enteritidis and the specific gene STM4497 of Salmonella typhimurium are cloned by conventional methods to construct a recombinant plasmid, and the plasmid is sequenced. After the sequencing results are correct, the Escherichia coli Top10 competent cells are transformed, expanded and cultured, the plasmid is extracted, and the enzyme-cleaved and purified DNA fragment is used as a positive control.
[0017] The present application also provides a method for detecting Salmonella enteritidis and / or Salmonella typhimurium, wherein the above-mentioned primer probe set or the above-mentioned kit is used to perform a dual fluorescence quantitative PCR reaction on the test sample, and the detection result is determined.
[0018] Preferably, the procedure of the dual fluorescence quantitative PCR reaction is: first stage pre-denaturation, 50°C / 2 min; second stage 95°C / 3 min; third stage 95°C / 15 s, 60°C / 30 sec, for a total of 40 cycles; fluorescence collection is performed during the 60°C extension in each cycle of the third stage.
[0019] The present application performs fluorescence PCR reaction and then reads the results. For a multi-channel PCR instrument, the FAM (465-510) channel and HEX (VIC) (533-580) channel are used to read the results. For an ABI instrument, the FAM and HEX channels are selected and the results are read without a fluorescence quenching group. Preferably, the test results are determined as follows:
[0020] Negative: There is no Ct value in both detection channels and no characteristic amplification curve, indicating that there is no Salmonella Enteritidis and Salmonella Typhimurium in the sample;
[0021] Positive: If only the FAM detection channel shows a characteristic amplification curve with a Ct value ≤ 36, while the HEX / VIC detection channel has no Ct value and no amplification curve, it indicates that Salmonella Enteritidis is present in the sample;
[0022] If only the HEX / VIC detection channel shows a characteristic amplification curve with a Ct value ≤ 36, while the FAM detection channel has no Ct value and no amplification curve, it indicates that Salmonella Typhimurium is present in the sample;
[0023] The Ct values of both detection channels were ≤36, and a typical amplification curve appeared, indicating that the sample contained both Salmonella Enteritidis and Salmonella Typhimurium.
[0024] For the detection method provided in this application, the negative control should show no Ct value and no amplification curve when reading data using both detection channels. The positive control should show corresponding characteristic amplification curves when reading data using both detection channels, and both Ct values should be ≤32. If the negative or positive control does not meet these conditions, the experiment will be considered invalid. Samples with a Ct value greater than 36 in any channel and a typical amplification curve are recommended to be retested. If the above results are still observed after retest, the sample is considered positive; otherwise, it is considered negative.
[0025] The present application has demonstrated through experiments that the detection kit and detection method provided by the present application can confirm in one step whether Salmonella Enteritidis and Salmonella Typhimurium are present in the sample to be tested.
[0026] This application selected the internal specific fragment SdfⅠ of the lygD gene of Salmonella Enteritidis and the specific gene STM4497 of Salmonella Typhimurium as target sequences, designed primers and probes respectively, established and optimized a dual fluorescence PCR detection kit for Salmonella Enteritidis and Salmonella Typhimurium, and achieved excellent technical results:
[0027] 1) Rapid: This method monitors PCR products in real time, and results are obtained immediately after the PCR is completed. It is more time-efficient than single-plex fluorescent PCR detection methods and has the advantages of being rapid and enabling identification in one step.
[0028] 2) Sensitivity: Due to the use of specific fluorescent probes and a highly sensitive fluorescent PCR instrument, this method is 100 to 1000 times more sensitive than traditional PCR methods. Testing of plasmid DNA with a known copy number showed that the kit's sensitivity can reach 10 copies / reaction.
[0029] 3) Specificity: Since not only specific primers but also specific probes are used, the specificity of this method is higher than that of ordinary PCR methods; there is no cross-reaction with Escherichia coli, Staphylococcus, Shigella, Listeria monocytogenes, Vibrio parahaemolyticus, etc., indicating good specificity.
[0030] 4) Stability: The results of the repeatability test showed that the established method has good stability; the repeatability test was performed on plasmid DNA of different concentrations and known copy number, and the coefficient of variation was less than 5%.
[0031] 5) Not easy to be contaminated: fully enclosed reaction, no need for PCR post-processing, safe operation.
