A five - fold fluorescence PCR detection reagent simultaneously for Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum and its application

Abstract

The present invention belongs to the field of biotechnology and relates to a five - plex fluorescence PCR detection reagent for Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum and its application. By using the five - plex fluorescence PCR detection reagent of the present invention, the purpose of simultaneously identifying and detecting Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum can be achieved. It has the advantages of simple operation, rapidity, high accuracy, high sensitivity and good repeatability, and is of great significance for the rapid and accurate detection of clinical animal salmonellosis and epidemic disease prevention and control.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a five - multiplex fluorescence PCR detection reagent for Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum and its application. Background Art

[0002] Salmonella is an important zoonotic pathogen and also an important food - borne pathogen, which can cause infections and diseases in humans and various animals, seriously affecting the healthy development of the aquaculture industry and public health safety. There are numerous Salmonella serotypes, and the antigenicity and pathogenicity of different serotype Salmonella are different, with different susceptibilities to humans and animals, and many serotype strains can cross - infect between humans and animals. Salmonella can also infect humans through contaminated livestock and poultry products such as meat, eggs, and milk, causing food poisoning. Among them, contaminated chicken and eggs are the main sources of Salmonella transmission, playing an important role in the spread of Salmonella and food contamination.

[0003] Salmonellosis is widely prevalent in the poultry industry in China. Salmonella infection can cause various acute or chronic diseases in poultry. The Salmonella serotypes prevalent in different regions are not exactly the same, and there are obvious differences in pathogenicity. Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum are the four main Salmonella serotypes causing avian salmonellosis. Once poultry is infected with Salmonella, it will cause growth restriction at least, and at most, the egg production rate, fertilization rate, hatching rate will decrease, and a large number of chicks will die after hatching, bringing serious losses to the poultry breeding industry.

[0004] Currently, the detection technologies for Salmonella mainly include bacterial isolation and culture and biochemical identification, immunological technologies, molecular biological technologies, etc. Traditional detection methods such as bacterial isolation and culture are time - consuming and laborious, with cumbersome operations and low detection efficiency, unable to meet the requirements of rapid detection; immunological technologies have low sensitivity and specificity, and it is difficult to distinguish some serotypes or biotypes of Salmonella; PCR technology has the advantages of strong specificity, high sensitivity, simplicity, and rapidity, and has been widely used in the diagnosis of multiple fields and various diseases. Fluorescence PCR technology has the characteristics of high accuracy and good reproducibility. Compared with conventional PCR, fluorescence PCR has a higher degree of automation, higher specificity and sensitivity, and rapid and efficient detection. Multiplex fluorescence PCR technology has more unique advantages in pathogen detection. It can simultaneously detect multiple pathogens and serotypes, and can accurately identify bacteria of different species and serotypes, especially suitable for rapid detection of multi - pathogen identification.

[0005] At present, the PCR technology for detecting Salmonella mainly focuses on the detection of a single pathogen, and most of them are conventional PCR. There are few methods for simultaneously detecting different serotypes of Salmonella, and there is no application of multiplex fluorescence PCR method that can simultaneously identify and detect Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum in clinical practice. Therefore, in order to solve the problem of difficult differential detection of salmonellosis caused by the limitations of traditional pathogen detection methods and control the occurrence and spread of salmonellosis, establishing a multiplex fluorescence PCR detection method that can simultaneously identify and detect Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum is of great significance for improving the monitoring ability of animal diseases and preventing public health risks. Summary of the Invention

[0006] On the one hand, the present invention provides a reagent, including primer-probe sets 1-5 for five-fold fluorescence PCR detection, including:

[0007] The primer-probe combination 1 for detecting the invA gene, including:

[0008] The primer pair shown in SEQ ID NO: 1 and 2 and the probe shown in SEQ ID NO: 3;

[0009] The primer-probe combination 2 for detecting the STM4495 gene, including:

[0010] The primer pair shown in SEQ ID NO: 4 and 5 and the probe shown in SEQ ID NO: 6;

[0011] The primer-probe combination 3 for detecting the Sdf I gene, including:

[0012] The primer pair shown in SEQ ID NO: 7 and 8 and the probe shown in SEQ ID NO: 9;

[0013] The primer-probe combination 4 for detecting the speC gene, including:

[0014] The primer pair shown in SEQ ID NO: 10 and 11 and the probe shown in SEQ ID NO: 12; and

[0015] The primer-probe combination 5 for detecting the glgC gene, including:

[0016] The primer pair shown in SEQ ID NO: 13 and 14 and the probe shown in SEQ ID NO: 15.

