CN109762914B - HRM serotyping method, target gene, specific amplification primer and kit for salmonella food-borne pathogenic bacteria - Google Patents

Abstract

The invention relates to the technical field of food safety detection, in particular to an HRM serotyping method, a target gene, a specific amplification primer and a kit for salmonella food-borne pathogenic bacteria. The HRM serotyping method for the salmonella food-borne pathogenic bacteria is characterized by comprising the following steps of: designing specific amplification primer pairs at two ends of a mutation region of the salmonella according to a ykgD gene sequence of the salmonella, wherein the ykgD gene sequence is shown as SEQ 1; extracting sample DNA, and amplifying by using the specific amplification primer pair obtained in the step one by adopting an HRM method; judging whether the sample contains salmonella or not through a fluorescence curve; the above methods do not relate to methods for the treatment and diagnosis of diseases. When the parting method is used for seroparting the salmonella strains, the detection speed is high, the result is accurate and reliable, the operation is simple, and the detection cost is low.

Description

HRM serotyping method, target gene, specific amplification primer and kit for salmonella food-borne pathogenic bacteria

Technical Field

The invention relates to the technical field of food safety detection, in particular to an HRM serotyping method, a target gene, a specific amplification primer and a kit for salmonella food-borne pathogenic bacteria.

Background

Salmonella bacteria (A), (B)Salmonella) Is the most important food-borne pathogenic bacteria in enterobacteriaceae, and the poisoning cases caused by salmonella are the first food poisoning in all parts of the world. The bacterium is a main pathogenic bacterium causing food poisoning and typhoid fever, is often transmitted by polluted food and water sources, and can cause diseases of people and livestock. The salmonella has various plant types, and the serotypes separated in the world at present have 2,500 types and 46 serogroups, and about 300 serotypes in China belong to 37 serogroups. However, the serotypes associated with human disease are mainly concentrated in groups A-E, with 70% of pathogenic Salmonella in groups A-D, most commonly Salmonella typhimurium and Salmonella enteritidis. The division of salmonella serotypes has important significance for classification and identification of strains and also has important significance for monitoring and controlling the spread of pathogenic bacteria.

The rationale for HRM technology has been developed primarily based on differences in the physical properties of nucleic acid molecules. Since the GC contents, GC distributions, and the like of different nucleic acid molecules are different, the double-stranded DNA molecules have the shape and position of their own melting curve when denatured by heating. The principle of the HRM technique is to differentiate samples according to the melting temperature and the melting shape of nucleic acid molecules. A saturated fluorescent dye LC Green was introduced at the time of PCR reaction, which was embedded into the DNA double strand during PCR amplification. When the DNA molecules embedded with the dye melt, the instrument collects the fluorescence signals and draws the melting curve of the DNA molecules. The melting curve of a DNA molecule is related to the properties of the DNA molecule, such as GC content, GC distribution, and fragment length, and different samples show different melting curve shapes and melting temperatures (Tm values) due to different base forces.

Due to the fact thatSequence differences exist in the genome of different serotypes of salmonella, subject to host and environmental influences, and these genes can be used for HRM typing. For example, foreign researchers Zeinzinger et al are based on SalmonellafljB、gyrBAndycfQa set of HRM serotyping methods is established for genes, which is published in journal of Applied and Environmental Microbiology, 2012, volume 78, stage 09, 3352, page 3360, entitled "One-step triple high-resolution culturing analysis for rapid identification and multiple amino sub-typing of frequent infectionSalmonella serovars ". However, this method uses three genesfljB、gyrBAndycfQare not specific to Salmonella, and genes with similar DNA sequences are present in other bacteria. Therefore, when typing is carried out by this method, it is necessary to determine the type of the strain as Salmonella, or else, other bacteria are mistaken for a certain Salmonella serotype. Furthermore, the multiplex PCR method is not only susceptible to interference of primer dimers, but also increases the probability of primer mismatching, leading to typing errors.

Disclosure of Invention

In order to overcome the defects of the prior art, the first purpose of the invention is to provide an HRM serotyping method of salmonella food-borne pathogenic bacteria, the second purpose of the invention is to provide HRM serotyping target genes of the salmonella food-borne pathogenic bacteria, the third purpose of the invention is to provide specific amplification primers for HRM serotyping of the salmonella food-borne pathogenic bacteria, and the fourth purpose of the invention is to provide a kit for HRM serotyping of the salmonella food-borne pathogenic bacteria. The method for serotyping the salmonella can shorten the typing time, has low cost and simple, quick and accurate result judgment.

In order to achieve the first object, the invention is realized by the following technical scheme:

the HRM serotyping method for the salmonella food-borne pathogenic bacteria is characterized by comprising the following steps of:

designing specific amplification primer pairs at two ends of a mutation region of the salmonella according to a ykgD gene sequence of the salmonella, wherein the ykgD gene sequence is shown as SEQ 1;

extracting sample DNA, and amplifying by using the specific amplification primer pair obtained in the step one by adopting an HRM method;

judging whether the sample contains salmonella or not through a fluorescence curve;

the above methods do not relate to methods for the treatment and diagnosis of diseases.

