CN110714089B - PCR primer of specific targeting flagellum hook gene flgE and application thereof in rapid detection of flagella and flagellum-free salmonella - Google Patents

Abstract

The invention discloses a specific targeting flagellum hook geneflgEThe PCR primer and the application thereof in the rapid detection of flagella and flagellum-free salmonella belong to the technical field of microbial detection. The specific targeting flagellum hook gene of the inventionflgEThe PCR primer comprises an upstream primerflgE-UP and downstream primersflgELO, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The primer targets flagellum gene specificallyflgEThe PCR rapid detection method for all salmonella including flagellate salmonella and nonflagellate salmonella has the advantages of rapid, sensitive and specific detection, simple operation and low cost, accurately detects the salmonella existing and polluted in a sample to be detected, can obtain a detection result within 24 hours, and provides effective technical support for monitoring and preventing and controlling salmonella infection of human beings, livestock and various animals in food, feed, environmental samples or excrement.

Description

PCR primer of specific targeting flagellum hook gene flgE and application thereof in rapid detection of flagella and flagellum-free salmonella

Technical Field

The invention relates to a PCR primer of a specific targeting flagellar hook gene flgE and application thereof in rapid detection of flagellated and nonflagellated salmonella, belonging to the technical field of microbial detection.

Background

Salmonella is widely distributed in nature and possesses numerous serotypes, and to date, there are 2600 more salmonella serotypes identified. Wherein a substantial portion of the serotype salmonella bacteria are capable of infecting humans and animals, including typhoid fever and food poisoning of human infections; in veterinary medicine, Salmonella infections causing pullorum disease, fowl typhoid and chicken paratyphoid disease, as well as Salmonella infections and diseases in many other animal species, severely compromise the development of poultry and animal husbandry. Meanwhile, the salmonella is also an important food-borne pathogenic microorganism, and has important public health significance. Therefore, the development of a rapid, accurate, convenient to operate and low-cost salmonella detection method has important significance for salmonella detection, monitoring, salmonella infection control and global human and animal safety maintenance.

The classical method based on bacterial culture, isolation, biochemical profiling and serotyping has long been recognized as the "gold standard" for salmonella detection. However, the conventional salmonella detection process is time-consuming and labor-consuming, and although the slide agglutination test based on the thalli antigen (O) and the flagellum antigen (H) is simple and rapid to complete detection, the detection sensitivity is low, false positive results are easy to generate, and the method requires naked eyes for observation, so that subjectivity and result misjudgment exist. Notably, conventional serotype detection methods are not economical because the large number of complex salmonella serotypes means that a large number of different single (multi-) factor detection sera must be prepared to complete the detection of the system, and commercial specific detection sera are often complex to prepare and expensive.

In recent years, Polymerase Chain Reaction (PCR) has been widely used for the detection of various pathogens as a sensitive, rapid and specific identification method. To date, several bacterial surface antigens (e.g., flagella and pili) as well as genes encoding, and regulatory genes for bacterial virulence factors have been used as targets for the identification of salmonella.

Bacterial flagella are one of the bacterial surface accessory structures, consisting of codes for a specific gene family in the bacterial chromosome, and in the bacterial genome, approximately 60 genes are involved in the synthesis and regulation of flagella. In salmonella, except pullorum disease and typhoid, all salmonella have flagella structure and function on the surface. Structurally, an intact flagella comprises a basal portion embedded in a cell membrane, a flagella hook free from bacteria, and a flagella filament. The rotary motion of the flagella filaments enables bacteria to have motility, benefit and avoid harm, swim to an environment which is beneficial to the bacteria, can endow the bacteria with motility, is also an important virulence factor of pathogenic bacteria, and plays an important role in pathogenic processes of the bacteria (such as adhesion and invasion to host cells, biofilm formation, intracellular survival and the like).

Flagella are both a bacterial surface antigen and an important virulence factor of pathogenic bacteria, and the flagella of salmonella are considered as salmonella detection targets with great application potential, for example, a flagellin protein coding gene (fliC) has been reported to be used for detecting salmonella. However, the methods reported to date, which use flagella-associated genes (e.g., fliC gene) as targets, are almost exclusively directed against salmonella of a single serotype. For example, there have been reports of PCR detection methods for salmonella choleraesuis (s.cholereasuis) and salmonella typhi (s.typhi), respectively, using fliC gene as a target.

