阅读说明：本技术 一种快速鉴定和定量肠炎沙门菌的探针法双重荧光定量pcr引物组及试剂盒 (Probe-method dual-fluorescence quantitative PCR primer group and kit for rapidly identifying and quantifying salmonella enteritidis ) 是由 焦新安 潘志明 熊丹 宋丽 周懿 刘博闻 顾丹 康喜龙 孟闯 于 2021-10-15 设计创作，主要内容包括：本发明属于生物技术检测领域,具体涉及一种快速鉴定和定量肠炎沙门菌的探针法双重荧光定量PCR引物组及试剂盒,所述PCR引物组中包括用于分别检测lygD基因和invA基因的引物对,进一步的,所述PCR引物组中包括1)第一引物对：包括核苷酸序列如SEQ ID NO:1所示的上游引物与核苷酸序列如SEQ ID NO:2所示的下游引物；2)第一探针引物：核苷酸序列如SEQ ID NO:3所示；3)第二引物对：包括核苷酸序列如SEQ ID NO:4所示的上游引物与核苷酸序列如SEQ ID NO:5所示的下游引物；4)第二探针引物：核苷酸序列如SEQ ID NO:6所示。所述试剂盒中包括所述引物组。本发明的试剂盒能快速高通量鉴定和定量肠炎沙门菌,可替代传统沙门菌血清学分型和生化鉴定等复杂程序。(The invention belongs to the field of biotechnology detection, and particularly relates to a probe-method dual-fluorescence quantitative PCR primer group and a kit for rapidly identifying and quantifying salmonella enteritidis, wherein the PCR primer group comprises primer pairs for respectively detecting a lygD gene and an invA gene, and further comprises 1) a first primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2; 2) a first probe primer: the nucleotide sequence is shown as SEQ ID NO. 3; 3) a second primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5; 4) a second probe primer: the nucleotide sequence is shown as SEQ ID NO. 6. The kit comprises the primer group. The kit can rapidly identify and quantify the salmonella enteritidis in high flux, and can replace the traditional salmonella serological typing, biochemical identification and other complex procedures.)

1. A PCR primer group for detecting salmonella enteritidis is characterized by comprising primer pairs for respectively detecting a lygD gene and an invA gene.

2. The PCR primer set according to claim 1, wherein any one or more of the following is included in the PCR primer set:

1) a first primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;

2) a first probe primer: the nucleotide sequence is shown as SEQ ID NO. 3;

3) a second primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5;

4) a second probe primer: the nucleotide sequence is shown as SEQ ID NO. 6.

3. The PCR primer set according to claim 2, wherein the first probe primer and the second probe primer are labeled with different fluorescent reporter groups at 5 'ends and different fluorescent quencher groups at 3' ends.

4. Use of the PCR primer set according to any one of claims 1 to 3 for the preparation of a product for detecting the lygD gene and the invA gene.

5. Use according to claim 4, wherein the lysD gene and invA gene detection product is a Salmonella enteritidis identification or quantification product.

6. The use according to claim 5, wherein the Salmonella enteritidis identification or quantification product is a kit.

7. A probe-method dual-fluorescence quantitative PCR detection kit for detecting salmonella enteritidis, which is characterized by comprising the PCR primer set of any one of claims 1 to 3.

8. The kit according to claim 7, wherein the kit further comprises one or more of water, Premix Ex TaqMaster, ROX Reference Dye II, positive control DNA, negative control DNA, and sample genomic DNA extraction reagent.

9. Use of the primer set of any one of claims 1 to 3 or the kit of claim 7 for identifying and quantitatively detecting Salmonella enteritidis.

10. The method for identifying the salmonella enteritidis is characterized by comprising the following steps of:

1) preparing a PCR reaction system: preparing a PCR reaction system by using the probe method dual-fluorescence quantitative PCR detection kit of claim 7 or 8 and the genomic DNA of a sample to be detected;

2) and (3) PCR reaction: carrying out PCR amplification on the prepared PCR reaction system;

3) and (4) analyzing results: the sample to be detected, which can simultaneously amplify the lysD gene and the invA gene, is the salmonella enteritidis, and the sample to be detected, which cannot simultaneously amplify the lysD gene and the invA gene, is not the salmonella enteritidis.

