A primer probe set for detecting salmonella enteritidis and application thereof

Abstract

The application belongs to the field of biology, and discloses a use of a SNP site for developing a detection kit for detecting Salmonella enteritidis, the SNP site being a 508th base of a group_25091 gene of the Salmonella enteritidis, and the base being a T base. The application uses the SNP site as a specific target, has higher specificity and sensitivity, and avoids interference of other serotypes. Meanwhile, the application also discloses a primer probe group for detecting the Salmonella enteritidis and related applications.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, and particularly relates to a primer probe set for detecting Salmonella enteritidis and application thereof. BACKGROUND

[0002] Salmonella enteritidis is a bacterium that poses a significant threat to food safety and the livestock industry. Currently, the commonly used detection methods are mainly based on bacterial culture and traditional biochemical detection techniques. These methods are not only time-consuming and labor-intensive, but also have certain limitations in accuracy and sensitivity. In order to solve the above problems, some researchers have begun to focus on detection methods based on molecular biology techniques, such as PCR amplification and sequencing technology, but the detection time and convenience still need to be optimized. Therefore, it is of great significance to find specific targets that can accurately distinguish Salmonella enteritidis from other different serotypes of Salmonella and establish molecular detection methods.

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

[0004] The patent application with the publication number CN 105936935 A, the subject of which is a PCR detection kit for rapid identification of specific serotypes of Salmonella, has the tcpS gene as the target gene;

[0005] The patent application with the publication number CN 119432860 A, the subject of which is a kit for detecting Salmonella enteritidis and its application, has the TtAgo protein gene as the target gene;

[0006] The patent application with the publication number CN 107201400 B, the subject of which is a five-plex PCR detection method and kit for detecting avian Escherichia coli, chicken typhoid Salmonella, chicken white dysentery Salmonella, etc., has the sdfⅠ gene as the target gene;

[0007] The patent application with the publication number CN 118813838 B, the subject of which is a five-plex fluorescence PCR detection kit for Salmonella, Salmonella enteritidis, Salmonella typhimurium, chicken white dysentery Salmonella, and chicken typhoid Salmonella, and its application, has the invA gene and the lygD gene as the target genes;

[0008] The patent application with the publication number CN 113755617 A, the subject of which is a probe method double fluorescent quantitative PCR primer set and kit for rapid identification and quantification of Salmonella enteritidis, has the invA gene and the sdfⅠ gene as the target genes;

[0009] In the above-mentioned schemes, a target gene is basically used as a specific detection object for detection.

[0010] The target genes specifically existing in Salmonella enteritidis have been widely developed and applied, but there are still many deficiencies in distinguishing different serotypes of Salmonella.

[0011] Therefore, the present project mainly takes specific SNP site targets as the research direction.

[0012] Based on this strategy, the applicant proposes the prior application CN117987579B, a primer and probe for detecting Salmonella pullorum, a detection system and use. This scheme realizes a new detection method that distinguishes most of the existing detection methods by screening specific SNP targets.

[0013] In the development of SNP sites, there are many difficulties. The first difficulty is the large number of SNP sites. SNPs exist widely in the genome, with an average of 1 SNP in every 300 base pairs. The sequence length of a general bacterium is more than 90,000 bp, which means that there are several hundred SNPs in a bacterium, significantly increasing the screening workload. The second difficulty is that there are too many serotypes of Salmonella, reaching more than 2,000 strains. It is a challenging and difficult task to screen a specific SNP by comparing these several hundred SNPs one by one. Finally, it is also necessary to repeatedly explore to develop primers and probes with high sensitivity and strong specificity based on the site.

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

[0015] Through analysis of the prior art, it can be seen that there are very few detection methods for distinguishing Salmonella enteritidis and different serotypes of Salmonella, such as CN 105936935 A. Therefore, the present application has important significance. SUMMARY

[0016] The purpose of the present application is to provide the use of the 508th base of the group_25091 gene of Salmonella enteritidis in the development of a kit for detecting Salmonella enteritidis. At the same time, the present application also discloses a primer probe set for detecting Salmonella enteritidis and related applications.

[0017] To achieve the above-mentioned purpose, the present application discloses:

[0018] The use of an SNP site for developing a detection kit for detecting Salmonella enteritidis, the SNP site being the 508th base of the group_25091 gene of Salmonella enteritidis, which is a T base.

[0019] Meanwhile, the application 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 as SEQ ID NO. 1.

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

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

[0023] In addition, the application also discloses a detection system containing the primer and the probe.

[0024] Finally, the application also discloses the use of the primer and the probe in preparing a kit for detecting Salmonella enteritidis.

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

[0026] 1. The SNP site is used as a specific target, and the specificity and sensitivity are higher, and the interference of other serotypes is avoided.

[0027] 2. The traditional serotype identification usually uses a culture method combined with a biochemical identification test, and the time consumption is long and the steps are complicated. The molecular detection method using the SNP site as a specific target greatly shortens the time required for identifying Salmonella enteritidis, and the result can be directly obtained in only 2 hours.