However, no studies have been reported so far for detecting Salmonella which do not produce flagella by a PCR method established using a flagella-related gene as a target gene, and no studies have been reported for detecting all Salmonella (with and without flagella). In view of the fact that the bacterial surfaces of the salmonella pullorum and the salmonella gallinarum have no flagella accessory structures, and the existence of flagella related genes in the genomes of the two bacteria is considered and analyzed for a long time for the salmonella with a flagella-free phenotype, so that the strategy and the implementation of identifying the salmonella without flagella by using specific flagella genes are greatly limited, and particularly all the salmonella (all the salmonella with flagella and without flagella) are identified.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a PCR primer of a specific targeting flagellar hook gene flgE and application thereof in quickly detecting flagellated salmonella and flagellaless salmonella.

The inventor of the application discovers that although the bacterial surfaces of the pullorum disease and the salmonella gallinarum do not have flagella structures through the analysis of the genome of the pullorum disease and the salmonella gallinarum, the genome of the pullorum disease and the salmonella gallinarum contains flagella related coding genes. Through sequence comparison analysis, the flagella related gene of the salmonella pullorum is found to have extremely high homology and similarity with flagella related genes of other salmonella flagellates (such as salmonella enteritidis). This finding provides the possibility to invent a method for simultaneously detecting all salmonella of both "flagella" and "nonflagellates" with specific flagella-related genes as targets.

On the basis, after comparing flagella related genes of various different types of bacteria, the inventor definitely verifies and selects the flagellin hook protein coding gene flgE as a target gene for detecting salmonella, and realizes the PCR (polymerase chain reaction) simultaneous and rapid detection of flagella and flagellaless salmonella. The key link of the implementation is that the inventor discovers two unique sequences in the gene sequence, the sequences are highly conserved in all salmonella, only exist in salmonella, but not exist in non-salmonella, and the primer is very suitable for designing the flgE specific primer of the targeted flagella gene for the rapid PCR identification of the salmonella.

To further determine the specificity and applicability of the method, the present inventors first verified that the flgE gene is indeed present in all Salmonella nonfilaginosa (pullorum and fowl typhoid) and that its flgE sequence has 100% homology with the flgE sequence of Salmonella flagellata (e.g., Salmonella enteritidis) by PCR amplification of a full-length fragment of the flgE gene and sequencing verification. This means that the PCR detection method for Salmonella based on the specific targeting flagella gene flgE is also suitable for the detection of pullorum disease and Salmonella gallinarum.

Based on the above-described results and verification, pullorum disease and salmonella gallinarum do not have a flagella structure on the bacterial surface, but have a full set of genes encoding flagella in their genome (although they do not encode a functional flagella structure on the bacterial surface, flagella do not occur on the bacterial surface), and have high homology with flagella-related genes of salmonella flagelliforme. That is, all salmonella (including flagellated and nonflagellated salmonella) chromosomal genomes possess flagella-associated genes. This is a prerequisite and key to the discovery and implementation of the present invention patent. The applicant noticed that although all salmonella have flagella related genes, the various flagella related genes are conserved in different salmonella species, after analysis and comparison, a salmonella specific segment exists in the flagella hook gene flgE, the segment exists only in salmonella, is highly conserved in salmonella, is definitely a specific target gene, and a suitable primer is designed in the segment to perform specific identification for specifically targeting all salmonella. This is a strong guarantee of the effectiveness of the invention of the present application.

Based on the discovery and the characteristics, the invention provides a PCR primer of a specific targeting flagellar hook gene flgE, which comprises an upstream primer flgE-UP and a downstream primer flgE-LO, wherein the nucleotide sequences are respectively as follows:

flgE-UP:5'-ACGGACCCTGTACCGTCTAAA-3' (shown as SEQ ID NO. 1),

flgE-LO:5'-TGATGTTCACCGTACCGCC-3' (shown in SEQ ID NO. 2).

