Primer probe group for detecting salmonella enteritidis and application thereof

Abstract

The invention belongs to the field of biology, and discloses application of a detection kit for detecting salmonella enteritidis developed by adopting an SNP (Single Nucleotide Polymorphism) site, the SNP site is the 508th basic group of a Group 25091 gene of the salmonella enteritidis, and the basic group is a T basic group. According to the present invention, the SNP site is adopted as the specific target, such that the high specificity and the high sensitivity are provided, the interference of other serotypes is avoided, and the invention further discloses the primer probe group for detecting the salmonella enteritidis and the related applications.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a primer probe set for detecting Salmonella enteritidis and an application thereof. Background Art

[0002] Salmonella Enteritidis is a bacterium that poses a significant threat to food safety and the aquaculture industry. Currently, commonly used detection methods are primarily based on bacterial culture and traditional biochemical assays. These methods are not only time-consuming and labor-intensive, but also have limitations in accuracy and sensitivity. To address these issues, some researchers have begun to focus on detection methods based on molecular biology techniques, such as PCR amplification and sequencing, but their detection time and convenience still need to be optimized. Therefore, it is of great significance to identify specific targets that can accurately distinguish Salmonella Enteritidis from other serotypes of Salmonella and establish molecular detection methods.

[0003] The relevant detection methods for Enteritidis Salmonella can be found in the following literature:

[0004] Publication number CN 105936935 A, the subject of the patent application is a PCR detection kit for rapid identification of specific serotypes of Salmonella, whose target gene is the tcpS gene;

[0005] Publication number CN 119432860 A, the subject of the patent application is a kit for detecting Salmonella Enteritidis and its application, the target gene of which is the TtAgo protein gene;

[0006] Publication number CN 107201400 B, the subject of the patent application is a five-plex PCR detection method and detection kit for avian Escherichia coli, Salmonella Gallinarum, Salmonella Pullorum, etc., the target gene of which is the sdfⅠ gene;

[0007] Publication number CN 118813838 B, the subject of the patent application is a five-fold fluorescent PCR detection reagent for Salmonella, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Pullorum, and Salmonella Gallinarum, and its application, the target genes of which are the invA gene and the lygD gene;

[0008] Publication number CN113755617A is a patent application for a probe-based dual fluorescence quantitative PCR primer set and kit for rapid identification and quantification of Salmonella enteritidis, with the target genes being the invA gene and the sdfⅠ gene.

[0009] In the above scheme, a target gene is basically detected as a specific detection object.

[0010] Target genes that are specific to Salmonella Enteritidis have been widely developed and applied, but these genes still have many deficiencies in distinguishing different serotypes of Salmonella.

[0011] Therefore, this project mainly focuses on specific SNP site targets as research direction;

[0012] Based on this strategy, the applicant proposed a prior application CN117987579B, which describes a primer and probe, a detection system, and uses for detecting Salmonella pullorum. This approach achieves a new detection method that distinguishes itself from most existing detection methods by screening for specific SNP targets.

[0013] The development of SNP sites presents numerous challenges. The first is the sheer number of SNPs, which are widespread throughout the genome, occurring at an average of one in every 300 base pairs. The average bacterial sequence is over 90,000 base pairs long, meaning that a single bacterium harbors hundreds of SNPs, significantly increasing the screening workload. A second challenge is the sheer number of Salmonella serotypes, exceeding 2,000 strains. Using these hundreds of SNPs to identify a specific SNP is a challenging and challenging task. Finally, developing highly sensitive and specific primers and probes for this site also requires repeated attempts.

[0014] In addition, developing targeted detection methods for SNP sites is not necessarily a successful task. In the serotype differentiation of many target bacteria, there may not be SNP sites that meet the above requirements.

[0015] An analysis of the existing technologies shows that there are very few detection methods for distinguishing between Salmonella enteritidis and Salmonella of different serotypes, such as CN 105936935 A. Therefore, the invention is of great significance. Summary of the Invention

[0016] The present invention aims to provide the use of base 508 of the group_25091 gene of Salmonella Enteritidis in developing a kit for detecting Salmonella Enteritidis. The present invention also discloses a primer probe set for detecting Salmonella Enteritidis and related applications.

