CN105256041B - The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application - Google Patents

Abstract

The present invention relates to a nucleotide specific to the O antigen of Aeromonas hydrophila (Aeromonas hydrophila) and its application, and the nucleotide includes 1) the nucleotides shown in SEQ ID NO: 1‑8 At least one; 2) At least one of the nucleotides complementary to the nucleotides shown in SEQ ID NO: 1‑8. These nucleotides can be used to prepare a PCR kit and a gene chip for detecting Aeromonas hydrophila. The nucleotide specific to the Aeromonas hydrophila O antigen of the present invention and the PCR kit and the gene chip containing the nucleotide have strong practicability, the preparation method of the PCR kit is simple, the detection period is short, the speed is fast, and the Strong operability, easy industrial production, but relatively low detection cost; high accuracy; high sensitivity.

Description

Nucleotides specific to Aeromonas hydrophila O44, O24, O25 and O28 and their application

technical field

The present invention relates to nucleotides specific to Aeromonas hydrophila O44, O24, O25 and O28 serotypes, in particular to individual genes in the O antigen gene cluster of Aeromonas hydrophilia O44, O24, O25 and O28 serotypes Specific nucleotides and their applications.

Background technique

Aeromonas hydrophila (Aeromonas hydrophila) belongs to the Vibriaceae Aeromonas genus, is a Gram-negative short bacillus, without spores and capsules, also known as Aeromonas hydrophila. Aeromonas hydrophila widely exists in many water bodies in nature and is the primary pathogenic bacteria of many aquatic animals. It is an opportunistic pathogenic bacterium and a typical human-animal-fish comorbid pathogen. It enters the body through the intestinal tract and can produce highly toxic exotoxins, causing fulminant hemorrhagic disease. In general, the stronger the adhesion of the bacteria to the intestinal tissue, the stronger the toxicity of the exotoxin it produces. There are many serotypes of Aeromonas hydrophila, and the symptoms are also different. The disease infected by Aeromonas hydrophila is generally serious, with multiple malignant transmission and high mortality.

Bacterial typing and identification methods mainly include traditional phenotypic methods, serological methods and molecular identification methods. However, with the development of molecular biology, there are certain problems in the traditional serotyping and identification methods. For example, the diagnostic method of serotyping requires a large amount of antiserum, and the antiserum is generally incomplete and insufficient. Antisera also present some difficulties in preparation and storage. On the other hand, the serotyping method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy. There are often cross-reactions between antisera produced by different O antigens. Therefore, establishing a serum identification method based on molecular biology technology has become a development direction.

The molecular identification of Aeromonas hydrophila has attracted more and more attention, and has become an important basis for the identification of Aeromonas hydrophila species and strain types, and many new bacteria have also been produced. For Vibrio, biochemical reactions are mainly the performance of various zymograms, which belong to the content of external forms, and the similarity of nucleic acid is the most fundamental and direct feature for defining Vibrio species. Therefore, the most effective and direct way to type and identify Aeromonas hydrophila should be triggered by the study of nucleic acid, and determine the identity of Aeromonas hydrophila by comparing the similarity of nucleic acid (including genome and nucleic acid fragments).

In recent years, more and more molecular techniques have been used for typing, identification, detection and disease diagnosis of pathogenic bacteria, including sequence analysis of transcribed spacer (ITS), random amplified length polymorphism (RAPD) analysis, ribosomal DNA restriction Sexual fragment length polymorphism (RFLP) analysis, etc. Molecular biology methods can not only be used for rapid serotyping screening of Aeromonas hydrophila, but stable identification results can make up for the shortcomings of phenotypic characterization methods. Compared with traditional detection technologies, these polymerase chain reaction (PCR)-based molecular detection technologies do not require the isolation and pure cultivation of pathogenic bacteria, and have the advantages of rapidity, sensitivity, and strong specificity.

Polymerase chain reaction technology (Polymerase chain reaction, referred to as PCR technology) is currently being recognized and promoted as a microbial detection technology. Compared with traditional methods, this technology has the advantages of high throughput, fast detection speed, strong specificity, and high sensitivity. Simply perform a simple pre-enrichment or enrichment process on the sample, and then prepare the bacterial DNA template by centrifugation and lysis, and then the target sequence can be amplified in the PCR process mediated by highly specific primers to detect whether the sample contains The purpose of pathogenic microorganisms to be tested. The PCR amplification process only takes one and a half hours. This has undoubtedly greatly improved the work speed and reduced the work cost for the inspection and quarantine department and clinical inspection.