Further, the upstream primer and the downstream primer specifically target two specific DNA segments of the flgE gene of the Salmonella flagellium hook, which are selected by bioinformatics analysis and multiple comparative analysis of flgE gene sequences of strains including Salmonella flagellata, Salmonella amastiformis and Salmonella nontyphimurium.

Further, the flgE gene sequences of the Salmonella flagellata and non-Salmonella strains are derived from GenBank published sequences; one part of the flgE gene sequence of the salmonella amastii strain is from a sequence published by GenBank, and the other part of the flgE gene sequence is from a nucleic acid sequence obtained by sequencing flgE genes of pullorum disease and typhoid chicken strains separated, collected and stored in a laboratory.

The invention also provides application of the PCR primer of the specific targeting flagellum hook gene flgE, which is used for PCR rapid detection of all salmonella including flagellate salmonella and flagellate salmonella.

The invention also provides a PCR rapid detection kit for the salmonella flagellate and the salmonella amastigotica, and the kit comprises the PCR primer.

Further, the kit also comprises PCR reaction buffer solution, dNTPs and rTaq DNA polymerase.

Further, the PCR rapid detection method of all Salmonella including Salmonella flagellata and Salmonella nonflagellata comprises the following steps:

and (3) taking a sample to be detected as a template, carrying out PCR amplification reaction by using the primer, carrying out agarose gel electrophoresis on an amplification product, placing the gel in an imaging system, observing whether an amplification strip with an expected size appears at 262bp of the sample to be detected, and judging whether the sample to be detected contains salmonella or is non-salmonella.

Further, when the sample to be detected is food, animal feed, an environmental sample or excrement, selective enrichment of salmonella is firstly carried out, then, the chromosomal DNA prepared by the enrichment liquid is used as a template for PCR amplification, and the result is judged to judge whether the sample contains salmonella within 24 hours.

Further, when the sample to be detected is a pure bacterial culture, the PCR amplification is directly carried out by taking the chromosome DNA prepared by the bacterial liquid as a template, and the result is judged, so that whether the sample to be detected is salmonella or not is identified within 3 hours.

Further, the reaction system of the PCR amplification is as follows: 2.5 mu L of 10 XPCR reaction buffer solution, 2 mu L of dNTPs with the concentration of 2.5mM, 1 mu L of upstream primer with the concentration of 10 mu M, 1 mu L of downstream primer with the concentration of 10 mu M, 1.5U of rTaq DNA polymerase and 1 mu L of DNA template, sterile double distilled water is added to make up the final volume to be 25 mu L, and a positive control (DNA template of different salmonella) and a negative control (DNA template of non-salmonella) are added;

the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 45s, extension at 72 ℃ for 45s, 30 cycles; extension at 72 ℃ for 10 min.

The invention has the following beneficial effects:

1. the primer disclosed by the invention can be used for quickly detecting all salmonella including flagellate salmonella and flagellate-free salmonella by virtue of specific targeting flagella gene flgE and PCR, is quick, sensitive and specific in detection, simple to operate and low in cost, and can be used for accurately detecting the salmonella existing and polluted in a sample to be detected (food, drinking water, dust, excrement sample, environment, animal feed and the like). The detection result can be obtained within 24 hours, and effective technical support is provided for monitoring salmonella in food and feed environments, preventing and controlling salmonella infection of human beings, livestock and various animals.

2. The detection method has the advantages that the cost of the required equipment and reagents is low, the synthesized primer can be stored for a long time at the temperature of-20 ℃, can be taken out at any time for use, and is convenient and rapid.

3. The method is suitable for large-batch detection, can be combined with the traditional detection method for use, is applied to the primary screening link after selective enrichment, can quickly and conveniently know whether the sample contains salmonella, greatly reduces the workload of detection work, saves labor force and shortens the working time of the whole detection link.

Drawings

FIG. 1 is a graph showing the results of experiments in example 1 of the present invention in which the presence of flgE gene was confirmed in all Salmonella nonfilamentosa (pullorum disease and Salmonella gallinarum) using the PCR method;

wherein, M: 2K plus DNA ladder. Lanes 1-7: and (3) detection results by taking different salmonella gallinarum genome DNAs as templates. Lanes 8-26: and (3) detection results by taking different salmonella pullorum genomic DNAs as templates. Lane 27: positive control, results of amplification using genomic DNA of a strain known to contain the flgE gene (salmonella enteritidis SE50336) as a template. Lane 28: the negative control was amplified using known non-salmonella genomic DNA as a template, and escherichia coli DH5 α genomic DNA as a template.