[0017] To achieve the above objectives, this application discloses:

[0018] The invention relates to a method for developing a detection kit for detecting Salmonella Enteritidis using a SNP site, wherein the SNP site is the 508th base of the group_25091 gene of Salmonella Enteritidis, and the base is a T base.

[0019] At the same time, the present invention also discloses a primer probe set, comprising an upstream primer, a downstream primer and a probe;

[0020] The sequence of the upstream primer is shown in SEQ ID NO.1;

[0021] The sequence of the downstream primer is shown in SEQ ID NO.2;

[0022] The sequence of the probe is shown in SEQ ID NO.3.

[0023] In addition, the present invention also discloses a detection system comprising the primers and probes described above.

[0024] Finally, the present invention also discloses the use of the primers and probes described above to prepare a kit for detecting Salmonella enteritidis.

[0025] This application has at least the following beneficial effects:

[0026] 1. Using SNP sites as specific targets has higher specificity and sensitivity, avoiding interference from other serotypes.

[0027] 2. Traditional serotype identification often relies on culture combined with biochemical tests, which is time-consuming and complex. The development of a molecular detection method using SNPs as specific targets has significantly shortened the time required for S. Enteritidis identification, providing direct results in just two hours.

[0028] 3. Combining the optimized DNA-RNA polymerase probe and the hydrolysis reaction activated by RNase HⅡ enzyme, the specificity of the SNP reaction is doubly guaranteed, making the detection of specific Enteritidis serotype Salmonella more accurate.

[0029] 4. The establishment of a molecular identification method based on SNP sites provides a new option for the detection of Enteritidis Salmonella and has broader application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the specific test result of Enteritidis serotype;

[0031] Figure 2 This is the amplification curve result of the plasmid template sensitivity test;

[0032] Figure 3 This is a standard curve of the logarithm of plasmid concentration and CT value.

[0033] Specific embodiment

[0034] Below in conjunction with embodiments of the present invention, the present invention is clearly and completely described, in description of the present invention, it should be noted that, in the embodiment, the unrecited specific conditions person, carry out according to the condition of normal condition or manufacturer's suggestion. Reagents therefor or instrument are not recited manufacturer person, are the conventional products that can be obtained by commercial purchase. When not making special instructions, used part in the embodiments of the present invention is all weight part.

[0035] Principles of the method

[0036] 1. Target screening:

[0037] ① The genome sequences of multiple serotypes of Salmonella were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / genome) to construct a library, including the full genome data of the isolates in our laboratory, a total of 2657 bacterial genome sequences, 2628 Salmonella strains (covering 26 serogroups and 112 serotypes of Salmonella) and 29 non-Salmonella strains.

[0038] ② Use Prokka software to annotate all gene sequences in the database. After annotation, use Roary software to perform pan-genome analysis. Convert the matrix file gene_presence_absence.csv generated after pan-genome analysis into a 0 / 1 format. Each column corresponds to the gene cluster presence of the strain, with a value of 1 indicating presence of the gene and a value of 0 indicating absence. Combining and sorting the presence patterns allows for screening of specific genes for the target bacteria.

[0039] ③ Screen out the specific genes of the target bacteria and use NCBI online BLAST to further compare them back to the nucleic acid database of all bacteria to verify whether the screened Enteritidis Salmonella is specific.

[0040] ④ By comparing the whole genome database to identify gene annotation fragments similar to the selected specific genes, Snapgene comparison is used to identify the specific SNP sites of the target bacteria. Other serotypes of Salmonella and strains of other species do not contain similar fragments of the gene, or the specific SNP sites of the gene fragment only have base differences in the Enteritidis serotype.

[0041] 2. Molecular Detection:

[0042] ① Based on the fragments with the screened SNP sites specific to Salmonella Enteritidis, specific primers were designed. DNA-RNA polymerase probes were designed based on the SNP sites. The probes were used in a qPCR reaction system without 5'→3' exonuclease-active Taq enzyme in combination with RNase HⅡ enzyme with RNA hydrolysis activity.

[0043] ② When the target SNP site in the amplified fragment of the test sample is complementary to the designed site RNA of the probe, the RNaseHⅡ enzyme is activated to cut the probe, thereby generating a fluorescent signal. When the two are not complementary, no fluorescent signal is generated, thereby achieving the purpose of specific detection of Enteritidis Salmonella.