[0028] 3. The specificity of the SNP reaction is double guaranteed by combining the optimized DNA-RNA polymerase probe and the Rnase HII enzyme activated hydrolysis reaction, and the detection of specific Salmonella enteritidis serotype is more accurate.

[0029] 4. The molecular identification method based on the SNP site provides a new choice for the detection of Salmonella enteritidis, and has a broader application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a specific experimental result of the enteritis serotype;

[0031] Figure 2 is an amplification curve result of a plasmid template sensitivity test;

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

[0033] DETAILED EMBODIMENT

[0034] The application will be clearly and completely described below in combination with the embodiments of the application. It should be noted that, in the description of the application, the specific conditions not noted in the embodiments are implemented according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are conventional products that can be commercially available. In the absence of special instructions, the parts used in the embodiments of the application are all by weight.

[0035] Method principle

[0036] I. Target screening aspect:

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

[0038] ② The Prokka software was used to annotate all gene sequences in the database, and the Roary software was used for pan-genome analysis after the annotation was completed. The matrix text gene_presence_absence.csv generated after the pan-genome analysis was converted into 0 / 1 form, each column corresponded to the gene cluster carrying condition of the strain, and the value 1 indicated that the gene was carried, and the value 0 indicated that the gene was not carried. The specific genes of the target bacteria can be screened by combining and sorting the carrying conditions.

[0039] ③ The specific genes of the target bacteria were screened out by NCBI online BLAST, which were further compared with the nucleic acid database of all bacteria to verify whether the screened Salmonella enteritidis had specificity.

[0040] ④ The similar gene annotation fragments to the screened specific genes were found by comparing in the whole genome database, and the specific SNP sites of the target bacteria were found by Snapgene comparison. The similar fragments of the gene or the specific SNP sites of the gene fragments were not contained in the strains of other serotypes of Salmonella and other species, or only the enteritidis serotype had base differences.

[0041] II. Molecular detection aspect:

[0042] ① According to the screened fragments with specific SNP sites of Salmonella enteritidis, specific primers were designed, DNA-RNA polymerase probes were designed according to the SNP sites, and Rnase HII enzyme with RNA hydrolysis activity was used in the qPCR reaction system without 5'→3' exonuclease activity Taq enzyme.

[0043] ②When the target SNP site and the designed site of the probe are complementary to each other in the amplified fragment of the test sample, RnaseHII enzyme is activated to cut the probe, thereby generating a fluorescence signal; when the two are not complementary, no fluorescence signal is generated, thereby achieving the purpose of specifically detecting Salmonella enterica.

[0044] Method validation

[0045] I. Target screening

[0046] The screened Salmonella enterica specific target group_25091 gene sequence is verified by NCBI-BLAST, and the specificity is good. The position is 4495658-4496554 bp in Salmonella enterica subsp. enterica serovar Enteritidis strain SE95. The SNP site is located at the 508th base of the group_25091 gene, and only Salmonella enterica has T base. In order to evaluate the specificity of the new SNP site target, we compared it with other specific detection targets of Salmonella enterica (Table 1 and Table 2);

[0047] Table 1 Distribution of Salmonella enterica specific detection targets

[0048]

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

[0050]

[0051]

[0052] It can be seen from the above analysis that the SNP site selected by the present application has high specificity.

[0053] II. qPCR detection method based on SNP site

[0054] The upstream and downstream primers and probes were designed based on the group_25091 gene and SNP site, and the sequences are shown in Table 2. The following materials were used to establish the basic system of the fluorescence quantitative PCR method: 10 μL of 10x Taq HS Mix, 0.5 μL of 1 mM of upstream and downstream primers, and 0.5 μL of 1 mM of probes, 0.01 uL of 25 mU of RNase H2 enzyme, 1 μL of DNA template, and deionized water to make up to 20 μL. The reaction program: 95 °C pre-denaturation for 2 min; 95 °C for 10 s, 60 °C for 20 s (signal collection), 72 °C for 30 s, 40 cycles. The Bio-Rad fluorescence quantitative PCR instrument was used for detection, and the CY5 mode was used to collect the fluorescence signal to establish the basic reaction system of the qPCR method 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 SNP site target, we selected different serotypes of Salmonella (determined by biochemical identification) and non-Salmonella strains commonly found in the environment, food, poultry products or animal feces in China, and used the kit to extract DNA as a template for qPCR reaction. Monitoring was performed under a fluorescence quantitative instrument. It can be seen that only Salmonella enteritidis produced fluorescence signal, and other serotypes of Salmonella and non-Salmonella strains did not produce fluorescence signal. Subsequently, the reaction product was sequenced, and the sequence of the SNP site was consistent with the expectation, further verifying the specificity of the new target for the enteritidis serotype Figure 1