FIG. 2 is a graph showing the effect of annealing temperature on the PCR reaction for specifically targeting flgE in example 2 of the present invention;

wherein, M: DL2000 DNA ladder. Lanes 1-12: the amplification effect is shown under the condition that the annealing temperature is 50-61 ℃.

FIG. 3 is a graph showing the effect of magnesium ion concentration on the response of a specific target flgE in example 2 of the present invention;

wherein, M: DL2000 DNA ladder. Lanes 1-11: different final concentrations of MgCl₂(0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0mM) PCR amplification system.

FIG. 4 shows the specific detection of the specific target flgE by the PCR method in example 3 of the present invention;

wherein, M is DL2000 DNA ladder. Lanes 1-31, results from amplification of Salmonella flagellata. Lanes 32-57 No Salmonella flagellata amplification results. Lanes 58-76: amplification results of non-salmonella strains. PC: positive control, result of amplification using known positive bacteria (SE50336) as template.

FIG. 5 is a graph showing the result of the minimum detection limit measurement of reference Salmonella genomic DNA by the PCR method for specifically targeting the flgE gene in example 4 of the present invention;

wherein, M is DL2000 DNA ladder. Lanes 1-7: amplification results using bacterial genomic DNA at different concentrations (183 ng/. mu.L, 18.3 ng/. mu.L, 1.83 ng/. mu.L, 183 pg/. mu.L, 18.3 pg/. mu.L, 1.83 pg/. mu.L, 183 fg/. mu.L) as templates.

FIG. 6 is a graph showing the results of the determination of the minimum detection limit of the number of reference Salmonella bacterial cells by the PCR method for specifically targeting the flgE gene in example 5 of the present invention;

wherein, M is DL2000 DNAA ladder. Lanes 1-6: to contain different cell numbers (10)⁶CFU,10⁵CFU,10⁴CFU,10³CFU,10²CFU and 10¹CFU) as a template.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1: primer design

The inventor discovers that the chromosome genomes of the salmonella pullorum and the salmonella gallinarum contain flagella related coding gene clusters although the surfaces of the salmonella gallinarum and the salmonella gallinarum do not have flagella accessory structures through comprehensive analysis on the genomes of the salmonella gallinarum and the salmonella gallinarum. Further, through sequence comparison analysis, specific flagella related genes in the chicken white diarrhea and chicken typhoid salmonella chromosomes have extremely high homology and similarity with flagella related genes of other flagella (such as salmonella enteritidis).

On the basis, after comparing flagella related genes of various different types of bacteria, the applicant selects the flagellin encoding gene flgE as a target spot for identifying salmonella, and realizes the simultaneous detection of flagella and nonflagenaria. The key reason for the implementation is that we found two unique sequences in the gene sequence, which are highly conserved in all salmonella and exist only in salmonella, but not in non-salmonella, and are very suitable for designing specific primers for rapid PCR identification of salmonella.

To further determine the specificity and applicability of the method, the present inventors first detected the existing nonflagellated salmonella (pullorum disease and fowl typhoid) in this laboratory by a PCR detection method of specifically targeted amplifying the full-length fragment of flgE gene, and confirmed that the flgE gene is indeed present in all nonflagellated salmonella (pullorum disease and fowl typhoid) (see FIG. 1). On the basis of confirming that all the salmonella amastigotis has the flgE gene, the inventor further sequences the obtained flgE gene fragments of pullorum disease and fowl typhoid fever, and confirms that the flgE gene sequence of the salmonella amastigotis has 100 percent of homology with the flgE sequence of the salmonella flagelligotis (salmonella enteritidis). The invention also discloses a nucleic acid sequence (SEQ ID No.3) of the flgE gene of the Salmonella pullorum CVCC523 strain obtained by sequencing the unit, and the flgE gene sequences of all strains of Salmonella nonfilamentosa are 100% identical and completely identical to the sequence disclosed by the invention (all other obtained flgE gene sequences are not listed) as confirmed by multiple alignments. It was also a very valuable finding that the flgE gene-based PCR identification method for Salmonella is equally applicable to the identification of nonflagellate pullorum and Salmonella gallinarum.