[0017] In some embodiments, both ends of the probe are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups labeled between the probes are different.

[0018] In some embodiments, the fluorophore is at least one of FAM, NED, Hex, VIC, TAMRA, ROX, Texas-Red, and CY5; and / or, the quencher is at least one of TAMRA, MGB, BHQ1, BHQ2, and BHQ3.

[0019] On the one hand, the present invention provides the use of the reagent, which is any one of the following a1-a10:

[0020] a1. Identifying the invA gene, STM4495 gene, Sdf I gene, speC gene, and / or glgC gene;

[0021] a2. Preparing a product for identifying the invA gene, STM4495 gene, SdfI gene, speC gene, and / or glgC gene;

[0022] a3. Identifying whether a test bacterium has the invA gene, STM4495 gene, Sdf I gene, speC gene, and / or glgC gene;

[0023] a4. Preparing a product for identifying whether a test bacterium has the invA gene, STM4495 gene, Sdf I gene, speC gene, and / or glgC gene;

[0024] a5. Identifying whether a test bacterium is Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and / or Salmonella gallinarum;

[0025] a6. Preparing a product for identifying whether a test bacterium is Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and / or Salmonella gallinarum;

[0026] a7. Identifying whether there are bacteria with the invA gene, STM4495 gene, Sdf I gene, speC gene, and / or glgC gene in a test sample;

[0027] a8. Preparing a product for identifying whether there are bacteria with the invA gene, STM4495 gene, SdfI gene, speC gene, and / or glgC gene in a test sample;

[0028] a9. Identifying whether there are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and / or Salmonella gallinarum in a test sample;

[0029] a10. Preparing a product for identifying whether there are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and / or Salmonella gallinarum in a test sample.

[0030] On the one hand, the present invention provides a kit containing the reagent, and the use of the kit is any one of the following b1 - b5:

[0031] b1. Identifying invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene;

[0032] b2. Identifying whether the bacteria to be tested have invA gene, STM4495 gene, SdfI gene, speC gene and / or glgC gene;

[0033] b3. Identifying whether the bacteria to be tested are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and / or Salmonella gallinarum;

[0034] b4. Identifying whether there are bacteria with invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene in the sample to be tested;

[0035] b5. Identifying whether there are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and / or Salmonella gallinarum in the sample to be tested.

[0036] On the one hand, the present invention provides the application of the described kit, which is any one of the following c1 - c5:

[0037] c1. Identifying invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene;

[0038] c2. Identifying whether the bacteria to be tested have invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene;

[0039] c3. Identifying whether the bacteria to be tested are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and / or Salmonella gallinarum;

[0040] c4. Identifying whether there are bacteria with invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene in the sample to be tested;

[0041] c5. Identifying whether there are Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and / or Salmonella gallinarum in the sample to be tested.

[0042] On the one hand, the present invention provides the application of the reagent in the preparation of a kit for multiplex fluorescence PCR detection of Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum.

[0043] On the one hand, the present invention provides a multiplex fluorescence PCR detection kit for Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum, including the reagents described above.

[0044] In some embodiments, the application is for the diagnosis and treatment of non-diseases.

[0045] On the one hand, the present invention provides a multiplex fluorescence PCR detection method for detecting a sample to be tested, including the following steps:

[0046] Using the nucleic acid of the sample to be tested as a template, contacting the nucleic acid of the sample to be tested with the reagents described above, or the kit, and making the following judgments after the fluorescence PCR reaction:

[0047] If only the invA gene is detected in the sample to be tested, the sample to be tested is Salmonella, or Salmonella exists in the sample to be tested;

[0048] If both the invA gene and the STM4495 gene are detected in the sample to be tested, the sample to be tested is Salmonella typhimurium, or Salmonella typhimurium exists in the sample to be tested;

[0049] If both the invA gene and the Sdf I gene are detected in the sample to be tested, the sample to be tested is Salmonella enteritidis, or Salmonella enteritidis exists in the sample to be tested;

[0050] If the invA gene, the speC gene and the glgC gene are detected simultaneously in the sample to be tested, the sample to be tested is Salmonella gallinarum, or Salmonella gallinarum exists in the sample to be tested;

[0051] If both the invA gene and the speC gene are detected in the sample to be tested and the glgC gene cannot be detected, the sample to be tested is Salmonella pullorum, or Salmonella pullorum exists in the sample to be tested.