[0032] 6) The successful establishment of a dual fluorescence PCR detection method for Salmonella Enteritidis and Salmonella Typhimurium will help strengthen Salmonella detection in duck farming. This provides valuable technical guidance for controlling the disease at its source, developing effective prevention and control measures, and effectively promoting the elimination of salmonellosis, promoting the healthy and sustainable development of regional industries, and supporting rural revitalization. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG1 is a diagram showing the sensitivity test results of (Salmonella enteritidis / Salmonella typhimurium dual fluorescence PCR detection kit) in the embodiment of the present application - Salmonella typhimurium.
[0034] FIG2 is a diagram showing the sensitivity test results of (Duck Salmonella Enteritidis / Salmonella Typhimurium Dual Fluorescence PCR Detection Kit) in the embodiment of the present application - Salmonella Enteritidis.
[0035] FIG3 is a diagram showing the specificity test results of the (Dual Fluorescence PCR Detection Kit for Duck Enteritidis / Salmonella Typhimurium) in the embodiment of the present application - Salmonella Typhimurium.
[0036] FIG4 is a diagram showing the specificity test results of the (Duck Salmonella Enteritidis / Salmonella Typhimurium Dual Fluorescence PCR Detection Kit) in the embodiment of the present application - Salmonella Enteritidis. DETAILED DESCRIPTION
[0037] The technical solutions provided in this application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of this application.
[0038] In the following examples, unless otherwise specified, all methods are conventional.
[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0040] Example 1
[0041] Design of primers and probes
[0042] 1. Primer and probe design principles
[0043] In order to ensure the conservation and specificity of primers and probes, specific sequences of the coding regions of the specific genes (fragments) of Salmonella Enteritidis and Salmonella Typhimurium were selected as target regions.
[0044] Most primer designs in this field are directly synthesized by software, and the optimal primer sequence is selected. The primer sequences involved in this application are the result of manual design based on software design; the primer probe sequences involved in the embodiments can ensure that no detection is missed, and can simultaneously perform rapid identification and detection of Salmonella Enteritidis and Salmonella Typhimurium.
[0045] 2. Primer and probe design
[0046] The complete gene sequences of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella fowltyphi were downloaded from NCBI for comparison. The Salmonella typhimurium-specific gene STM4497 and the specific sequence SdfI of the lygD gene coding region of Salmonella enteritidis were found. 26 specific sequences of the STM4497 coding region of the Salmonella typhimurium-specific gene and 26 specific sequences of the lygD gene coding region of Salmonella enteritidis were downloaded. Primer probes were designed using Prime 5.0 primer design software. After preliminary screening, manual design was performed. Based on the multiple sequence alignment, specific primer probes were designed. Some Salmonella typhimurium reference strains and some Salmonella enteritidis reference strains used for primer probe design are shown in Tables 1 and 2.
[0047] Table 1 Some reference strains of Salmonella typhimurium used for primer and probe design
[0048] Table 2 Some reference strains of Salmonella Enteritidis used for primer and probe design
[0049] 3. Primer and probe design features
[0050] Primers are approximately 20 bases long, with a GC content of 50%-60%. They contain no secondary structure or repetitiveness within the primers, no complementary sequences within or between primers, and a melting temperature (Tm) difference of less than 2°C between primers. Probes are approximately 25 bases long, with a Tm approximately 5°C higher than that of the primers. The names and sequence sources of other primers and probes used for screening and comparison are shown in Table 3.
[0051] Table 3 Names and sequences of primers and probes used for screening comparison
[0052] The manually designed primer-probe set had similar TM values for both Salmonella Enteritidis and Salmonella Typhimurium, achieving consistent reaction parameters and similar sensitivity when simultaneously detecting both bacteria. BLAST analysis revealed no base mismatches. However, software-designed primers for both Salmonella Enteritidis and Salmonella Typhimurium yielded inconsistent optimal reaction parameters for dual detection. BLAST analysis revealed base mismatches, and the probe TM values were very close to the primer TM values, preventing optimal results. Therefore, the final nucleotide sequences of the primers and probes are shown in Table 4:
[0053] Table 4 Nucleotide sequences of primers and probes
[0054] Example 2
[0055] Establishment of a dual fluorescence PCR reaction system for Salmonella Enteritidis and Salmonella Typhimurium
[0056] Based on multiple sequence alignment, a primer-probe set was designed that could simultaneously and rapidly identify and detect Salmonella Enteritidis and Salmonella Typhimurium. A fluorescence PCR detection method was initially established and optimized using the Taguchi method to find the primers, probes, and Mg 2+ The optimal range of concentration was adjusted accordingly to obtain the primers, probes, and Mg 2+The optimal conditions for this detection method were determined by determining the specific concentration of the primers. The reaction system, totaling 25 μL, consisted of a fluorescent PCR reaction solution, an enzyme mixture, and a template. The fluorescent PCR reaction solution consisted of 1.5 volumes of a 10 μmol / L upstream primer for S. Enteritidis, 1.5 volumes of a 10 μmol / L downstream primer for S. Enteritidis, and 0.5 volumes of a 10 μmol / L probe for S. Enteritidis; 1 volume of a 10 μmol / L upstream primer for S. Typhimurium, 1 volume of a 10 μmol / L downstream primer for S. Typhimurium, and 0.3 volumes of a 10 μmol / L probe for S. Typhimurium; 10 volumes of 2× buffer, 2 volumes of 25 mmol / L MgCl2, 2 volumes of 2.5 mmol / L dNTPs, and 0.2 volumes of diethyl pyrocarbonate-treated water.