In combination with the above findings and further experimental verification, primer sequences were designed as follows:

flgE-UP:5'-ACGGACCCTGTACCGTCTAAA-3' (shown as SEQ ID NO. 1),

flgE-LO:5'-TGATGTTCACCGTACCGCC-3' (shown in SEQ ID NO. 2).

Example 2: establishment of FlgE gene-based salmonella PCR detection method

1. Reagents and materials required:

reagent: rTaq DNA polymerase, dNTPs, MgCl₂Purchased from Takara corporation.

Strain: salmonella enteritidis Standard strain CMCC (B)50336, offered by professor Pyrin university of Yangzhou.

Preparing a template:

and taking a single colony of a sample to be detected on an agar culture dish to inoculate the single colony in a liquid LB culture medium. Culturing at 37 ℃ for 18h, putting 1.5mL of the bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 12000rpm for 10min, washing twice with sterile PBS, centrifuging at 12000rpm for 10min, resuspending the precipitate with 200 mu L of sterile double-distilled water, centrifuging at 12000rpm for 10min in a boiling water bath, and taking the supernatant to be used as a template for PCR reaction.

PCR amplification method:

the following PCR system was configured:

10 XPCR reaction buffer 2.5 uL, dNTPs (2.5mM)2 uL, upstream primer (10 uM) 1 uL, downstream primer (10 uM), 1 uL, rTaq DNA polymerase 1.5U, template 1 uL, sterile double distilled water to make up to the final volume of 25 uL. A positive control (DNA template of different salmonella) and a negative control (DNA template of non-salmonella) are arranged at the same time. The components are sequentially added into a PCR reaction tube, fully and uniformly mixed, the reaction solution is gathered at the bottom of the tube by low-speed instantaneous centrifugation, and the PCR reaction tube is placed in a PCR instrument.

The following PCR amplification parameters were set:

the reaction conditions are as follows: firstly, pre-denaturation is carried out for 4min at 94 ℃; then denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 45s for 30 cycles; final extension at 72 ℃ for 10 min.

3. Agarose gel electrophoresis detection of PCR products:

after the PCR reaction, 5. mu.L of the PCR product was mixed with 1. mu.L of 6 Xloading buffer solution and added to the well. Sample 5. mu.L of DNA marker. Electrophoresis at 100V for about 1 h. The results were observed with a uv gel imager.

4. And (4) observation and judgment:

if the positive control shows an amplified band only at 262bp, and the negative control shows no band at 262bp, the test is established, in this case, if the sample to be tested shows a band at 262bp, the sample contains salmonella, or the test strain is salmonella; correspondingly, if the sample to be detected has no amplification band at 262bp, the sample does not contain salmonella or the strain to be detected is non-salmonella.

5. Optimum annealing temperature condition exploration

On the basis of the basic reaction conditions and parameters, the annealing temperature is optimized, other conditions are unchanged, the annealing temperature in the reaction parameters is only changed, PCR amplification is carried out by using different annealing temperatures (50-61 ℃), the amplified products are identified by agarose gel electrophoresis, and the amplification effects under different annealing temperature conditions are compared. As shown in FIG. 2, the flgE gene band of 262bp can be amplified at different annealing temperatures (50 ℃ -61 ℃).

6. Optimum magnesium ion concentration condition groping

In the determination of annealingOn the basis of temperature, the final concentration of magnesium ions in the reaction system is optimized, other conditions are unchanged, only the final concentration of the magnesium ions in the reaction system is changed, the reaction systems containing the magnesium ions with different final concentrations are used for PCR amplification, and the amplification effects are compared. As shown in FIG. 3, the experiment shows that the reaction system is in MgCl₂The concentration range of 0.4-2.0mM can amplify the targeted gene fragment, and the optimal amplification effect can be achieved under the condition of 2.0mM enzyme ions.

Example 3: specific detection of PCR method for specific targeting of flgE gene

1. Bacterial strains

The strains used in the present invention are shown in Table 1.