[0052] In some embodiments, the method is for the diagnosis and treatment of non-diseases.

[0053] In some embodiments, the "sample" or "sample to be tested" may include clinical samples, isolated nucleic acids, or isolated microorganisms. In some embodiments, the "sample" or "sample to be tested" may be a segment of isolated nucleic acid, an isolated bacterium to be tested, a culture of a strain, an extract of a strain culture, or the untreated original sample itself.

[0054] On the one hand, a detection system for identifying whether a sample to be tested is infected with Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and / or Salmonella gallinarum according to the present invention, characterized in that the detection system includes the following components:

[0055] 1) Detection components for invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene;

[0056] 2) Data processing components;

[0057] 3) Result output components;

[0058] The detection components for invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene contain the said reagents and / or the said kits.

[0059] In some embodiments, the detection objects of the detection components are fluorescence signals, "S"-shaped amplification curves and / or Ct values.

[0060] In some embodiments, the data processing components are configured to:

[0061] According to whether the invA gene, STM4495 gene, Sdf I gene, speC gene and / or glgC gene in the test sample detected by the detection components are detected or not, determine whether the test sample contains Salmonella and / or different serotypes of Salmonella;

[0062] The judgment criteria of the data processing components are:

[0063] If only the invA gene is detected in the test sample, the test sample contains or is suspected to contain Salmonella;

[0064] If both the invA gene and the STM4495 gene are detected in the test sample, the test sample contains or is suspected to contain Salmonella typhimurium;

[0065] If both the invA gene and the Sdf I gene are detected in the test sample, the test sample contains or is suspected to contain Salmonella enteritidis;

[0066] If the invA gene, speC gene and glgC gene are detected simultaneously in the test sample, the test sample contains or is suspected to contain Salmonella gallinarum;

[0067] If both the invA gene and the speC gene are detected in the test sample and the glgC gene cannot be detected, the test sample contains or is suspected to contain Salmonella pullorum.

[0068] In some embodiments, if the Ct value of any gene in the sample to be tested satisfies 38 < Ct value ≤ 40 and a specific amplification curve appears, it is judged as suspicious, and nucleic acid needs to be re-extracted and tested again. If the repeated test results meet the above nucleic acid positive judgment criteria, it is judged as positive for the corresponding target bacterium nucleic acid; if the repeated test results show that the Ct value of the gene corresponding to the target bacterium to be tested > 40 or there is no Ct value, it is judged as negative for the corresponding nucleic acid; if the Ct value of any one of the repeated test genes satisfies 38 < Ct value ≤ 40, it is judged as suspicious and further analysis needs to be performed by other methods such as sequencing.

[0069] The five - fold fluorescence PCR detection reagent of the present invention can be used to simultaneously identify and detect Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and Salmonella gallinarum, and has the advantages of simple operation, rapidity, high accuracy, high sensitivity, and good repeatability, which is of great significance for the rapid and accurate detection and epidemic disease prevention and control of animal salmonellosis.

[0070] Compared with the prior art, the advantages of the present invention are as follows: The five - fold fluorescence PCR detection reagent and its application provided by the present invention can simultaneously complete the identification and detection of Salmonella genus and four serotypes of Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and Salmonella gallinarum in one fluorescence PCR amplification reaction, and have the advantages of simple operation, rapidity, specificity, sensitivity, stability, etc., overcoming the problems of single existing detection method, time - consuming and laborious, poor specificity, and low sensitivity, and improving the accuracy and efficiency of salmonellosis detection. Brief Description of the Drawings

[0071] Figure 1 It is the five - fold fluorescence PCR amplification diagram of the target Salmonella in Example 1.

[0072] Figure 2 It is the amplification diagram of the non - Salmonella specificity test of the five - fold fluorescence PCR in Example 2.

[0073] Figure 3 It is the amplification diagram of the non - target Salmonella specificity test of the five - fold fluorescence PCR in Example 3.