11. The authentication method of claim 10, further comprising one or more of the following features:

1) the sample to be tested is selected from food, water and poultry;

2) the PCR reaction system prepared in the step 1) comprises a reaction system of a sample to be detected, a reaction system of positive control and a reaction system of negative control;

3) the PCR reaction system is a 25 mu L system, and comprises: 2 × Premix Ex Taq Master 12.5 μ L, lysgD-F240 nM, lysgD-R240 nM, lysgD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂Supplementing O to 25 mu L;

4) the conditions of the PCR reaction were: (a) 30s at 95 ℃; (b) 5s at 95 ℃; (c) 34s at 60 ℃, and the steps (b) to (c) are circulated for 40 times.

12. A method for quantitatively detecting Salmonella enteritidis, which is characterized by comprising the following steps:

1) preparing a PCR reaction system of a standard curve: the probe-method dual-fluorescence quantitative PCR detection kit of claim 8, wherein the positive control DNA is subjected to gradient dilution, and the positive control DNA subjected to gradient dilution is respectively used as a template to prepare a reaction system with a standard curve;

2) preparing a PCR reaction system of a sample to be detected: preparing a PCR reaction system of a sample to be detected by using the probe method dual-fluorescence quantitative PCR detection kit of claim 8 by using the genome DNA of the sample to be detected as a template;

3) preparing a negative control PCR reaction system: preparing a negative control PCR reaction system by using the negative control DNA in the probe-method dual-fluorescence quantitative PCR detection kit of claim 8 as a template;

4) and (3) PCR reaction: carrying out PCR amplification on each prepared PCR reaction system;

5) and (4) analyzing results: and substituting the amplification signals of each PCR reaction system into the standard curve to calculate the content of the salmonella enteritidis in the sample to be detected.

Technical Field

The invention relates to the field of biotechnology detection, in particular to a probe-method dual-fluorescence quantitative PCR primer group and a kit for rapidly identifying and quantifying salmonella enteritidis.

Background

Salmonellosis is one of the infectious diseases of great public health significance and one of the main causes of food-borne bacterial enteritis, and most cases of salmonella infection are caused by eating contaminated food or water, such as pork, beef, poultry, and eggs. Of the many serotypes of salmonella, salmonella enteritidis is one of the most important pathogens causing serious infections in humans. After the salmonella enteritidis infects human bodies, gastroenteritis, diarrhea, vomiting and even serious systemic infection can be caused. Salmonella enteritidis can survive in egg white and contaminate egg products, causing a significant economic and health burden.

Traditional salmonella serotyping is based on Kauffman-White identification by specific antisera to bacterial surface O and H antigens. Although this method is widely used, it is relatively expensive, labor intensive and time consuming due to the need for traditional culture-based methods. Therefore, the accurate identification method is important for preventing and controlling salmonellosis, and the establishment of the rapid and accurate identification method for epidemic salmonella serotypes can promote routine monitoring and public health safety. Polymerase Chain Reaction (PCR) is widely studied for its rapidity, high throughput, high sensitivity and specificity. Different from the traditional PCR, the probe method fluorescence quantitative PCR method does not need to confirm the existence of bacterial pathogens through gel electrophoresis analysis, can carry out real-time quantitative analysis on data, and has great significance for timely identifying the salmonella enteritidis to the effective prevention and control of salmonellosis.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a probe-method dual-fluorescence quantitative PCR primer set and a kit for rapidly identifying and quantifying salmonella enteritidis, which are used for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a PCR primer set for detecting salmonella enteritidis, wherein the PCR primer set comprises primer pairs for detecting a lysd gene and an invA gene, respectively.

Preferably, any one or more of the following is included in the PCR primer set:

1) a first primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;

2) a first probe primer: the nucleotide sequence is shown as SEQ ID NO. 3;

3) a second primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5;

4) a second probe primer: the nucleotide sequence is shown as SEQ ID NO. 6.