[0044] Method validation

[0045] 1. Target Screening

[0046] The selected S. Enteritidis-specific target gene group_25091 was validated by NCBI-BLAST and demonstrated good specificity. Its location is between bp 4495658 and 4496554 in the standard enteritis bacterium, Salmonella enterica subsp. enterica serovar Enteritidis strain SE95. The SNP site is located at base 508 of the group_25091 gene, where only S. Enteritidis has a T base. To evaluate the specificity of the new SNP site target, we compared it with other S. Enteritidis-specific detection targets (Tables 1 and 2).

[0047] Table 1 Distribution of specific detection targets for Salmonella Enteritidis

[0048]

[0049] Table 2 Mutation site information of group_25091 gene of different serotypes of Salmonella

[0050]

[0051]

[0052] From the above analysis, it can be seen that the SNP sites selected in the present invention are highly specific.

[0053] 2. qPCR detection method based on SNP sites

[0054] Upstream and downstream primers and probes were designed based on the group_25091 gene and SNP locus; their sequences are shown in Table 2. A basic fluorescence quantitative PCR system was established using the following materials: 10 μL 10× Taq HS Mix, 0.5 μL 1 mM upstream and downstream primers and 0.5 μL 1 mM probe, 0.01 μL 25 mU RNase H2 enzyme, 1 μL DNA template, and deionized water to 20 μL. The reaction procedure was as follows: 95°C pre-denaturation for 2 min; 40 cycles of 95°C for 10 s, 60°C for 20 s (signal acquisition), and 72°C for 30 s. Detection was performed using a Bio-Rad fluorescence quantitative PCR instrument, using the CY5 mode to collect fluorescence signals. This established a basic qPCR system for Salmonella Enteritidis.

[0055] Table 2 Primer and probe sequence information

[0056]

[0057] Experimental results

[0058] In order to prove the specificity of the new target of SNP site, we selected different serotypes of Salmonella (the serotype was determined by biochemical identification) and non-Salmonella strains from common domestic environments, foods, poultry products or animal feces, and used the kit to extract DNA as a template for qPCR reaction. Monitoring was carried out under a fluorescence quantitative instrument. It can be seen that only Enteritidis Salmonella produces fluorescent signals, while other serotypes of Salmonella and non-Salmonella strains do not produce fluorescent signals. Subsequently, by sequencing the reaction products, the SNP sites of their sequences were consistent with expectations, further verifying the specificity of the new target for Enteritidis serotypes ( Figure 1 );

[0059] Figure 1 These are the specific test results for Enteritidis serotypes, including 1-26: different serotypes of Salmonella in the genus Salmonella; 1-3: S. Enteritidis standard bacteria ATCC13076; 4-5: S. Enteritidis isolates; 5-26: Pullorum, Agona, Typhimurium, São Paulo, Derby, Indiana, Xugochengron, Montevideo, Thompson, Brundenloop, Mbandaka, Rawson, Livingstone, Newport, Kentucky, Albany, Corvallis, Wetevreden, London, Turkey, and Cerro isolates; 27-30: Non-Salmonella bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, Campylobacter, and Listeria isolates; 31: Negative control group;

[0060] The sensitivity of the SNP probe for detecting Salmonella enteritidis in the qPCR method was also evaluated. It can be seen that the concentration of the plasmid template amplified by this method within 40 cycles was 5.73×10 0 copies / μL to 5.73×10 5copies / μL, the reaction was able to proceed normally, but the reaction could not detect the concentration of 5.73×10 -1 copies / μL of plasmid template, which means the minimum detection limit of the reaction is 5.73×10 0 copies / μL( Figure 2 At the same time, the linear fitting equation of the logarithm of the plasmid copy number and the time when the fluorescence signal reaches the threshold value (CT value) is y=-3.0168x+40.327(R 2 =0.9913)( Figure 3 ), showing a good linear relationship between the two, proving that this method can be used for the quantitative detection of Salmonella Enteritidis.

[0061] Figure 2 is the amplification curve result of the plasmid template sensitivity test, where a is 5.73×10 5 copies / μL; b: 5.73×10 4 copies / μL; c: 5.73×10 3 copies / μL; d: 5.73×10 2 copies / μL; e: 5.73×10 1 copies / μL; f: 5.73×10 0 copies / μL; g: 5.73×10 -1 copies / μL; h: negative control.

