CN112375835A - Loop-mediated isothermal amplification detection method for 4O antigen serotypes of vibrio fluvialis and application - Google Patents
Loop-mediated isothermal amplification detection method for 4O antigen serotypes of vibrio fluvialis and application Download PDFInfo
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Abstract
The invention relates to a loop-mediated isothermal amplification (LAMP) detection method for O antigen serotyping of vibrio fluvialis and application thereof. In the invention, specific genes, namely wzy, in O antigen gene clusters of Vibrio fluvialis O1, O4, O14 and O17 are used as target genes, 4 LAMP primers for O antigen serotype classification of Vibrio fluvialis O1, O4, O14 and O17 are designed and screened respectively, and a corresponding detection reaction system is established. The LAMP method is used for detecting the vibrio fluvialis and performing O antigen typing on the vibrio fluvialis, and has the advantages of simplicity in operation, rapidness, high efficiency and the like.
Description
Technical Field
The invention relates to a loop-mediated isothermal amplification (LAMP) technology for serotype strains of vibrio fluvialis O1, O4, O14 and O17 and a preparation method thereof. The invention also relates to a detection method by using the LAMP technology.
Background
River vibrio is gram-negative bacteria, is one of main inhabitants of marine environment, widely exists in rivers and water areas of the sea outlet environment, is one of main bacterial pathogens of human beings and aquatic organisms, can cause diseases of various cultured animals such as fish, shrimps, shellfish and the like, and brings serious economic loss to the breeding industry. River vibrio, which also causes severe epidemic diarrhea in humans through various foods, is a pathogenic vibrio next to vibrio cholerae and vibrio parahaemolyticus in vibrio, and is considered as a novel pathogen of zoonosis worldwide.
Loop-mediated Isothermal Amplification (LAMP) can amplify nucleic acid in a short time (usually within one hour) under the condition of Isothermal temperature (60-65 ℃), and is a simple, convenient, rapid, accurate and low-price gene Amplification method. Compared with the conventional PCR, the method has the characteristics of simplicity and rapidness without the processes of thermal denaturation, temperature circulation and the like of the template. The technology can be comparable to or even superior to the PCR technology in the indexes such as sensitivity, specificity, detection range and the like, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has detection cost far lower than that of fluorescent quantitative PCR.
The technical principle is as follows: the temperature of 60-65 ℃ is the intermediate temperature of renaturation and extension of double-stranded DNA, and the DNA is in a dynamic equilibrium state at about 65 ℃. Thus, DNA synthesis at this temperature is possible. The use of 4 specific primers relies on a highly active strand-displacing DNA polymerase. So that strand displacement DNA synthesis is continuously self-circulating.
Amplification is in two stages: stage 1 is the initial stage, in which either primer undergoes base-pairing extension to the complementary portion of the double-stranded DNA, the other strand dissociates and becomes single-stranded. The F2 sequence of the upstream inner primer FIP is firstly combined with the template F2c, and is extended forward under the action of strand displacement type DNA polymerase to start strand displacement synthesis. The outer primer F3 binds to and extends from template F3c, displacing the entire FIP-ligated complementary single strand. F1c on FIP and F1 on this single strand are complementary structures. Self base pairing forms a ring structure. Using the strand as a template, the downstream primers BIP and B3 sequentially initiate synthesis similar to FIP and F3 to form a single strand with a dumbbell-shaped structure. Starting immediately with the F1 segment at the 3' end. DNA synthesis and extension are carried out by taking the self as a template to form a stem-loop structure. This structure is the initial structure of the LAMP gene amplification cycle.
Stage 2 is the amplification cycle stage. FIP binds to the F2c region of the stem-loop using the stem-loop structure as a template. Strand displacement synthesis is started, and a loop structure is also formed in the dissociated single-stranded nucleic acid. The B1 segment at the 3' end is taken as a starting point, the self is taken as a template, DNA synthesis extension and strand displacement are formed, 2 pieces of DNA with different lengths and new stem loop structures are formed, B2 on the BIP primer is hybridized with the DNA, a new round of amplification is started, and the length of the product DNA is doubled. 2 circular primers LF and LB are added into the reaction system, and are respectively combined with the stem-loop structure to start strand displacement synthesis. In cycles, the final product of amplification is a mixture of DNAs with different stem-loop structures and different lengths, and the product DNA is an alternating inverted repeat sequence of the amplified target sequence.