The invention also provides application of the PCR primer group in preparation of a detection product of the lygD gene and the invA gene.

The invention also provides a probe-method dual-fluorescence quantitative PCR detection kit for detecting salmonella enteritidis, and the kit comprises the PCR primer group.

The invention also provides application of the primer group or the kit in identification and quantitative detection of salmonella enteritidis.

The invention also provides an identification method of salmonella enteritidis, which comprises the following steps:

1) preparing a PCR reaction system: preparing a PCR reaction system by using the probe method dual-fluorescence quantitative PCR detection kit and the genome DNA of a sample to be detected;

2) and (3) PCR reaction: carrying out PCR amplification on the prepared PCR reaction system;

3) and (4) analyzing results: the sample to be detected, which can simultaneously amplify the lysD gene and the invA gene, is the salmonella enteritidis, and the sample to be detected, which cannot simultaneously amplify the lysD gene and the invA gene, is not the salmonella enteritidis.

The invention also provides a method for quantitatively detecting salmonella enteritidis, which comprises the following steps:

1) preparing a PCR reaction system of a standard curve: carrying out gradient dilution on positive control DNA in the probe-method dual-fluorescence quantitative PCR detection kit, and respectively using the positive control DNA subjected to gradient dilution as a template to prepare a reaction system of a standard curve;

2) preparing a PCR reaction system of a sample to be detected: preparing a PCR reaction system of a sample to be detected by using the probe method dual-fluorescence quantitative PCR detection kit by using the genome DNA of the sample to be detected as a template;

3) preparing a negative control PCR reaction system: preparing a negative control PCR reaction system by taking negative control DNA in the probe method dual-fluorescence quantitative PCR detection kit as a template;

4) and (3) PCR reaction: carrying out PCR amplification on each prepared PCR reaction system;

5) and (4) analyzing results: and substituting the amplification signals of each PCR reaction system into the standard curve to calculate the content of the salmonella enteritidis in the sample to be detected.

As mentioned above, the probe-method dual-fluorescence quantitative PCR primer group and the kit for rapidly identifying and quantifying salmonella enteritidis have the following beneficial effects: the invention firstly detects the distribution of the lygD gene and the invA gene in different serotype salmonella and other bacteria by two pairs of specific primers through a probe method dual fluorescence quantitative PCR technology. The kit can rapidly identify and quantify the salmonella enteritidis in high flux, can replace the traditional salmonella serological typing, biochemical identification and other complex procedures, and provides a new method with simplicity, rapidness and good repeatability for the monitoring and laboratory diagnosis of the salmonella enteritidis.

Drawings

FIG. 1 shows the dual fluorescent quantitative PCR standard curve established by the probe method based on the lysD gene and invA gene and the sensitivity analysis thereof according to the present invention; wherein the lygD gene is a specific gene of the salmonella enteritidis, and the invA gene is a reference gene for identifying the salmonella. The detection sensitivity of the method is determined by detecting the genomic DNA of the salmonella enteritidis C50041 with different concentrations. (A) Detecting an amplification curve of the salmonella enteritidis by a probe method dual fluorescence quantitative PCR; (B) and (3) establishing a dual-fluorescence quantitative PCR lygD and invA gene standard curve by a probe method.

FIG. 2 shows the detection and quantitative analysis of Salmonella enteritidis in mouse viscera by probe-based dual fluorescent quantitative PCR; wherein, the bacterial counts of the liver and the spleen are respectively detected by a plate counting method, and the bacterial counts of the viscera are respectively calculated by the established probe method double fluorescence quantitative PCR standard curve.

Detailed Description

The invention firstly provides a PCR primer group for detecting salmonella enteritidis, wherein the PCR primer group comprises primer pairs for respectively detecting a lygD gene and an invA gene.