[0062] Figure 3 This is a standard curve of the logarithm of plasmid concentration and CT value.

[0063] The results of the reproducibility test showed that in each concentration gradient group, the inter-group coefficient of variation of the reaction ranged from 0.45% to 2.44%, and the intra-group coefficient of variation ranged from 0.06% to 2.53%. All coefficients of variation were less than 5%, indicating that the method has good stability (Table 3).

[0064] Table 3 Results of coefficient of variation in repeatability evaluation

[0065] Plasmid concentration (copies / μL) Intra-group coefficient of variation Intergroup coefficient of variation <![CDATA[5.73×10 0 ]]> 1.94％ 0.45％ <![CDATA[5.73×10 1 ]]> 2.53％ 1.94％ <![CDATA[5.73×10 2 ]]> 0.76％ 0.86％ <![CDATA[5.73×10 3 ]]> 0.06％ 1.00％ <![CDATA[5.73×10 4 ]]> 1.12％ 0.64％ <![CDATA[5.73×10 5 ]]> 0.23％ 2.44％

[0066] Finally, the clinical application effect of molecular identification of Salmonella Enteritidis was evaluated.

[0067] Chicken embryos and meat samples were collected from hatcheries, slaughterhouses, and poultry markets in Guangdong Province. Suspected colonies were cultured and selected according to the national standard for Salmonella testing (GB 4789.4-2016). Genomic nucleic acid was extracted using a kit and then verified using the molecular identification method of the present invention. Both traditional isolation and identification methods (GB4789.4-2016) and the national standard PCR detection method (GBT40049-2021) were used to verify whether they were Salmonella Enteritidis.

[0068] The test results of clinical samples are shown in Table 4.

[0069] As shown in the table, 33 strains of Enteritidis Salmonella were isolated from 400 dead embryo samples using traditional isolation and identification methods, with an isolation rate of 8.25%. Both this method and the national standard PCR detection method detected 33 strains of Enteritidis Salmonella, with sample detection rates of 8.25% and 8.25%, respectively, for a 100% concordance rate among the three methods. Further validation with clinical samples demonstrated that this qPCR method has good specificity and is simpler to use than traditional methods.

[0070] Table 4 Clinical test results

[0071]

[0072]

[0073] Note: +: positive test result; -: negative test result

[0074] It can be seen from the above experiments that the present invention has the following advantages:

[0075] 1. The present invention discovered a specific SNP site that is highly specific in different serotypes of Salmonella;

[0076] The discovery is difficult and has profound implications;

[0077] First, it is very difficult to separate SNP sites from a large number of serotypes;

[0078] Secondly, different bacterial strains will produce different SNP sites when combined, and each bacterial strain contains a large number of SNP sites, which will make site screening almost impossible; the reason why the present application was able to screen out effective target sites is that the applicant has been conducting research on Salmonella before, with a deep research depth and a sufficient self-constructed database, which can shorten the screening time and accurately screen out the target sites.

[0079] Finally, by screening SNP sites, the difficulty of detection can be reduced. Using SNP sites as specific targets to establish molecular detection methods can greatly shorten the time required for Enteritidis Salmonella identification, and results can be obtained directly in just 2 hours.

[0080] 2. The present invention combines an optimized DNA-RNA polymerase probe with a hydrolysis reaction activated by RNase HⅡ enzyme, doubly ensuring the specificity of the SNP reaction, making the detection of specific enteritis serotype Salmonella more accurate.

[0081] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and it is intended that equivalents falling within the claims be included. All variations within the meaning and scope of the elements are encompassed by the present invention. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. The invention relates to a method for developing a detection kit for detecting Salmonella enteritidis using a single nucleotide polymorphism (SNP) site, wherein the SNP site is the 508th base of the group_25091 gene of Salmonella enteritidis, which is a T base.

2. A primer probe set, characterized in that: including upstream primers, downstream primers and probes; The sequence of the upstream primer is shown in SEQ ID NO.1; The sequence of the downstream primer is shown in SEQ ID NO.2; The sequence of the probe is shown in SEQ ID NO.

3.

3. A detection system, characterized in that: Contains the primers and probes as claimed in claim 2.

4. Use of the primers and probes as claimed in claim 2 in preparing a kit for detecting Salmonella enteritidis.

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