[0074] Figure 4 is the amplification diagram of the five - fold fluorescence PCR sensitivity test in Example 4, where: Figure 4a : invA; Figure 4b : STM4495; Figure 4c : SdfⅠ; Figure 4d : speC; Figure 4e : glgC.

[0075] Figure 5 is the standard curve diagram of the five - fold fluorescence PCR sensitivity test in Example 4, where: Figure 5a : invA; Figure 5b : STM4495; Figure 5c : SdfⅠ;Figure 5d : speC; Figure 5e : glgC.

[0076] Figure 6 It is the amplification diagram of the quintuple fluorescence PCR repeatability test in Example 5. Detailed implementation manners

[0077] The technical solutions of the present invention will be further described below through specific embodiments. The specific embodiments do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0078] Unless clearly stated otherwise in the context, the singular forms "a", "an", "the" used in the specification and claims include their plural forms.

[0079] The term "comprising" is used in this specification or claims and is intended to include additional elements or steps in a manner similar to the way the term "including" is interpreted when used as a transitional word in the claims. In addition, with respect to the use of the term "or" (e.g., A or B), it is intended to mean "A or B or both". When it is intended to mean "only A or B but not both", the term "only A or B but not both" is used. Therefore, the use of the term "or" herein is inclusive rather than exclusive. When the terms "and" and "or" are used together, as in "A and / or B", this indicates A or B and A and B.

[0080] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0081] The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0082] In the following examples, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0083] General experimental materials and methods

[0084] 1 Materials

[0085] 1.1 Strains

[0086] Salmonella enteritidis DK6, Salmonella typhimurium ATCC 14028, Salmonella pullorum CVCC 526, Salmonella gallinarum ATCC 9184, Salmonella anatum CVCC 3758, Salmonella meleagridis BNCC 186368, Salmonella arizonae CMCC 47001, Salmonella dublin CVCC 3760, Salmonella choleraesuis CVCC 503, Salmonella bovismorbificans BNCC 186363, Escherichia coli ATCC 25922, Pasteurella multocida CVCC 1662, Proteus vulgaris CVCC 1969, Klebsiella pneumoniae BNCC102997, Shigella sp. ATCC 25931, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 6538.

[0087] 1.2 Reagents

[0088] Bacterial culture medium, a product of Beijing Land Bridge Technology Co., Ltd.; Bacterial genomic DNA extraction kit, a product of Tiangen Biochemical Technology (Beijing) Co., Ltd.; Fluorescent PCR premix (2×qPCR mix), a product of ABI; Other conventional reagents are all of analytical grade.

[0089] 1.3 Instruments

[0090] Fluorescent PCR instrument (QuantStudio 7), a product of ABI, USA; High-speed refrigerated centrifuge (5430R), a product of Eppendorf, Germany; Nucleic acid extractor (TGuide S32), a product of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0091] 2 Methods

[0092] 2.1 Primers and Probes

[0093] Specific primers and probes were designed for the Salmonella genus-specific invA gene, Salmonella typhimurium STM4495 gene, Salmonella enteritidis SdfⅠ gene, Salmonella pullorum and Salmonella gallinarum speC gene, and Salmonella gallinarum glgC gene. According to the BLAST sequence specificity check results, single-reaction specificity verification, and multiplex cross-specificity verification results of the primers and probes, the optimal primer and probe sets were determined. The primer and probe sequences are shown in Table 1. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0094] Table 1 Primer and Probe Sequences

[0095]

[0096]

[0097] 2.2 Cultivation of Strains

[0098] It is carried out according to the conventional procedures for bacterial isolation and culture.

[0099] 2.3 Extraction of bacterial genomic DNA

[0100] Take 1 mL of bacterial culture solution, and extract genomic DNA according to the instructions of the bacterial genomic DNA extraction kit.