In one embodiment, the PCR primer set includes any one or more of:

1) a first primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;

2) a first probe primer: the nucleotide sequence is shown as SEQ ID NO. 3;

3) a second primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5;

4) a second probe primer: the nucleotide sequence is shown as SEQ ID NO. 6.

The first primer pair is used for detecting the lysD gene, the second primer pair is used for detecting the invA gene, the lysD gene only exists in salmonella enteritidis, the invA gene only exists in salmonella, and the lysD and invA double genes can accurately identify whether a sample to be detected is salmonella enteritidis.

One end of each of the first probe primer and the second probe primer is respectively marked with a fluorescent reporter group, and the other end of each of the first probe primer and the second probe primer is respectively marked with a fluorescent quenching group.

Preferably, the first probe primer and the second probe primer are labeled with different fluorescent reporter groups at the 5 'end and different fluorescent quencher groups at the 3' end.

Specifically, the fluorescent reporter group is selected from one of the following: FAM, HEX, VIC, CY3, ROX, 610, TEXAS RED, CY 5.

Specifically, the fluorescence quenching group corresponds to a fluorescence reporter group, and can quench the corresponding fluorescence reporter group. For example, when the fluorescence reporter is FAM, the fluorescence quencher is selected from one of the following: BHQ1, Dabcyl, TAMRA, MGB.

Preferably, the fluorescence reporter groups marked at the 5 'ends of the first probe primer and the second probe primer are CY5 and FAM fluorescent groups respectively, and the fluorescence quencher groups marked at the 3' ends are BHQ2 and TAMRA respectively.

In one embodiment, the PCR primer set includes a first primer pair, a first probe primer, a second primer pair, and a second probe primer.

The specific base sequence of the primer pair exemplified above may be replaced with 1 or more bases or 1 or more bases may be added to the 3 'end or 5' end, as long as the specific recognition region can be specifically recognized under the conditions for carrying out PCR (preferably, annealing and self-annealing do not occur between primers used in a single reaction vessel). The number of the plurality is, for example, 2 to 3. When 1 or more bases are added to the primer, it is preferable to add the base to the 5' end of the primer.

The identity of a nucleotide sequence obtained by substituting 1 or more nucleotides in a specific nucleotide sequence of the primer set exemplified above for other nucleotides is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more, with the nucleotide sequence before substitution (i.e., the nucleotide sequence represented by the sequence number).

The length of each primer is not particularly limited as long as it can specifically recognize the corresponding specific recognition region and hybridization does not occur between the primers, and is preferably 15 bases or more and 40 bases or less. The lower limit of the length of the primer is more preferably 16 bases or more, still more preferably 17 bases or more, and still more preferably 18 bases or more. More preferably, the upper limit of the length of the primer is 39 bases or less, still more preferably 38 bases or less, and still more preferably 37 bases or less.

The invention also provides application of the PCR primer group in preparation of a detection product of the lygD gene and the invA gene.

In one embodiment, the lygD gene and invA gene detection product is a salmonella enteritidis identification or quantification product.

In one embodiment, the salmonella enteritidis identification or quantification product is a kit.

The invention also provides a probe-method dual-fluorescence quantitative PCR detection kit for detecting salmonella enteritidis, and the kit comprises the PCR primer group.

Other conventional reagents required for fluorescent quantitative PCR can also be included in the kit. The kit also comprises one or more of water, Premix Ex Taq Master, ROX Reference Dye II and a sample genome DNA extraction reagent. Since the common PCR reagents can be purchased separately or configured by themselves through the market, the reagents can be assembled into the kit according to the actual needs of customers, and can be assembled into the kit for convenience.

The kit of the present invention may include the above reagents packaged separately, or may be a prepared fluorescent quantitative PCR detection solution containing the primer set. The PCR detection solution can be prepared by itself, or can be obtained by directly adding a commercial general PCR detection solution without primers into the primer group. In one embodiment, the PCR assay solution has a concentration of lygD-F and lygD-R of 240nM, a concentration of lygD-P of 100nM, a concentration of invA-F and invA-R of 200nM, and a concentration of invA-P of 80nM, respectively.