From both domestic and international perspectives, it is very important to quickly and accurately identify serotypes and provide effective technical support for the prevention and control of Aeromonas hydrophila.

Contents of the invention

The object of the present invention is to provide a kind of nucleotide specific to Aeromonas hydrophila O antigen, it is characterized in that described nucleotide has:

1) At least one of the nucleotides shown in SEQ ID NO:1-8;

2) At least one of the nucleotides complementary to the nucleotides shown in SEQ ID NO: 1-8; said SEQ ID NO: 1-8 is as follows:

The present invention also provides a PCR kit, including PCR primers, dNTP, buffer and DNA polymerase, the PCR primers are at least one of the nucleotides shown in SEQ ID NO: 1-8. The kit also includes the following reagents: 30 μl of 10 mM dNTP; 50 μl of 10×enzyme-specific reaction buffer; 5 μl of 5 U/μl heat-resistant DNA polymerase; 10 μl of primer mixture; 10 μl of positive control substance; 10 μl of negative control substance; _ddH2O 5ml.

Wherein said PCR primer is preferably at least one of said nucleotides shown in SEQ ID NO: 1-8.

The present invention further discloses a nucleotide of SEQ ID NO: 1-8 specific to Aeromonas hydrophila O antigen in the preparation of a PCR kit, gene chip or microarray for detecting Aeromonas hydrophila O antigen aspects of application.

The hydrophilic Aeromonas described in the present invention can be sampled from crude extracts of tap water, sewage, seawater cultures, fruits and vegetables, disease specimens or pure cultures of Aeromonas hydrophilics.

The collected extracted genome of Aeromonas hydrophila was prepared by conventional methods.

For the PCR kit for Aeromonas hydrophila, the entire detection step includes sample pretreatment-amplification-electrophoresis detection results. The primers and reagents required for the PCR reaction system have been added to the amplification tube in advance, and the user only needs to add the pretreated sample into the amplification tube to start the amplification reaction, and the detection work can be completed simply and quickly.

The present invention also provides a gene chip, comprising a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, wherein the oligonucleotide probe comprises the above-mentioned nucleotide; wherein the nucleotide Preferred are the nucleotides shown in SEQ ID NO: 1-8.

The present invention also provides a microarray, which includes the above-mentioned nucleotides; wherein the nucleotides are preferably nucleotides as shown in SEQ ID NO: 1-8. The application of the gene chip for the detection of hydrophilic aeromones and the microarray for the detection of hydrophilic aeromones. The Aeromonas hydrophila is used to detect various mixed infections such as acute gastroenteritis, trauma infection, intestinal infectious disease, and hospital infection caused by polluted water sources and uncooked and unwashed food.

Compared with the prior art, the nucleotide specific for Aeromonas hydrophila O antigen disclosed by the present invention has the following advantages:

(1) Strong practicability: a PCR reaction system established by the present invention can detect Aeromonas hydrophila, provide specific primers used in serotyping detection, and use the PCR method to detect clinical specimens.

(2) High accuracy: the present invention obtains a band for each sample through the PCR reaction of each serotype-specific gene of Aeromonas hydrophila, and compares the obtained target fragment with the known length to obtain The serotype to which Aeromonas hydrophila belongs is obtained.

(3) The detection cost is relatively low: it can be popularized and applied in food hygiene supervision, environmental monitoring, Shangpin monitoring, inspection and quarantine and other fields, and provide a technical model for other different pathogenic bacteria detection combinations.

The above is only the operation and implementation method of the present invention, and does not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still belong to within the scope of the technical solutions of the present invention.

Description of drawings:

Fig. 1 shows the electrophoresis result figure of other serotype standard bacterial strains of Aeromonas hydrophila detected by O44 serotype specific primers of the present invention, the screening of wzx gene P1 and P2 primers, the target band is 151bp, and all the other serotypes do not have any specific bands The strain information is shown in Table 2;

Fig. 2 shows the identification electrophoresis result figure of O44 serotype-specific primer species specificity of the present invention, wherein detected 6 strains of Vibrio and 1 strain of Salmonella, 1 strain of Escherichia coli, all without any band, specific bacterial strain information is shown in Table 2;

Fig. 3 shows that O24 serotype wzz gene P3 and P4 primers of the present invention detect other serotype standard strain electrophoresis results of Aeromonas hydrophilic, the screening of wzz gene P3 and P4 primers, the target band is 194bp, and all the other serotypes do not have any strip. Specific strain information is shown in Table 2;