TABLE 1 test strains and related information

Wherein the salmonella enteritidis domestic standard strain CMCC (B)50336 (hereinafter referred to as SE50336) is offered by professor of Pyrun an of Yangzhou university; salmonella enteritidis T48, T64, Salmonella typhimurium W32, T14, U27, W2, A12, Salmonella typhi W33, Salmonella choleraesuis U80, U81, U82 and Salmonella gallinarum U20 were offered by professor Liujunin university of Harbin medicine, Salmonella pullorum standard strains CVCC523, CVCC526, CVCC535 and CVCC540 were purchased from China veterinary culture collection center (CVCC), and Escherichia coli CE7 was offered by professor Chengping of university of agriculture of Nanjing. Staphylococci were from the animal veterinary institute of the agroforestry academy of sciences of Beijing. Other strains were kept by the laboratory. The Salmonella enteritidis domestic standard strain SE50336 is used as a reference strain in the embodiment related to the invention and is used for evaluating a positive standard reference strain in a minimum detection limit determination test and an artificial simulated contaminated feed test of the PCR method for the specific targeted flgE gene disclosed by the patent of the invention.

2. Specific detection of PCR method for specific targeting of flgE gene

Templates for each of the strains in Table 1 above were prepared as described in example 2.

Using the above strain template, PCR analysis was performed according to the PCR reaction conditions and parameters established in example 2. As shown in FIG. 4, the results indicate that the method of the present invention can achieve not only the detection of Salmonella flagellata (lanes 1-31) but also the detection of Salmonella nonflagenaria (lanes 32-57). In the current test range, the method can specifically detect salmonella, but cannot amplify any band for non-salmonella.

Example 4: determination of minimum detection limit of PCR method for specific targeting flgE gene on bacterial genome DNA

To determine the sensitivity of the PCR assay specifically targeting flgE, genomic DNA of Salmonella enteritidis strain SE50336 was first extracted and diluted in 10-fold gradient with sterile double distilled water (in this example, the genomic template concentration was diluted from 18.3 ng/. mu.L to 18.3 fg/. mu.L) and then used as the DNA template for the PCR reaction. The aim of this experiment was to determine the lowest DNA content that could be detected by the method for specifically targeting flgE. As can be seen from FIG. 5, the minimum detection limit of the method for bacterial genomic DNA was 18.3 pg/. mu.L.

Example 5: determination of minimum detection limit of specific targeting flgE gene PCR method on reference salmonella bacterial cell number

To determine the minimum number of bacterial cells that can be detected by PCR methods specifically targeting flgE. mu.L of the Salmonella enteritidis SE50336 strain broth was inoculated into 3mL of fresh sterile liquid LB and grown to mid-log phase at 37 ℃ with shaking at 200 rpm for about 1.5 h. The culture was first adjusted to an optical density of 1.0 (OD) at 600nm_{600 nm}1.0) and then serially diluted 10-fold in PBS. mu.L of each dilution was used directly as a template for PCR. Subsequently, 100. mu.L of each dilution was plated on LB agar plates, and then cultured overnight at 37 ℃ to determine Colony Forming Units (CFU) of SE50336, thereby judging bacterial cells detectable by the method for specifically targeting flgEThe minimum number of (c). As can be seen from FIG. 6, the minimum detection limit for the number of bacterial cells in the present method is 100CFU per response.

Example 6: the evaluation of the detection limit of the PCR method of the specific targeting flgE gene on the salmonella in the artificial simulated polluted feed is to evaluate the detection capability of the PCR method of the specific targeting flgE disclosed by the invention on the salmonella in the feed.

1. Preparation of bacterial liquid of salmonella enteritidis domestic standard strain CMCC (B)50336

Taking a salmonella enteritidis domestic standard strain CMCC (B)50336 culture plate, picking a single colony in 8mL LB culture solution under the aseptic condition, and culturing for 12h at 37 ℃ and 180 rpm. The culture solution was transferred to a 50mL centrifuge tube, centrifuged at 4000 rpm for 10min, and then the cells were washed repeatedly with sterile PBS solution 3 times to resuspend the bacterial solution. The bacterial concentration of the bacterial suspension was counted by plate counting and then diluted to a concentration of 10⁶、10⁵、10⁴、10³、10²、10¹100CFU/mL for use.