In one embodiment, the kit may further comprise a positive control DNA. The positive control DNA is DNA containing salmonella enteritidis genome.

In one embodiment, the kit may further comprise a negative control DNA. The negative control DNA is the DNA of Salmonella non-enteritis.

The kit provided by the invention adopts a probe method dual fluorescent quantitative PCR detection technology to detect the lygD gene and the invA gene, and whether a detection object belongs to salmonella enteritidis can be analyzed and judged according to amplification and detection conditions. Therefore, the design of the primer is one of the keys of the kit of the invention.

The invention also provides application of the primer group or the kit in identification and quantitative detection of salmonella enteritidis. The primer group or the kit can rapidly identify whether a sample to be detected is salmonella enteritidis and the content of the salmonella enteritidis.

The invention also provides an identification method of salmonella enteritidis, which comprises the following steps:

1) preparing a PCR reaction system: preparing a PCR reaction system by using the probe method dual-fluorescence quantitative PCR detection kit and the genome DNA of a sample to be detected;

2) and (3) PCR reaction: carrying out PCR amplification on the prepared PCR reaction system;

3) and (4) analyzing results: the sample to be detected, which can simultaneously amplify the lysD gene and the invA gene, is the salmonella enteritidis, and the sample to be detected, which cannot simultaneously amplify the lysD gene and the invA gene, is not the salmonella enteritidis.

The sample to be detected is selected from substances easily polluted by salmonella enteritidis. In one embodiment, the sample to be tested is selected from food, water, poultry, and the like. The identification method is a method for non-disease diagnostic purposes.

Specifically, the PCR reaction system prepared in step 1) comprises a reaction system of a sample to be detected, a reaction system of positive control and a reaction system of negative control.

In one embodiment, the PCR reaction system is a 25 μ L system comprising: 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L.

Other volumes of PCR reaction system can be changed proportionally according to the 25. mu.L system.

In one embodiment, the conditions of the PCR reaction are: (a) 30s at 95 ℃; (b) 5s at 95 ℃; (c) 34s at 60 ℃, and the steps (b) to (c) are circulated for 40 times.

The invention also provides a method for quantitatively detecting salmonella enteritidis, which comprises the following steps:

1) preparing a PCR reaction system of a standard curve: carrying out gradient dilution on positive control DNA in the probe-method dual-fluorescence quantitative PCR detection kit, and respectively using the positive control DNA subjected to gradient dilution as a template to prepare a reaction system of a standard curve;

2) preparing a PCR reaction system of a sample to be detected: preparing a PCR reaction system of a sample to be detected by using the probe method dual-fluorescence quantitative PCR detection kit by using the genome DNA of the sample to be detected as a template;

3) preparing a negative control PCR reaction system: preparing a negative control PCR reaction system by taking negative control DNA in the probe method dual-fluorescence quantitative PCR detection kit as a template;

4) and (3) PCR reaction: carrying out PCR amplification on each prepared PCR reaction system;

5) and (4) analyzing results: and substituting the amplification signals of each PCR reaction system into the standard curve to calculate the content of the salmonella enteritidis in the sample to be detected.

In one embodiment, the PCR reaction system is a 25 μ L system comprising: 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L.

Other volumes of PCR reaction system can be changed proportionally according to the 25. mu.L system.

In one embodiment, the conditions of the PCR reaction are: (a) 30s at 95 ℃; (b) 5s at 95 ℃; (c) 34s at 60 ℃, and the steps (b) to (c) are circulated for 40 times.

The method for quantitatively detecting salmonella enteritidis is a method for non-disease diagnosis purposes.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

EXAMPLE 1 preparation of the kit

Designing and synthesizing a primer: respectively taking a lygD gene (nucleotide sequence is shown as SEQ ID NO.7) and an invA gene (nucleotide sequence is shown as SEQ ID NO.8) as templates, designing and analyzing primers, and selecting an optimal detection primer pair and a probe primer according to the condition of a genome DNA sequence, wherein the lygD primer is used for amplifying a partial sequence (111bp), and the probe primer is marked by Cy5 and is used for detecting salmonella enteritidis; the invA primer amplifies partial sequences (105bp), the probe primer is a FAM label and is used for detecting the salmonella, and the nucleotide sequences of two pairs of detection primers and probe primers are shown in Table 1.