Fig. 4 shows the identification electrophoresis results of species specificity of P3 and P4 primers of O24 serotype wzz gene of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected without any bands. Specific strain information is shown in Table 2;

Fig. 5 shows the electrophoresis result figure of other serotype standard bacterial strains of Aeromonas hydrophile detected by O25 serotype wzy gene P5 and P6 primers of the present invention, the screening of wzy gene P5 and P6 primers, the target band is 180bp, and all the other serotypes do not have Any band, see Table 2 for specific strain information;

Fig. 6 represents the identification of O25 serotype wzy gene P5 and P6 primer species specificity of the present invention; Electrophoresis result figure, wherein detected 6 strains of Vibrio and 1 strain of Salmonella, 1 strain of Escherichia coli, all without any band, concrete bacterial strain The information is shown in Table 2;

Fig. 7 shows the electrophoresis result figure of other serotype standard strains of Aeromonas hydrophila detected by O28 serotype GT gene P7 and P8 primers of the present invention, the screening of GT gene P7 and P8 primers, the target band is 167bp, and all the other serotypes do not have any strip. Specific strain information is shown in Table 2;

Fig. 8 shows the electrophoresis results of species-specific identification of O28 serotype GT genes P7 and P8 primers of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected without any bands. Specific strain information is shown in Table 2;

Figure 9 shows the electrophoresis results of the corresponding serotypes amplified with O44, O24, O25 and O28 specific primers respectively, and the specific strain information is shown in Table 2;

Among them, O44, O24, O25, 028, and the negative control (-) indicate the amplification results of the corresponding serotype primers, and the first swimming lane H and the last swimming lane are 2000bp ladder markers.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions are generally followed, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). The Aeromonas hydrophila O24, O25, O28 and O44 in this experiment were from Japan Collection of Microorganisms (JCM).

Embodiment 1 : the extraction of genome

Aeromonas hydrophila was cultivated in nutrient broth medium at 37°C, the bacteria were collected, and the genome was extracted. The specific steps are as follows:

The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg/ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg/ml proteinase K, 15ul10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg/ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant, and then extract twice with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. Precipitate DNA with 2 times the volume of ethanol in the supernatant, roll out the DNA with glass wool and wash the DNA with 70% ethanol, and finally resuspend the DNA in 30ul TE. Genomic DNA was detected by 0.4% agarose gel electrophoresis.

Embodiment 2: sequence deciphering

Extract the genomes of the standard strains of each serotype of Aeromonas hydrophila, and use Solexa pair-end sequencing technology to perform whole-genome sequencing on the genomes of each serotype of Aeromonas hydrophila to obtain the sequence of the serotype, using Blast and PSI-Blast Sequence alignment was carried out, TMHMM 2.0 program was used for transmembrane structure prediction, and ClustalW program was used for sequence alignment and screening of conserved and specific gene fragments, and finally the O antigen gene cluster sequence and deciphering results of each serotype of Aeromonas hydrophila were obtained.

Embodiment 3 : primer design

The O antigen gene cluster sequences of each serotype of Aeromonas hydrophila were self-tested by our laboratory. Through comparative analysis, we selected gene-specific segments with relatively low identity and similarity values in the Blast comparison results to design primers. Among them, the identity value and similarity value of the wzx gene comparison result of O44 serotype were 67% and 78%; the identity value and similarity value of the wzz gene comparison result of O24 serotype were 87% and 93%; the wzy gene ratio of O25 serotype The result identity value and similarity value are 79% and 88%; the GT gene comparison result identity value and similarity value of O28 serotype are 65% and 79%; so each serotype selects the above-mentioned corresponding gene as the serotype respectively For specific target genes, specific primers are designed for the specific segments of the genes of each serotype.

Primer design is a core part of this invention. Import the above genes into Primer Premier 5 for primer design, design a pair of primers for each gene, and have a single target band.

After the primers are designed, BLAST is performed in Genbank. The designed primers should not have too high sequence similarity with other related bacteria, so as to ensure that the primers can only amplify at their predetermined positions, and not with other related bacteria or bacteria. Relative bacteria in the environment where the specimen was collected did not produce a positive reaction. This point is very important to avoid the generation of non-specific bands and the success or failure of the experiment.

The designed primers are shown in Table 1:

Table 1 Primer sequences for PCR

Embodiment 4 : the screening of specific primer

Standard strains of Aeromonas hydrophila O44, O24, O25 and O28 serotypes, 6 other Vibrio strains, 1 Salmonella strain and 1 Escherichia coli strain were collected to verify the specificity of the primers, the strain numbers and See Table 2 for sources.