2. Preparation of chicken feed

Taking sterile and antibiotic-free chicken feed, respectively weighing 25g of the feed under the sterile condition, and placing the feed into 8 sterile and dry beakers (7 dilutions of bacterial liquid, each dilution corresponds to one beaker, and the 8 th beaker is used as negative control).

3. Salmonella enteritidis domestic standard strain CMCC (B)50336 artificial simulated contaminated feed sample preparation

The bacterial suspension of each dilution gradient was added to each beaker in sequence according to the inoculation amount of 1ml per beaker, and 1ml of sterile double distilled water was added to the 8 th beaker (negative control). Mixing thoroughly under aseptic condition.

4. Enrichment culture of artificial simulated polluted feed sample

Respectively transferring all the prepared feed samples into corresponding 8 flasks which are filled with 225ml of sterile peptone water (BPW) in advance, and carrying out pre-enrichment for 6h at 37 ℃; then inoculating 1ml of enrichment liquid into 9ml of selenite-cystine (SC) culture solution according to the proportion, respectively inoculating into the SC culture solution, and selectively enriching for 12h at 37 ℃. The template was prepared as described in example 2. Using the above template, PCR analysis was performed under the PCR reaction conditions and parameters established in example 3, and the test results are shown in Table 2.

TABLE 2 detection results of different feed samples

Sample (I)	Initial Salmonella content	PCR detection result of specific targeting flgE gene after enrichment
			Sample
1	106/25g	+
			Sample 2	105/25g	+
Sample 3	104/25g	+
			Sample 4	103/25g	+
Sample No. 5	102/25g	+
			Sample No. 6	101/25g	+
Sample 7	100/25g	-
			Sample 8 (negative control)	-	-

According to the results, the minimum detection limit of the salmonella bacteria number in the simulated artificially contaminated chicken feed sample is 10CFU/25 g.

Example 7: application of PCR (polymerase chain reaction) method for specific targeting flgE (fluorogenic kinase) gene to detection of sample from chick embryo

In order to evaluate the detection capability of the PCR method of the specific targeting flgE disclosed by the invention on clinical samples, 247 chicken embryo samples collected clinically are detected, and meanwhile, the traditional culture identification method is taken as a reference method.

247 samples were aseptically collected from healthy and dead chick embryos from a farm in Guangxi. The sample was first pre-enriched in peptone water (BPW) at 37 ℃ for 6 hours, and then selectively enriched in selenite-cystine (SC) medium at 37 ℃ for 12 hours.

After the pre-enrichment and selective enrichment procedures are finished, all samples are divided into duplicate, and salmonella is detected by two methods respectively. One sample is subjected to selective enrichment culture solution detection on all samples by using a PCR method of specific targeting flgE, and the result of PCR amplification of the specific targeting flgE is recorded. And performing subsequent identification on the other sample according to an improved national standard (GB 4789.4-2010) detection process, namely culturing and culturing the sample on Xylose Lysine Deoxycholate (XLD) agar and MacConkey (MAC) agar respectively, and performing serotype test and identification by using a slide agglutination test by referring to a White-Kauffmann-Le Minor (WKLM) antigen table and using a commercial salmonella O and H antiserum detection kit (Tianjin biochip, China Tianjin). The test results of the standard detection method were recorded and compared with those of the PCR method for specific targeting flgE, and the results of the two detection methods are shown in Table 3.

TABLE 3 comparison of the consistency of PCR method for specific targeting of flgE with standard method for detecting Salmonella

Tests show that the coincidence rate of the detection result of the PCR method for the specific targeting flgE and a standard identification method is 100%, and meanwhile, the result can be obtained within 1 day by using the PCR method for the specific targeting flgE, while the standard detection method usually needs 5 days or more.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