TABLE 1 nucleotide sequences of dual fluorescent quantitative PCR detection primers and probe primers by probe method

The primer pair can be packaged independently or can be prepared into PCR detection solution. In the PCR detection solution, 240nM of lysD-F/R (the concentrations of lysD-F and lysD-R are 240nM, respectively) and 100nM of lysD-P and 200nM of invA-F/R (the concentrations of invA-F and invA-R are 200nM, respectively) and 80nM of invA-P were used as the amounts of the above primer pairs.

That is, the kit of the present invention may contain the primer set packaged independently, or may contain a PCR detection solution containing the primer set.

Further, the kit further contains sterile water (ddH)₂O)、Premix Ex Taq Master、ROX Reference Dye II。

Example 2 specific identification of the kit for detecting Salmonella enteritidis

The distribution characteristics of the lygD gene and the invA gene in different bacteria are identified by a probe method dual fluorescence quantitative PCR method by adopting two pairs of detection primers and probe primers in the kit of example 1 and taking genomes of different serotypes of salmonella and other non-salmonella bacteria as templates respectively.

The PCR reaction system was (25. mu.L): 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L.

The PCR program is 95 ℃ for 30 s; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.

Acquiring amplification signals through a fluorescent quantitative PCR instrument, wherein PCR results show that two amplification signals, namely, lygD and invA, can be acquired by taking salmonella enteritidis genome as a template; only one signal, the invA gene, was amplified using the genome of other serotypes of salmonella than enteritis as template (table 2). It is shown that whether the unknown bacteria are salmonella enteritidis can be rapidly identified by using the specific lysD and invA amplification primers and probe primers in the kit of example 1 through a probe method dual fluorescence quantitative PCR method. Namely, the lysD gene only exists in the salmonella enteritidis, the invA gene only exists in the salmonella, and the dual-gene detection of the lysD and invA can accurately identify the salmonella enteritidis.

TABLE 2 specificity identification of dual fluorescent quantitative PCR detection of Salmonella enteritidis by probe method

Example 3 sensitivity identification of kit for detecting Salmonella enteritidis

The two pairs of detection primers and probe primers in the kit described in example 1 are adopted to sequentially dilute the salmonella enteritidis C50041 genome by 10 times, and the sensitivity of the kit for detecting the salmonella enteritidis genomic DNA is identified by taking the diluted genome as a template.

The PCR reaction system was (25. mu.L): 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L.

The PCR program is 95 ℃ for 30 s; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.

Amplification signals of DNA templates with different concentrations are collected through a fluorescent quantitative PCR instrument, and a dual fluorescent quantitative PCR standard curve based on a probe method of the lygD gene and the invA gene is established. The PCR results show that the dual PCR detection kit based on lygD and invA can detect the Salmonella enteritidis of 4 copies/system (FIG. 1).

Example 4 quantitative determination of Salmonella enteritidis in mouse organs with the kit

The two pairs of detection primers and probe primers in the kit of the embodiment 1 are adopted to detect the salmonella enteritidis in the internal organs of the mice, so that the salmonella enteritidis can be quickly and accurately detected and quantified. The method comprises the following specific steps:

will be 5X 10⁴And (3) perfusing the CFU salmonella enteritidis to infect the mice, collecting the livers and spleens of the mice 3 days after infection, homogenizing the organs, and detecting the bacterial-carrying quantity of the organs by a plate counting method. Meanwhile, diluting the homogeneous solution, extracting bacterial genome DNA, and detecting the content of salmonella enteritidis genome through the established kit.

The PCR reaction system was (25. mu.L): 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L.

The PCR program is 95 ℃ for 30 s; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.