[0057] The amplification conditions of this detection method were finally determined as follows: first stage pre-denaturation, 50℃ / 2min; second stage 95℃ / 3min; third stage 95℃ / 15s, 60℃ / 30sec, for a total of 40 cycles; fluorescence collection was carried out during the 60℃ extension of each cycle in the third stage.
[0058] After the fluorescence PCR reaction is completed, if there is no Ct value in both detection channels and no characteristic amplification curve, it indicates that there is no Enteritidis Salmonella and Salmonella Typhimurium in the sample; if the Ct values of both detection channels are ≤32.0 and both show typical amplification curves, it indicates that there are Enteritidis Salmonella and Typhimurium in the sample; if only the FAM detection channel shows a characteristic amplification curve and the Ct value is ≤36, while the HEX / VIC detection channel has no Ct value and no amplification curve, it indicates that Salmonella Enteritidis is present in the sample; if only the HEX / VIC detection channel shows a characteristic amplification curve and the Ct value is ≤36, while the FAM detection channel has no Ct value and no amplification curve, it indicates that Salmonella Typhimurium is present in the sample; if the Ct values of both detection channels are ≤36 and a typical amplification curve appears, it indicates that the sample contains both Enteritidis and Salmonella Typhimurium.
[0059] For the detection method provided in this application, the negative control should show no Ct value and no amplification curve when reading data using both detection channels. The positive control should show corresponding characteristic amplification curves when reading data using both detection channels, and both Ct values should be ≤32. If the negative or positive control does not meet these conditions, the experiment will be considered invalid. Samples with a Ct value greater than 36 in any channel and a typical amplification curve are recommended to be retested. If the above results are still observed after retest, the sample is considered positive; otherwise, it is considered negative.
[0060] Example 3
[0061] The assembly of the Salmonella Enteritidis / Salmonella Typhimurium dual fluorescence PCR detection kit is shown in Table 5.
[0062] Table 5 Kit composition list
[0063] Example 4
[0064] Sensitivity test of dual fluorescence PCR detection kit for Salmonella enteritidis / Salmonella typhimurium
[0065] DEPC-treated water was used to detect the positive control samples of Salmonella Enteritidis and Salmonella Typhimurium (copy number 0.2×10 5 After nucleic acid was extracted from the sample, the sample was diluted to 1:10, 1:100, 1:1000, 1:10000, and 1:100000 to serve as sensitivity control samples. The sensitivity control samples of Salmonella Enteritidis and Salmonella Typhimurium (copy number of 10 copies / uL) were diluted to 1:10, 1:10, 1:10000, and 1:100000 to serve as sensitivity control samples. 4 , 10 3 , 10 2 , 10 1 , 10 0 The test results of the control sample diluted 1:10, 1:100, and 1:1000 were all positive, 1:10000 was positive, and 1:100000 was negative, indicating that the detection limit of the kit can reach 1:10000, that is, 10 1 copies / reaction, see Figures 1 and 2.
[0066] Example 5
[0067] Specificity test of dual fluorescence PCR detection kit for Salmonella enteritidis / Salmonella typhimurium
[0068] Escherichia coli, Staphylococcus, Shigella, Listeria monocytogenes, and Vibrio parahaemolyticus were detected to verify the specificity of the kit. The specific detection method was the same as in Example 2. The results showed that there was no positive cross reaction, as shown in Figures 3 and 4.
[0069] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.