[0101] 2.4 Establishment of a five - plex fluorescence PCR reaction system

[0102] Using the standard strains of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum (such as the corresponding strains in 1.1) as templates, after extracting genomic DNA, perform single - plex fluorescence PCR respectively to verify the specificity of each primer - probe group, determine amplification conditions such as the concentration of each primer - probe, annealing temperature, and cycle number, and establish a single - plex fluorescence PCR amplification system for 5 Salmonella genes; on the basis of single - plex fluorescence PCR, conduct cross - verification of the specificity of the primer - probe groups, combine the 5 primer - probe groups in pairs (10 combinations) for duplex fluorescence PCR experiments, test and verify the DNA mixtures of 2 target Salmonella strains respectively, and optimize the primer - probe concentration and annealing temperature; then mix the 5 primer - probe groups and amplify them simultaneously in one reaction system, test the specificity when 2, 3, and 4 target target DNAs co - exist respectively, and through screening the concentrations between the components of the 5 primer - probe groups (primer 0.1 μmol / L - 1.0 μmol / L, probe 0.1 μmol / L - 0.5 μmol / L), further optimize the primer - probe concentrations of each group in the 5 - plex fluorescence PCR primer - probe mixed reaction, and optimize amplification parameters such as the overall annealing temperature and cycle number of the system through a temperature gradient experiment (57 °C - 62 °C) to determine the optimal five - plex fluorescence PCR reaction system and cycle amplification program.

[0103] In the embodiment of the present invention, "five - plex": includes Salmonella genus and 4 serotypes. Any Salmonella strain can be used as a detection template for the Salmonella genus, that is, these 4 strains of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum can be used as detection templates for their respective serotypes and also as detection templates for the Salmonella genus.

[0104] Composition of the five - plex fluorescence PCR reaction system (25 μL): 2×qPCR mix 12.5 μL, 0.75 μL each of the upstream and downstream primers of 10 μmol / L invA, 0.5 μL of 10 μmol / L invA probe; 1 μL each of the upstream and downstream primers of 10 μmol / L STM4495, 0.5 μL of 10 μmol / L STM4495 probe; 0.25 μL each of the upstream and downstream primers of 10 μmol / L SdfⅠ, 0.25 μL of 10 μmol / L SdfⅠ probe; 0.5 μL each of the upstream and downstream primers of 10 μmol / L speC, 0.5 μL of 10 μmol / L speC probe; 0.75 μL each of the upstream and downstream primers of 10 μmol / L glgC, 0.5 μL of 10 μmol / L glgC probe; 3 μL of DNA solution, add water to make up to 25 μL.

[0105] Five - plex fluorescence PCR amplification program: 95℃ for 10 min; 95℃ for 15 s, 58℃ for 1 min, a total of 45 cycles, and collect fluorescence signals at the second step (58℃ for 1 min) of each cycle. The settings of reporter fluorescence and quenching fluorescence are shown in Table 1.

[0106] 2.5 Judgment criteria

[0107] If the Ct value of the FAM channel of the test sample is ≤ 38 and a specific amplification curve appears, it is judged as positive for Salmonella nucleic acid.

[0108] If the Ct values of both the FAM channel and the NED channel of the test sample meet the criteria of Ct value ≤ 38 and a specific amplification curve appears, it is judged as positive for Salmonella typhimurium nucleic acid.

[0109] If the Ct values of both the FAM channel and the Texas Red channel of the test sample meet the criteria of Ct value ≤ 38 and a specific amplification curve appears, it is judged as positive for Salmonella enteritidis nucleic acid.

[0110] If the Ct values of both the FAM channel and the CY5 channel and the VIC channel of the test sample meet the criteria of Ct value ≤ 38 and a specific amplification curve appears, it is judged as positive for Salmonella gallinarum nucleic acid.

[0111] If the Ct values of both the FAM channel and the CY5 channel of the test sample meet the criteria of Ct value ≤ 38 and a specific amplification curve appears, and there is no amplification in the VIC channel, it is judged as positive for Salmonella pullorum nucleic acid.

[0112] If the Ct value of the corresponding channel of the target bacterium to be detected in the test sample is > 40 or there is no Ct value and no specific amplification curve, it is judged as negative for the nucleic acid of the corresponding pathogenic bacterium.

[0113] If the Ct value of any channel of the sample to be tested is 38 < Ct value ≤ 40 and a specific amplification curve appears, it is judged as suspicious, and nucleic acid needs to be extracted again for re - testing. If the repeated test results meet the above nucleic acid positive judgment criteria, it is judged as positive for the corresponding target bacteria nucleic acid; if the repeated test results show that the Ct value of the corresponding channel of the target bacteria to be tested is > 40 or there is no Ct value, it is judged as negative for the corresponding nucleic acid; if the Ct value of any channel in the repeated test is 38 < Ct value ≤ 40, it is judged as suspicious and further analysis needs to be carried out by other methods (such as sequencing).