The amplification signals of the salmonella DNA templates of different internal organs are collected by a fluorescent quantitative PCR instrument, the content of the salmonella in the internal organs is calculated by a standard curve and compared with a plate counting method, and the result shows that the two detection and counting methods have no significant difference (figure 2). The PCR result shows that the dual PCR detection kit based on the lygD and the invA can accurately and rapidly detect the content of the salmonella enteritidis in the viscera.

Example 5 application of kit in detection of Salmonella enteritidis in chick embryo

By adopting the reagent kit in the embodiment 1, the salmonella enteritidis in 70 dead chick embryos can be detected, and the salmonella enteritidis can be detected quickly and accurately. The method comprises the following specific steps:

1) isolation and classical serotype identification of Salmonella

In the test, a sample is collected from a chicken farm in Jiangsu, the sample is processed, enriched, separated and physiologically and biochemically identified by referring to a method (Li Y, et al. Food Control, 2016; Cai Y, et al. int J Food Microbiol,2016) established in the prior art, and 27 strains of salmonella containing 23 strains of salmonella enteritidis are co-separated and identified by the traditional method.

2) Probe method double-fluorescence quantitative PCR method for detecting salmonella enteritidis in sample

Collecting 70 dead chick embryo livers and yolks, respectively carrying out pre-enrichment in BPW enrichment solution, carrying out enrichment for 6h at 37 ℃ and 100rpm, extracting genomes of each sample by using a bacterial genome extraction kit, amplifying a lygD gene and an invA gene by using the genomes as templates, wherein a PCR reaction system is (25 mu L): 2 × Premix Ex Taq Master 12.5 μ L, lygD-F240nM, lygD-R240 nM, lygD-P100 nM, invA-F200 nM, invA-R200 nM, invA-P80 nM, DNA template 2.5 μ L, ROX Reference Dye II 0.25 μ L, ddH₂And O is supplemented to 25 mu L. The PCR program is 95 ℃ for 30 s; the Salmonella enteritidis can be simultaneously amplified by 40 cycles of 95 ℃ for 5s and 60 ℃ for 34s, and the genes of lygD and invA can be simultaneously amplified. The results showed that 26 out of 70 chick embryo samples could simultaneously detect the lygD and invA genes, which are Salmonella enteritidis (Table 3).

TABLE 3 Probe-method double fluorescent quantitative PCR identification of Salmonella enteritidis contamination in chick embryo

And SE: salmonella enteritidis; SW: -weiteff leidenden salmonella; SL: salmonella london.

In this example, the serotype identification and biochemical experiment identification method is adopted to screen out salmonella enteritidis from 70 chick embryo samples, and at least 2 days are required. By adopting the detection kit provided by the embodiment 1 of the invention, 26 salmonella enteritidis strains can be accurately screened out only in 7 hours, and the traditional salmonella separation and serotype identification method identifies 23 salmonella enteritidis strains, which shows that the established probe-method dual-fluorescence quantitative PCR has higher sensitivity compared with the traditional method.

In conclusion, compared with the traditional serotype identification method, the kit disclosed by the invention has the advantages that:

the conventional serotype identification needs to purchase a specific salmonella serotype identification kit, so that the price is high, the steps are complicated, and particularly, when a large sample is separated from specific salmonella serotypes (such as salmonella enteritidis), a large amount of time (at least two days) is consumed, the workload is huge, and the result is judged by naked eyes, so that human errors possibly exist; the detection method of the kit is simple to operate, the whole process from sample collection to bacterium identification can be completed within seven hours, the sensitivity is higher than that of the traditional salmonella separation and identification method, and the quantitative analysis can be performed on the salmonella in a specific sample.

Therefore, the probe-method dual-fluorescence quantitative PCR detection kit for rapidly identifying and quantifying salmonella enteritidis is beneficial to simplifying the traditional step of salmonella serotype identification, and provides a new method for rapidly identifying salmonella enteritidis in a large number of samples.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> Yangzhou university

<120> probe-method dual-fluorescence quantitative PCR primer group and kit for rapidly identifying and quantifying salmonella enteritidis

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