Claims
1. A dual fluorescence PCR detection primer probe set for Salmonella enteritidis and Salmonella typhimurium, It is characterized in that The nucleotide sequence of the detection primer pair for duck enteritis Salmonella is shown as SEQ ID NO.1-SEQ ID NO.2, and the nucleotide sequence of the detection probe for duck enteritis Salmonella is shown as SEQ ID NO.3; the nucleotide sequence of the detection primer pair for Salmonella typhimurium is shown as SEQ ID NO.4-SEQ ID NO.5, and the nucleotide sequence of the detection probe for Salmonella typhimurium is shown as SEQ ID NO.
6.
2. The primer probe set according to claim 1, It is characterized in that The detection probe is a probe modified with a fluorescent marker and a quenching group.
3. The primer probe set according to claim 2, It is characterized in that The detection probe of duck enteritidis Salmonella is modified with a FAM fluorescent marker and a BHQ1 quenching group.
4. The primer probe set according to claim 2, It is characterized in that The detection probe for Salmonella typhimurium is modified with a HEX fluorescent marker and a BHQ1 quenching group.
5. Use of the primer probe set according to any one of claims 1 to 4 in preparing a product for detecting Salmonella enteritidis and / or Salmonella typhimurium.
6. The use according to claim 5, It is characterized in that The types of products include reagents or kits.
7. A kit for detecting Salmonella enteritidis and / or Salmonella typhimurium, It is characterized in that The kit comprises the primer-probe set according to any one of claims 1 to 4.
8. The kit according to claim 7, It is characterized in that The kit also includes a fluorescent PCR reaction solution, Taq DNA polymerase, and a positive control or a negative control.
9. The kit according to claim 8, It is characterized in that The PCR reaction solution comprises 1.5 volumes of an upstream primer of Salmonella Enteritidis with a concentration of 10 μmol / L, 1.5 volumes of a downstream primer of Salmonella Enteritidis with a concentration of 10 μmol / L, and 0.5 volumes of a probe of Salmonella Enteritidis with a concentration of 10 μmol / L; 1 volume of an upstream primer of Salmonella Typhimurium with a concentration of 10 μmol / L, 1 volume of a downstream primer of Salmonella Typhimurium with a concentration of 10 μmol / L, and 0.3 volumes of a probe of Salmonella Typhimurium with a concentration of 10 μmol / L; 10 volumes of 2× buffer, 2 volumes of 25 mmol / L MgCl 2 , 2 volumes of dNTP with a concentration of 2.5 mmol / L and 0.2 volumes of water treated with diethyl pyrocarbonate.
10. The kit according to claim 8, It is characterized in that The concentration of the Taq DNA polymerase was 5 U / μL.
11. The kit according to claim 8, It is characterized in that The negative control was autoclaved diethyl pyrocarbonate treated water.
12. The kit according to claim 8, It is characterized in that The positive control is a DNA fragment of the identification target of Salmonella Enteritidis and Salmonella Typhimurium.
13. The kit according to claim 12, It is characterized in that The identification target of the positive control Salmonella Enteritidis is the lygD gene-specific fragment SdfI.
14. The kit according to claim 12, It is characterized in that The identification target of the positive control Salmonella typhimurium is the specific gene STM4497.
15. A method for detecting Salmonella enteritidis and / or Salmonella typhimurium, It is characterized in that The primer probe set described in any one of claims 1 to 4 or the kit described in any one of claims 7 to 14 is used to perform dual fluorescence quantitative PCR reaction detection on the sample to be tested, and the detection result is determined.
16. The method according to claim 15, It is characterized in that The procedure of the dual fluorescence quantitative PCR reaction is: pre-denaturation in the first stage, 50°C / 2min; 95°C / 3min in the second stage; 95°C / 15s and 60°C / 30sec in the third stage, for a total of 40 cycles; fluorescence collection is performed during the 60°C extension in each cycle of the third stage.
17. The method according to claim 15, It is characterized in that The test result is determined as follows: Negative: There is no Ct value in both detection channels and no characteristic amplification curve, indicating that there is no Salmonella Enteritidis and Salmonella Typhimurium in the sample; Positive: If only the FAM detection channel shows a characteristic amplification curve and the Ct value is ≤36, while the HEX / VIC detection channel has no Ct value and no amplification curve, it means that Salmonella Enteritidis is present in the sample; If only the HEX / VIC detection channel shows a characteristic amplification curve with a Ct value ≤ 36, while the FAM detection channel has no Ct value and no amplification curve, it indicates that Salmonella Typhimurium is present in the sample; The Ct values of both detection channels were ≤36, and a typical amplification curve appeared, indicating that the sample contained both Salmonella Enteritidis and Salmonella Typhimurium.