[0114] Example 1 Five - plex fluorescence PCR amplification of target Salmonella

[0115] The DNA mixed templates of 4 target Salmonella strains, Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum, were amplified by the five - plex fluorescence PCR method described in 2.4 above. The results showed that the genus - specific gene invA of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum obtained characteristic S - shaped amplification curves; the STM4495 gene of Salmonella typhimurium, the SdfⅠ gene of Salmonella enteritidis, the speC gene of Salmonella pullorum and Salmonella gallinarum, and the glgC gene of Salmonella gallinarum were specifically amplified respectively, and all obtained characteristic S - shaped amplification curves. There was no non - specific amplification among the 5 groups of primer - probes ( Figure 1 ).

[0116] Example 2 Non - Salmonella specificity test

[0117] Four target Salmonella standard strains, Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum, and 7 non - Salmonella bacteria: Escherichia coli, Pasteurella multocida, Proteus, Klebsiella pneumoniae, Shigella, Pseudomonas aeruginosa, and Staphylococcus aureus reference strains were selected for five - plex fluorescence PCR specificity verification.

[0118] The amplification results showed that only the 4 target Salmonella strains (DNA mixed templates) showed corresponding specific amplifications, while the 7 non - Salmonella bacteria had no amplifications, indicating that the five - plex fluorescence PCR method established in the present invention has high specificity and can specifically detect Salmonella( Figure 2 ).

[0119] Example 3 Non - target Salmonella specificity test

[0120] Six other serotypes of Salmonella other than the 4 target Salmonella strains, Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum, namely Salmonella anatum, Salmonella meleagridis, Salmonella arizonae, Salmonella dublin, Salmonella choleraesuis, and Salmonella bovis morbificans, were selected for five - plex fluorescence PCR specificity verification.

[0121] The amplification results showed that positive amplification of the invA gene of Salmonella genus occurred in all 6 different serotypes of Salmonella, while specific amplification did not occur for the STM4495 gene, SdfⅠ gene, speC gene, and glgC gene, indicating that the established multiplex fluorescence PCR method had high specificity. The STM4495, SdfⅠ, speC, and glgC genes did not amplify for non-target Salmonella (Salmonella other than the 4 target Salmonella strains of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum), and Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum could be specifically detected and identified ( Figure 3 ).

[0122] Example 4 Sensitivity Test

[0123] The strains were enriched and cultured, genomic DNA was extracted, and the concentrations of genomic DNA of the 4 target Salmonella strains of Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum were measured using an ultraviolet spectrophotometer, and their concentrations were adjusted to 100 ng / μL respectively, and then serially diluted 10-fold to: 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 1×10 -1 pg / μL, 1×10 -2 pg / μL, 1×10 -3 pg / μL, and multiplex fluorescence PCR amplification was performed respectively to determine the lowest DNA concentration required to detect 5 target genes and draw a standard curve.

[0124] The results showed that the lowest DNA concentrations required for the established multiplex fluorescence PCR method to detect 5 target genes of Salmonella were 1×10 -2 pg / μL (invA), 1×10 -1 pg / μL (STM4495), 1×10 -1 pg / μL (SdfⅠ), 1×10 -2 pg / μL (speC), 1×10 -2 pg / μL (glgC)( Figures 4a - 4e ).

[0125] The R 2 values of the standard curve were 0.997 (invA), 0.998 (STM4495), 0.99 (SdfⅠ), 0.997 (speC), 0.993 (glgC), and the amplification efficiencies were 99.103% (invA), 94.227% (STM4495), 94.619% (SdfⅠ), 96.006% (speC), 91.478% (glgC)( Figures 5a - 5e) It indicates that there is a good linear relationship between the concentration of the DNA to be measured and the Ct value, and the established Salmonella multiplex fluorescence PCR method has high sensitivity and high amplification efficiency.