The serological detection method can effectively distinguish strains with different pathogenicity in one species/genus, and has important significance for the identification of pathogenic bacteria and epidemiological investigation. The diversity of O antigens of different serotypes of the same bacterium is determined by the genetic diversity of the genes encoding the various enzymes that synthesize the O antigens, which are often located in clusters on fixed sites on the genome, called O antigen gene clusters. In earlier researches, the information of the O antigen gene cluster of the vibrio fluvialis is found to be decoded, so that the molecular serology detection of the vibrio fluvialis can be realized by adopting a molecular biological method and taking a specific gene in the gene cluster as a target.
Disclosure of Invention
In order to achieve the purpose, the invention discloses an LAMP primer for O antigen serotyping of Vibrio fluvialis strains O1, O4, O14 and O17, which comprises a FIP primer, a F3 primer, a BIP primer and a B3 primer. It is characterized in that 4 DNA fragments are respectively selected from wzy genes of Vibrio fluvialis O1, O4, O14 and O17, and the nucleotide sequence of the DNA fragments is shown as SEQ ID NO 1-SEQ ID NO 16.
The LAMP primers are mainly designed aiming at six different regions of a target gene, and 4 primers are designed based on 6 different sites such as F3c, F2c and Flc regions at the 3 'end of the target gene 3 and Bl, B2 and B3 regions at the 5' end.
Primer) Inner FIP (Forward: the upstream inner primer consists of an F2 region and an F1C region, wherein the F2 region is complementary with an F2c region at the 3 'end of the target gene, and the F1C region has the same sequence as the Flc region at the 5' end of the target gene 5.
F3 primer: upstream Outer Primer) Outer (Forward, consisting of the F3 region, and is complementary to the F3c region of the target gene.
The BIP primer is as follows: downstream Inner Primer) Inner (Backward, consisting of B1C and B2 regions, B2 region is complementary to B2c region at 3 'end of target gene 3, B1C region has the same sequence as Blc region at 5' end of target gene 5.
B3 primer: downstream Outer Primer) Outer (Backward, consisting of the B3 region, complementary to the B3c region of the target gene.
The invention also provides a loop-mediated isothermal amplification (LAMP) reaction system which comprises the following components:
the invention further discloses the application of the loop-mediated isothermal amplification (LAMP) reaction system in the detection of O antigen serotype of Vibrio fluvialis strain, wherein the Vibrio fluvialis refers to a crude DNA extract of a pure culture of a sample obtained by separating the Vibrio fluvialis strain from any environment suitable for the life of Vibrio fluvialis.
The experimental result shows that the loop-mediated isothermal amplification (LAMP) reaction system can be used for accurately detecting the serotype strains of the vibrio fluvialis O1, O4, O14 and O17.
The invention mainly provides a technical means for detecting 4O antigen serotypes of vibrio fluvialis by using a loop-mediated isothermal amplification (LAMP) method. The positive effects are as follows:
(1) the technical means for detecting the vibrio fluvialis O antigen serotype by utilizing a loop-mediated isothermal amplification (LAMP) method is disclosed for the first time, and an effective method is provided for the clinical detection and epidemiological monitoring of the vibrio fluvialis.
(2) The operation is simple and convenient, expensive reaction equipment is not needed, and the reaction can be finished in the environment of a common water area.
(3) The detection time is short: by using the technical means, the detection can be completed within about 1.5 hours after the genomic DNA crude extract is obtained.
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FIG. 1, LAMP reaction specificity detection of O1 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are respectively added into the LAMP system of O1, and no amplified band appears in electrophoresis detection except the genome of O1, which indicates that the LAMP system of Vibrio fluvialis O1 has good specificity;
FIG. 2, LAMP reaction specificity detection of O4 system: the O4 LAMP system is added with the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 respectively, and no amplified band appears in electrophoresis detection except the genome of O4, which shows that the LAMP system specificity of Vibrio fluvialis O4 is good.
FIG. 3, LAMP reaction specificity detection of O14 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are added into the LAMP system of O14 respectively, and no amplified band appears in electrophoresis detection except the genome of O14, which indicates that the LAMP system of Vibrio fluvialis O14 has good specificity.