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acggaccctg taccgtctaa aacgcccttt agcgtgagtg atgcggattc gtataacaaa 540

aaaggcaccg tcaccgttta tgacagccag ggtaatgccc atgacatgaa cgtctatttt 600

gtgaaaacca aagataatga atgggctgtg tacacccatg acagcagcga tcctgcagcc 660

actgcgccaa caacggcgtc cactacgctg aaattcaatg aaaacgggat tctggagtct 720

ggcggtacgg tgaacatcac caccggtacg attaatggcg cgacagcggc caccttctcc 780

ctcagcttcc ttaactccat gcagcagaac accggggcta ataacatcgt cgccaccaat 840

caaaacggct ataagccggg cgacctggtg agctaccaga ttaacaacga cggcaccgtg 900

gttggcaact actccaacga gcaggagcag gtgctggggc agattgtgct ggctaacttc 960

gccaacaacg aaggtctggc atcccagggc gataacgtct gggcggcgac gcaggcttcc 1020

ggggttgcgc tgctggggac tgccggttcc ggcaacttcg gtaagctgac gaacggcgcg 1080

ctggaagcct ctaacgtgga tttgagtaaa gagctggtga atatgatcgt cgcgcagcgt 1140

aactaccagt cgaatgcgca gaccatcaaa actcaggacc agatcctcaa tacgctggtt 1200

aacctgcgct aa 1212

Claims (8)

1. Specific targeting flagellin hook gene for detecting flagella and nonflagellate salmonellaflgEThe PCR primer of (1), characterized by comprising an upstream primerflgE-UP and downstream primersflgELO, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, of said Salmonella flagellata and non-Salmonella strainsflgEThe gene sequence is from GenBank published sequence; of said Salmonella nonfilamentosa strainsflgEOne part of the gene sequence is from GenBank published sequence, and the other part is from pullorum disease and typhoid fever strain separated, collected and stored in laboratoryflgEThe resulting nucleic acid sequence was sequenced.

2. The specifically targeted flagellin hook gene of claim 1flgEThe PCR primer of (1), wherein the upstream primer and the downstream primer specifically target the salmonella flagellin hook gene respectivelyflgEBy bioinformatics analysis, multiple comparative analysis, including against Salmonella flagellata, Salmonella flagellata and non-Salmonella strainsflgETwo salmonella-specific DNA segments selected from the gene sequence.

3. A kit for PCR rapid detection of Salmonella flagellata and Salmonella nonfilamentosa, characterized in that the kit comprises the PCR primer of claim 1.

4. The kit for the PCR rapid detection of Salmonella flagellata and Salmonella nonflagellate as claimed in claim 3, wherein the kit further comprises PCR reaction buffer, dNTPs and rTaq DNA polymerase.

5. The specifically targeted flagellin hook gene of claim 1flgEThe use of the PCR primer of (1) for the preparation of a reagent for the PCR rapid detection of all Salmonella including Salmonella flagellate and Salmonella nonflagellate, characterized in that the PCR rapid detection method of all Salmonella including Salmonella flagellate and Salmonella nonflagellate comprises: taking a sample to be detected as a template, carrying out PCR amplification reaction by using the primer as claimed in claim 1, carrying out agarose gel electrophoresis on an amplification product, placing the gel in an imaging system, observing whether an amplification strip with an expected size appears at 262bp of the sample to be detected, and judging whether the sample to be detected contains salmonella.

6. The use of claim 5, wherein when the sample to be tested is food, animal feed, environmental sample or feces, selective enrichment of Salmonella is performed first, then PCR amplification is performed using chromosomal DNA prepared from the enrichment fluid as a template, and the result is judged to determine whether the sample contains Salmonella within 24 hours.

7. The use of claim 5, wherein when the sample to be tested is a pure culture of bacteria, the PCR amplification is directly performed by using chromosomal DNA prepared from a bacterial solution as a template, and the determination of whether the sample to be tested is Salmonella is completed within 3 hours.

8. The use according to claim 5, wherein the reaction system of the PCR amplification is: 2.5 muL of 10 XPCR reaction buffer solution, 2 muL of dNTPs with the concentration of 2.5mM, 1 muL of upstream primer with the concentration of 10 muM, 1 muL of downstream primer with the concentration of 10 muM, 1.5U of rTaq DNA polymerase and 1 muL of DNA template, and sterile double distilled water is added to complement the final volume to 25 muL, and a positive control and a negative control are carried out;

the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 45s, extension at 72 ℃ for 45s, 30 cycles; extension at 72 ℃ for 10 min.

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