[0126] Example 5 Repeatability Test

[0127] The multiplex fluorescence PCR targeting 5 Salmonella target genes was subjected to a repeatability test. The DNA mixed templates of 4 target Salmonella strains, namely Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, and Salmonella gallinarum, with the same concentration were repeatedly measured 3 times to detect their repeatability and stability. The results showed that the 3 repeated amplification curves of the 5 target genes were basically consistent. The coefficient of variation CV of the Ct values was 0.43% (invA), 0.2% (STM4495), 0.42% (SdfⅠ), 0.88% (speC), and 0.67% (glgC) respectively, and the CV was less than 5%, indicating that the established Salmonella multiplex fluorescence PCR method had good repeatability and stability ( Figure 6 ).

[0128] Example 6 Clinical Application Evaluation

[0129] Clinical samples were collected to evaluate the established method.

[0130] A total of 100 samples were collected from large-scale chicken farms in Yanqing and Shunyi, Beijing. The pathogen identification of the 31 isolated pathogenic bacteria was carried out by the Salmonella multiplex fluorescence PCR method, and at the same time, the Salmonella conventional PCR was used for detection. The Salmonella conventional PCR used included: the double PCR for Salmonella pullorum and Salmonella gallinarum, and the single PCR for Salmonella, Salmonella enteritidis, and Salmonella typhimurium.

[0131] Salmonella PCR: ① Primers: upstream primer 5′-ACTGGCGTTATCCCTTTCTCTGCTG-3′, downstream primer 5′-ATGTTGTCCTGCCCCTGGTAAGAGA-3′; ② PCR system: ddH2O 27.8 μL, 10×buffer 5 μL, 2.5 mM dNTP 4 μL, upstream and downstream primers (5 μmol / L) 2.5 μL each, Taq DNA polymerase (5 U / μL) 0.7 μL, DNA template 7.5 μL; ③ PCR reaction conditions: pre-denaturation at 94°C for 5 min; 94°C for 1 min, 56°C for 1 min, 72°C for 1.5 min, 32 cycles; 72°C for 5 min.

[0132] Dual PCR for Salmonella pullorum and Salmonella gallinarum: ① Primers: glgC upstream primer 5′-GATCTGCTGCCAGCTCAA-3′, glgC downstream primer 5′-GCGCCCTTTTCAAAACATA-3′, speC upstream primer 5′-CGGTGTACTGCCCGCTAT-3′, speC downstream primer 5′-CTGGGCATTGACGCAAA-3′; ② PCR system: 34.75 μL of ddH2O, 5 μL of 10× buffer, 4 μL of 2.5 mM dNTP, 1 μL each of upstream and downstream primers (10 μmol / L), 0.25 μL of Taq DNA polymerase (5 U / μL), 2 μL of DNA template; ③ PCR reaction conditions: pre-denaturation at 95°C for 5 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s; 72°C for 7 min.

[0133] PCR for Salmonella enteritidis: ① Primers: upstream primer 5′-ATGCGTAAATCAGCATCTG-3′, downstream primer 5′-TTAGTTTTGATACTGCTGAAC-3′; ② PCR system: 37.5 μL of ddH2O, 5 μL of 10× buffer, 1 μL of 10 mM dNTP, 0.5 μL each of upstream and downstream primers (10 μmol / L), 0.5 μL of Taq DNA polymerase (5 U / μL), 5 μL of DNA template; ③ PCR reaction conditions: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min; 72°C for 10 min.

[0134] PCR for Salmonella typhimurium: ① Primers: upstream primer 5′-TTGTTCACTTTTTACCCCTGAA-3′, downstream primer 5′-CCCTGACAGCCGTTAGATATT-3′; ② PCR system: 38.5 μL of ddH2O, 5 μL of 10× buffer, 1 μL of 10 mM dNTP, 1 μL each of upstream and downstream primers (10 μmol / L), 0.5 μL of Taq DNA polymerase (5 U / μL), 3 μL of DNA template; ③ PCR reaction conditions: pre-denaturation at 95°C for 5 min; 35 cycles of 94°C for 40 s, 56°C for 40 s, 72°C for 50 s; 72°C for 10 min.

[0135] The results of the five-fold fluorescence PCR identification of Salmonella in 31 strains of pathogenic bacteria were completely consistent with those of the conventional PCR detection. Both methods detected 10 strains of Salmonella and 21 strains of non-Salmonella, including 2 strains of Salmonella enteritidis, 1 strain of Salmonella typhimurium, 3 strains of Salmonella pullorum, and 4 strains of Salmonella gallinarum.