In the first step, the specific amplification primer consists of an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is shown as SEQ 8, and the base sequence of the downstream primer is shown as SEQ 9.

In the second step, in the HRM method, the HRM serotyping system (20 μ L) is specifically:

mu.L of 2 × buffer and 2.4. mu.L of MgCl were taken₂Taking 1 μ L of each of the upstream and downstream primers, taking 2 μ L of the DNA template, and finally adding H₂O was 3.6. mu.L.

In the second step, in the HRM method, the amplification parameters of the HRM serotyping system are specifically as follows:

pre-denaturation at 94 ℃ for 5min, after which amplification cycles were started, the procedure for each cycle was: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30 s; 35 cycles in total; after the circulation is finished, extending for 10min at 72 ℃, and cooling to 10 ℃ to finish the circulation.

In order to achieve the second object, the invention is realized by the following technical scheme:

the HRM serotyping target gene of the salmonella food-borne pathogenic bacteria has a base sequence shown in SEQ 1.

In order to achieve the third object, the invention is realized by the following technical scheme:

the specific amplification primer for HRM serotyping of the salmonella food-borne pathogenic bacteria comprises an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is shown as SEQ 8, and the base sequence of the downstream primer is shown as SEQ 9.

In order to achieve the fourth object, the invention is achieved by the following technical scheme:

the kit for HRM serotyping of salmonella food-borne pathogenic bacteria comprises a DNA template, a specific amplification primer, 2 × buffer and MgCl₂The heterologous amplification primer consists of an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is shown as SEQ 8, and the base sequence of the downstream primer is shown as SEQ 9.

Compared with the prior art, the invention has the following beneficial effects: when the parting method is used for seroparting the salmonella strains, the detection speed is high, the result is accurate and reliable, the operation is simple, and the detection cost is low; the detection method of the invention can be used for detecting food samples; the target DNA sequence is obtained by comparing genomics and bioinformatics screening and performing biological verification, and has the advantages of single specificity, reliable detection result and simple result judgment.

Drawings

FIG. 1 shows the typing results of 7 Salmonella standard strains with the bcfC primer.

FIG. 2 shows the results of typing 7 Salmonella standard strains with the ykgC primer pair.

FIG. 3 shows the results of typing 7 Salmonella standard strains with the ykgD primer pair.

FIG. 4 is the results of typing of Salmonella typhimurium ATCC14028 in food.

Detailed Description

Example 1:

step one, according to salmonellabcfC、ykgC、ykgDGene sequence design primer

By comparing genomic and bioinformatic analyses, unique gene sequences were found in salmonella genomic DNA sequencesbcfC、ykgCAndykgDand the two types of the salmonella are respectively used as the typing targets of the salmonella (shown as SEQ 1, SEQ 2 and SEQ 3). The nucleic acid base sequences of the genes have certain difference in different salmonella serotypes;

the DNA nucleic acid sequence is input into Primer design software Primer Premier 5.0 to design primers, and the upstream and downstream Primer sequences are respectively positioned at two ends of a gene mutation region, and the sequences are as follows (the primers are from Dalibao bioengineering Co., Ltd):

bcfCan upstream primer: 5 '-GCAGAATGCGGGCCTTAG-3', (SEQ 4)

bcfCA downstream primer: 5 '-CGCGCGTCCGTATGAAT-3', (SEQ 5)

ykgCAn upstream primer: 5 '-GAACAGTCCGCCAGTATG-3', (SEQ 6)

ykgCA downstream primer: 5 '-GCAGGTAATCATCCACGAT-3', (SEQ 7)

ykgDAn upstream primer: 5 '-GATTCGCTCGCATAACCTACC-3', (SEQ 8)

ykgDA downstream primer: 5 '-GTGATTCGCTGGCATACCGT-3' (SEQ 9).

Step two, HRM serotyping method system and reaction parameters

HRM reaction (20. mu.L) was: mu.L of 2 × buffer and 2.4. mu.L of MgCl were taken₂Taking 1 μ L of each of the upstream and downstream primers, taking 2 μ L of the DNA template, and finally adding H₂O was 3.6. mu.L.

Then, after centrifuging the reaction tube, putting the reaction tube into a PCR instrument, and performing the reaction according to the following parameters:

pre-denaturation at 94 ℃ for 5min, after which amplification cycles were started, the procedure for each cycle was: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30 s; 35 cycles in total; after the circulation is finished, extending for 10min at 72 ℃, and cooling to 10 ℃ to finish the circulation.

Step three, parting

2 mu L of DNA solution of salmonella typhimurium ATCC14028, salmonella enteritidis ATCC13076, salmonella verolol ATCC15611, salmonella paratyphi A CMCC50093, salmonella turkey CICC21511, salmonella dublin CICC21497 and salmonella idelberg CICC21487 are taken as reaction templates and added into an HRM system for amplification reaction. The extraction process is as follows: respectively inoculating the 7 standard strains into 50 mL of LB liquid culture medium, enriching the strains for 8 h at 37 ℃, and then taking 1mL of bacterial liquid and putting the bacterial liquid into a 1.5 mL centrifuge tube; then centrifuging at 3,000 r/min for 10min, taking supernatant, centrifuging at 12,000 r/min for 5min, and collecting thalli. Resuspending the cells in sterile double distilled water, centrifuging, adding 100 μ L sterile ultrapure water, boiling in boiling water bath for 15 min, immediately taking out, and standing at-20 deg.C for 30 min. Thawing at 37 deg.C, centrifuging at 12,000 r/min for 5min, and collecting supernatant and standing at-20 deg.C.