Table 2 Strains used for specific detection

The PCR system used for gene identification primer screening is 0.4 μl of 5 μM primers, 2.5 μl of 10× enzyme-specific reaction buffer, 0.25 μl of 10 mM dNTP, 0.2 μl of 5 U/μl thermostable DNA polymerase, and 3 μl of the sample template to be tested to 0.2 ml of thin-walled PCR tubes, and finally make up to 25 μl of ddH ₂ O. All primers obtained positive results in their corresponding serotypes, and no PCR product bands were obtained in other groups.

The reaction cycle parameters on the PCR instrument in this step include the denaturation, renaturation, temperature and time of extension, and the number of cycles of DNA, specifically:

One cycle of the pre-treatment process necessary for the denaturation to reach the required temperature in the early stage is 95 ° C for 5 minutes;

The denaturation temperature and time are 95°C, 45 seconds;

Refolding temperature and time are 50°C/55°C, 1 minute;

The extension temperature and time are 72°C, 1 minute;

The number of cycles of denaturation, renaturation, and extension is 35 cycles;

The temperature and time for one cycle to stabilize the amplification product are 72°C for 5 minutes

Among them, O44 was amplified at 50°C, O24 was amplified at 50°C, O25 was amplified at 55°C, and O28 was amplified at 55°C.

The above steps electrophoresis amplify the product in the electrophoresis equipment, and the specific steps for recording the results are:

Take 2-5 μl of the amplification product and mix it with 6× bromophenol blue loading buffer at a volume ratio of 1:1;

Load the mixture on a 1.0% agarose gel;

Perform agarose gel electrophoresis at 120v constant voltage for about 20 minutes, and use DL2000 Marker as a control;

Observe and record the results.

After the basic PCR reaction, the work of primer screening is basically completed, and the necessary length adjustment has little influence on the overall reaction conditions. The sequences of the primers used in the present invention are all summarized in Table 1.

Table 1 Primer sequences for PCR

Embodiment 5: Preparation and application of PCR detection kit

1. The composition of the PCR kit:

dNTP (10mM) 30μl;

10×Buffer (10×Enzyme Specific Reaction Buffer) 50μl;

Taq polymerase (5U/μl thermostable DNA polymerase) 5μl;

PCR primer mix (5μM) 10μl;

Positive control substance (KP) 10μl;

Negative control substance (KN) 10μl;

ddH ₂ O 5ml;

Each kit can be used to detect 10 samples.

Among them, 10×Buffer, dNTP, and Taq polymerase were provided by Treasure Bioengineering Co., Ltd.; the primer mixture was a self-designed sequence provided to Shanghai Yingjun Biotechnology Company for synthesis; positive control substances, negative control substances and ddH ₂ O were prepared by ourselves .

2. Instruments and equipment

Among them, 10×Buffer, dNTP, and Taq polymerase were provided by Shanghai Sangong; the primer mixture was a self-designed sequence provided to Shanghai Yingjun Biotechnology Company for synthesis; positive control substances, negative control substances and ddH2O were prepared by us. Experimental equipment PCR instrument (also known as DNA thermal cycle amplification instrument), electrophoresis equipment (including electrophoresis instrument and electrophoresis tank), gel imager, -20 ℃ refrigerator, high-speed centrifuge, micropipette and 0.2ml PCR thin film wall pipe.

3. Specific examples of the use of PCR kits

The PCR detection method that uses above-mentioned PCR kit to detect Aeromonas hydrophila comprises the steps:

(1) Extract the template of the environmental sample to be tested;

(2) Add, dNTP, 10×Buffer, Taq polymerase, primers, sample template to be tested and ddH ₂ O into the PCR thin-walled tube and mix well;

(3) Amplify the mixture mixed in the thin-walled PCR tube on a PCR machine;

(4) Electrophoresis amplifies the product in the electrophoresis equipment, and records the result;

(5) Analyze and judge the results.

The environmental sample template in the above step (1) is the crude extract of the culture of tap water, polluted water, seawater, etc., or the crude extract of the pure culture of Aeromonas hydrophila, or pure DNA, or positive Controls and negative controls.