[0136] Compared with the conventional method, the present invention can simultaneously complete the differential detection of Salmonella and four serotypes, namely Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum, in a single fluorescence PCR amplification reaction. It has high sensitivity and specificity, is easy to operate, fast, stable, saves reagents, consumes less samples, overcomes the problems of the existing detection methods such as being single, time-consuming and laborious, poor specificity and low sensitivity, and improves the accuracy and efficiency of Salmonella disease detection.

Claims

1. A reagent, characterized in that, Including primer-probe sets 1-5 for five-plex fluorescence PCR detection: Primer-probe combination 1 for detecting the invA gene: The primer pair shown in SEQ ID NO: 1 and 2 and the probe shown in SEQ ID NO: 3; Primer-probe combination 2 for detecting the STM4495 gene: The primer pair shown in SEQ ID NO: 4 and 5 and the probe shown in SEQ ID NO: 6; Primer-probe combination 3 for detecting the Sdf I gene: The primer pair shown in SEQ ID NO: 7 and 8 and the probe shown in SEQ ID NO: 9; Primer-probe combination 4 for detecting the speC gene: The primer pair shown in SEQ ID NO: 10 and 11 and the probe shown in SEQ ID NO: 12; and Primer-probe combination 5 for detecting the glgC gene: The primer pair shown in SEQ ID NO: 13 and 14 and the probe shown in SEQ ID NO:

15.

2. The reagent according to claim 1, characterized in that, Both ends of the probe are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups labeled between the probes are different.

3. The reagent according to claim 2, characterized in that, The fluorescent group is at least one of FAM, NED, Hex, VIC, TAMRA, ROX, Texas-Red and CY5; and / or, the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

4. Use of the reagent according to any one of claims 1-3 in the preparation of a product for identifying whether Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum exist in a test sample.

5. A kit containing the reagent according to any one of claims 1-3.

6. A five - plex fluorescence PCR detection method for a sample to be tested, characterized in that, Including the following steps: Using the nucleic acid of the test sample as a template, contacting the nucleic acid of the test sample with the reagent according to any one of claims 1-3 or the kit according to claim 5, and making the following judgments after fluorescence PCR reaction: If only the invA gene is detected in the test sample, then Salmonella exists in the test sample; If both the invA gene and the STM4495 gene are detected in the test sample, then Salmonella typhimurium exists in the test sample; If both the invA gene and the Sdf I gene are detected in the test sample, then Salmonella enteritidis exists in the test sample; If both the invA gene, the speC gene and the glgC gene are detected in the test sample, then Salmonella gallinarum exists in the test sample; If both the invA gene and the speC gene are detected in the test sample and the glgC gene cannot be detected, then Salmonella pullorum exists in the test sample; The method is used for non-disease diagnosis and treatment.

7. A detection system for identifying the presence of Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and Salmonella gallinarum in a sample to be tested, characterized in that, The detection system includes the following components: 1) Detection components for the invA gene, STM4495 gene, Sdf I gene, speC gene and glgC gene; 2) Data processing components; 3) Result output components; The detection components for the invA gene, STM4495 gene, Sdf I gene, speC gene, and glgC gene contain the reagent described in any one of claims 1-3 or the kit described in claim 5.

8. The system according to claim 7, wherein The detection object of the detection component is a fluorescence signal.

9. The system according to claim 7, wherein The data processing component is configured to: Judge whether the test sample contains Salmonella and different serotypes of Salmonella according to the detection results of the invA gene, STM4495 gene, Sdf I gene, speC gene, and glgC gene in the test sample detected by the detection component; The judgment criterion of the data processing component is: If only the invA gene is detected in the test sample, the test sample contains Salmonella; If both the invA gene and the STM4495 gene are detected in the test sample, the test sample contains Salmonella typhimurium; If both the invA gene and the Sdf I gene are detected in the test sample, the test sample contains Salmonella enteritidis; If the invA gene, speC gene, and glgC gene are detected in the test sample at the same time, the test sample contains Salmonella gallinarum; If both the invA gene and the speC gene are detected in the test sample and the glgC gene cannot be detected, the test sample contains Salmonella pullorum.

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