[0059] Figure 1 is the specificity experiment result of enteritidis serotype, wherein 1-26: different serotypes of Salmonella in Salmonella; 1-3: S. Enteritidis standard strain ATCC13076; 4-5: S. Enteritidis isolates; 5-26: chicken white diarrhea, Agona, murine typhoid, Sao Paulo, Derby, Indiana, Ogo Shenglong, Montevideo, Tomson, Brundellup, Mbandaka, Rose, Livingston, Newport, Kentucky, Albany, Covallis, Vetaifred, London, turkey, Cerro isolates; 27-30: non-Salmonella bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, Campylobacter, 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 plasmid template concentration amplified in 40 cycles of this method was 5.73 x 10 0 copies / μL to 5.73 x 10 5 ​copies / μL, the reactions could be carried out normally, but the reaction could not detect the plasmid template with the concentration of 5.73×10 -1 copies / μL, which indicated that the minimum detection limit of the reaction was 5.73×10 0 copies / μL. Figure 2 Meanwhile, the linear fitting equation between the logarithm of the plasmid copy number and the time (CT value) for the fluorescence signal to reach the threshold value was y=-3.0168x+40.327 (R 2 Figure 3 =0.9913), which showed a good linear relationship between them, proving that this method could be used for the quantitative detection of Salmonella enteritidis.

[0061] Figure 2 The amplification curve results of the plasmid template sensitivity test, wherein a: 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 The standard curve graph of the logarithm of the plasmid concentration and the CT value.

[0063] The repeatability test results showed that in each concentration gradient group, the inter-group coefficient of variation of the reaction was in the range of 0.45% to 2.44%, and the intra-group coefficient of variation was in the range of 0.06% to 2.53%, all of which were less than 5%, which indicated that this method had good stability (Table 3)

[0064] Table 3 Coefficient of variation results in repeatability evaluation

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

[0066] Finally, the clinical application effect evaluation of Salmonella enteritidis molecular identification was carried out.

[0067] ​Chicken embryo and meat samples were collected from hatcheries, slaughterhouses and poultry markets in Guangdong Province. The samples were cultured according to the national standard method for Salmonella detection (GB 4789.4-2016) and suspected colonies were selected. The genomic nucleic acid was extracted using a kit and verified using the molecular identification method of the application. Traditional isolation and identification methods (GB 4789.4-2016) and national standard PCR detection methods (GBT 40049-2021) were used to identify and verify whether they were Salmonella enteritidis.

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

[0069] As shown in the table, from the 400 collected dead embryo samples, 33 strains of Salmonella enteritidis were isolated using traditional isolation and identification methods, with an isolation rate of 8.25%. Using the method and the national standard PCR detection method, 33 strains of Salmonella enteritidis were detected, with a sample detection rate of 8.25% and 8.25%, respectively. The coincidence rate of the three methods was 100%. Further verification of the clinical samples showed that the qPCR method has good specificity and is more convenient to operate than traditional methods.

[0070] Table 4: Clinical detection results

[0071]

[0072]

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

[0074] From the above experiments, the application has the following advantages:

[0075] 1. The application discovers a specific SNP site, which has high specificity in different serotypes of Salmonella;

[0076] This discovery is difficult and has profound significance;

[0077] Firstly, it is difficult to isolate SNP sites from a large number of serotypes;

[0078] Secondly, different combinations of bacteria can produce different SNP sites, and each bacterium contains a large number of SNP sites, which makes it almost impossible to screen sites. The reason why the present application can screen effective target sites is that the applicant has been researching Salmonella before, with deep research depth and sufficient self-constructed database, which can shorten the screening time and accurately screen the target sites.

[0079] Finally, by screening SNP sites, the detection difficulty can be reduced, and a molecular detection method can be established by using the SNP sites as specific targets, so that the time required for identification of Salmonella enteritidis can be greatly shortened, and the result can be directly obtained in only 2 hours.

[0080] 2. The application combines the optimized DNA-RNA polymerase probe and the Rnase HII enzyme activated hydrolysis reaction, which double guarantees the specificity of the SNP reaction, so that the detection of specific enteritis serotype Salmonella is more accurate.

[0081] It will be obvious to a person skilled in the art that the application is not limited to the details of the above-described exemplary embodiments, but that the application can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, the scope of the application being defined by the appended claims rather than by the above description, and it is intended to fall within the meaning and range of equivalent elements of the claims. All variations falling within the meaning and range of the essential features of the claims should be included in the application. Any reference signs in the claims should not be considered as limiting the claims involved.

Claims

1. Use of primers and probes for detecting SNP site, which is the 508th base of group_25091 gene of Salmonella enterica subsp. enterica serovar Enteritidis strain SE95, and the base is T, in the preparation of a kit for detecting Salmonella enterica subsp. enterica serovar Enteritidis strain SE95.

2. A primer probe set for detecting Salmonella enteritidis, characterized by, The primers and probes comprise an upstream primer, a downstream primer and a probe; 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; The primer and probe set is suitable for a qPCR detection system; The qPCR detection system is used for detecting SNP site, which is the 508th base of group_25091 gene of Salmonella enterica subsp. enterica serovar Enteritidis strain SE95, and the base is T, and the position of the group_25091 gene of Salmonella enterica subsp. enterica serovar Enteritidis strain SE95 is 4495658-4496554 bp.

3. A detection system characterized in that, The kit contains the primer and probe set of claim 2.

4. Use of the primer and probe set of claim 2 in the preparation of a kit for detecting Salmonella enterica subsp. enterica serovar Enteritidis strain SE95.

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