As shown in FIG. 1, inbcfCIn the result of typing 7 salmonella standard strains by gene primer pairs, the HRM curves roughly divide the strains into two types, wherein the HRM curves of the a6, B6, C6, D6, E6 and F6 types are similar, so that 7 common serotype strains are difficult to distinguish by the curves. The numbering information of the strains used is illustrated in the figures.

The templates of the HRM curves in fig. 1 are respectively: salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076, Salmonella veralol ATCC15611, Salmonella paratyphi A CMCC50093, Salmonella huoshimurium CICC21511, Salmonella dublin CICC21497, and Salmonella idelbergi CICC 21487.

As shown in FIG. 2, inykgCIn the results of typing 7 salmonella standard strains by gene primer pairs, the HRM curves classify the strains into three types, wherein the HRM types of two strains of a7, B7, D7 and G7 are very similar, so that the 7 common serotype strains cannot be clearly distinguished by the curves. The numbering information of the strains used is illustrated in the figures.

The templates of the HRM curves in fig. 2 are respectively: salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076, Salmonella veralol ATCC15611, Salmonella paratyphi A CMCC50093, Salmonella huoshimurium CICC21511, Salmonella dublin CICC21497, and Salmonella idelbergi CICC 21487.

As shown in FIG. 3, inykgDThe HRM curves of all serotype strains are different in the typing results of 7 salmonella standard strains by gene primer pairs, so that 7 common serotype strains can be distinguished by the curves. The numbering information of the strains used is illustrated in the figures.

The templates of the HRM curves in fig. 3 are: salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076, Salmonella veralol ATCC15611, Salmonella paratyphi A CMCC50093, Salmonella huoshimurium CICC21511, Salmonella dublin CICC21497, and Salmonella idelbergi CICC 21487.

Step four, establishing a HRM typing method

From the typing results, the third primer pair (C)ykgDGene primer) can obviously distinguish 7 salmonella serotypes, so that the gene primer can be selectedykgDThe gene is a typing target gene and comprises the following primers:

an upstream primer: 5 '-GATTCGCTCGCATAACCTACC-3', (SEQ 8)

A downstream primer: 5 '-GTGATTCGCTGGCATACCGT-3', (SEQ 9)

The established HRM typing method can be used for typing salmonella typhimurium, salmonella enteritidis, salmonella verolol, salmonella paratyphi A, salmonellosis turkey, salmonella dublin and salmonella idelberg of salmonella.

Example 2:

10 portions of milk samples were taken, and 3 portions of the milk samples were inoculated with Salmonella typhimurium ATCC14028, and the food samples were tested and typed using the test method of example 1. The results show that the HRM serotyping method not only can accurately detect the Salmonella typhimurium, but also the strain typing results are consistent with the results of example 1 in graph (as shown in FIG. 4).

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> scientific and technical research institute for inspection and quarantine in Zhejiang province

Northwest A & F University

<120> HRM serotyping method, target gene, specific amplification primer and kit for salmonella food-borne pathogenic bacteria

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Claims (3)

1. The HRM serotyping method of the salmonella food-borne pathogenic bacteria is used for detecting food samples and typing salmonella typhimurium, salmonella enteritidis, salmonella verolol, salmonella paratyphi A, salmonella turkey, salmonella dublin and salmonella idelberg of the salmonella; the method is characterized by comprising the following steps:

designing specific amplification primer pairs at two ends of a mutation region of the salmonella according to a ykgD gene sequence of the salmonella, wherein the ykgD gene sequence is shown as SEQ 3;

extracting sample DNA, and amplifying by using the specific amplification primer pair obtained in the step one by adopting an HRM method;

judging whether the sample contains salmonella or not through a fluorescence curve;

the specific amplification primer in the first step consists of an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is shown as SEQ 8, and the base sequence of the downstream primer is shown as SEQ 9.

2. The HRM serotyping method for Salmonella food-borne pathogenic bacteria according to claim 1, wherein in the second step, the HRM serotyping system in the HRM method is as follows:

20 μ L of HRM reaction: mu.L of 2-buffer and 2.4. mu.L of MgCl2 were taken, 1. mu.L of each of the upstream and downstream primers, 2. mu.L of DNA template, and 3.6. mu.L of H2O was added.

3. The HRM serotyping method for Salmonella food-borne pathogenic bacteria according to claim 1 or 2, wherein in the second step, the HRM serotyping system amplification parameters in the HRM method are specifically as follows:

pre-denaturation at 94 ℃ for 5min, after which amplification cycles were started, the procedure for each cycle was: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30 s; 35 cycles in total; after the circulation is finished, extending for 10min at 72 ℃, and cooling to 10 ℃ to finish the circulation.

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