The extraction method of the environmental sample template in the above step (1) is:

Take 1.5ml of the culture, centrifuge at 12000rpm for 1 minute, and remove the supernatant;

Take 500 μl of ddH ₂ O to resuspend the pellet, centrifuge at 8000 rpm for 5 minutes, remove the supernatant, and dry;

Take 100 μ lddH ₂ O to resuspend the precipitate, and bathe in boiling water at 100°C for 10 minutes;

After placing on ice for 10 minutes, centrifuge at 12000rpm for 2 minutes;

⑤ Take 3μl middle layer supernatant as PCR template

The reaction cycle parameters on the PCR instrument in the above step (3) include the temperature and time of DNA denaturation, renaturation, extension, and number of cycles, specifically:

One cycle of the pre-treatment process necessary for the denaturation to reach the required temperature in the early stage is 95 ° C for 5 minutes;

The denaturation temperature and time are 95°C, 45 seconds;

Refolding temperature and time are 50°C/55°C, 1 minute;

The extension temperature and time are 72°C, 1 minute;

The number of cycles of denaturation, renaturation, and extension is 35 cycles;

The temperature and time for one cycle to stabilize the amplification product was 72°C for 5 minutes.

The above step (4) electrophoresis amplifies the product in the electrophoresis equipment, and the specific steps for recording the results are:

Take 2-5 μl of the amplification product and mix it with 6× bromophenol blue loading buffer at a volume ratio of 5:1;

Load the mixture on a 1.0% agarose gel;

Perform agarose gel electrophoresis at 120v constant voltage for about 20 minutes, and use DL2000 Marker as a control;

Observe and record the results.

The results show: the results are shown in the attached figure, the PCR product results in bright bands and a single band.

In the present invention, by configuring an industrially produced PCR kit capable of detecting Aeromonas hydrophilia, the components needed for the PCR detection method are combined together, and when used, the sample to be tested is extracted, and at the same time, it undergoes a relatively simple process. Fast, sensitive, and simple detection can be carried out by operating the sequence. The dosage and concentration of each component in the kit are all experimental results. The test equipment used to detect Aeromonas hydrophila with this kit is simple and the detection cost is low. .

The purpose of using positive and negative control substances is to control the entire operation process in order to make accurate judgments. If it contains the O antigen type of Aeromonas hydrophila, the band at the same position as the positive control can be observed from the electrophoresis results; The same does not have this band.

The amount of reagents in the kit used for a detection test of the present invention is shown in the following table 3, and the amount of DNA template is 3 μl

Table 3 The amount of reagents in the kit used in a detection test

The thermostable DNA polymerase in the present invention is rTaq enzyme.

The above-mentioned positive control substances are samples that have been determined to be each type of O antigen of Aeromonas hydrophila, and the negative control substances are samples that have been determined not to be Aeromonas hydrophila.

If the PCR kit uses the bacterial suspension of Aeromonas hydrophila for PCR amplification, the result is consistent with the result obtained by using the extracted DNA as a template. There is no difference in sensitivity and specificity. In this way, the extraction step of template DNA can be omitted, and the operation method can be simplified. At the same time, compared with conventional biochemical detection methods, the sample to be tested in this method can be directly the culture solution of clinical samples, or the detection can be performed by simply separating and culturing the test sample, thus saving manpower and material resources.

4. Provision of samples to be tested

The standard strains of cholera O44, O24, O25 and O28 serotypes, 6 other Vibrio strains, 1 Salmonella strain and 1 Escherichia coli strain were collected to verify the specificity of the primers. The strain numbers and sources are shown in Table 2.

SEQUENCE LISTING

<110> Nankai University

<120> Nucleotides specific to Bacillus cholerae O44, O24, O25 and O28 and their application

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Claims (3)

1. one group of aeromonas hydrophila O44, O24, O25 and O28 specific detection primer, it is characterised in that the specific detection draws Object is：SEQ ID NO:Nucleotide shown in 1-8；Nucleotide therein is used to prepare detection aeromonas hydrophila O44, O24, O25 and O28 PCR kits；Aeromonas hydrophila sampling in tap water, sewage, seawater culture crude extract, melon In fruit vegetable, the crude extract of the pure culture of sick body sample or aeromonas hydrophila.

2. one kind includes one group of aeromonas hydrophila O44, O24, O25 and O28 specific detection primer as described in claim 1 PCR kit, including PCR primer, dNTP, buffer solution and archaeal dna polymerase, the PCR primer are SEQ ID NO:Shown in 1-8 Nucleotide.

3. one kind is being made including one group of aeromonas hydrophila O44, O24, O25 and O28 specific detection primer as described in claim 1 It is ready for use on the application in terms of detection aeromonas hydrophila microarray；The wherein described detection aeromonas hydrophila refer to detection by It is really felt with acute gastroenteritis, trauma infection contamination, enteric infectious disease, hospital caused by not cooked, unwashed food in polluted source Contaminate a variety of mixed type infection.