FIG. 4, LAMP reaction specificity detection of O17 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are added into the LAMP system of O17 respectively, and no amplified band appears in electrophoresis detection except the genome of O17, which indicates that the LAMP system of Vibrio fluvialis O17 has good specificity.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
Example 1
Design of primers
1. Screening for specific genes
The O antigen processing genes wzy, wzx, wzm and wzt are highly serotype determinative and have therefore been widely used as target genes for the serotyping of many gram-negative bacterial molecules. Wzy is selected as a specific gene from Vibrio fluvialis O1, O4, O14 and O17.
According to the invention, all genes in the gene cluster are compared by the all _ vs _ all _ blast method, and the matching number of specific genes is inevitably far smaller than that of conserved genes. The above method is combined to find a specific gene and design a primer for the specific gene.
2. Design of primers
The LAMP primer is designed by taking the selected specific gene of the river vibrio as a template.
Jp/e:// primerxplorwzyLAMP primers are designed according to the gene sequence. Primer) Inner FIP (Forward: an upstream inner primer, which consists of an F2 region and an F1C region, wherein the F2 region is complementary with an F2c region at the 3 'end of the target gene, and the F1C region has the same sequence as the Flc region at the 5' end of the target gene 5; f3 primer: upstream Outer Primer) Outer (Forward, consisting of the F3 region, and complementary to the F3c region of the target gene; the BIP primer is as follows: downstream Inner Primer) Primer Inner (Backward, consisting of B1C and B2 regions, B2 region is complementary to B2c region at 3 'end of target gene, B1C region has the same sequence as Blc region at 5' end of target gene; b3 primer: downstream Outer Primer) Primer Outer (Backward, consisting of the B3 region, complementary to the B3c region of the target gene. The number and sequence information for each primer is listed in table 2.
The length of the B3 and F3 primers is about 20 nt, and the Tm is between 55 and 60 ℃; the length of BIP and FIP primers is about 50 nt. The primers were synthesized by GENEWIZ (tianjin, china).
TABLE 2 primers used for LAMP
Example 2
Extraction of sample nucleic acid (crude extract of DNA isolated from pure culture of sample obtained in any environment suitable for Vibrio fluvialis life)
1. The sample to be tested is diluted with sterile water, typically at a dilution factor of 1: 10 (e.g. 10g solid sample or 10ml liquid sample in 90ml sterile water) to make a bacterial liquid mother liquor (liquid only).
The mother liquor was re-diluted with sterile water for 5 gradients: 1X 10‐1、1×10‐2、1×10‐3、1×10‐4、1×10‐5And uniformly coating each gradient bacterial liquid on an LB solid culture medium respectively for culture at 37 ℃. When a single colony grows on the plate, the colony is picked up and inoculated in LB liquid medium and cultured at 37 ℃ overnight at 180rpm (the inoculation operation is carried out in a clean bench).
2. Sample treatment: adding 1 mL of bacterial liquid cultured overnight into a 1.5 mL centrifuge tube, centrifuging at room temperature of 8000 rpm for 1 min, discarding the supernatant, and collecting the thallus. Adding 400 mu L Buffer digest, shaking and mixing uniformly, and carrying out water bath at 65 ℃ for 1 h until the cells are completely cracked.
In the water bath process, the mixture is inverted and uniformly mixed once every 10 min, so that the sample can be promoted to crack, and the mixed solution becomes clear and transparent and is completely cracked;
3. adding 200 mu L Buffer PB, fully reversing and uniformly mixing, and placing in a refrigerator at-20 ℃ for 5 min;
4. centrifuging at room temperature of 10000 rpm for 5 min, and transferring the supernatant (500-550 mu L) into a new 1.5 mL centrifuge tube;
5. adding isopropanol with equal volume, reversing for 5-8 times to mix thoroughly, standing at room temperature for 2-3 min, centrifuging at room temperature 10000 rpm for 5 min, and removing supernatant;
6. adding 1 mL of 75% ethanol, rinsing by inversion for 1-3 min, centrifuging at 10000 rpm for 2 min, and removing the supernatant;
7. repeating the step 5 once;
8. opening the cover and inverting for 5-10 min at room temperature until the residual ethanol is completely volatilized;
9. the obtained DNA was used in 50-100 µL ddH2Dissolving O, and keeping in a refrigerator at-20 ℃ for later use;
10. and (5) determining the concentration to 300 ng/muL.
Example 3
Specificity detection
The nucleic acid solution extracted in example 2 was used as a template for LAMP reaction, and LAMP reaction systems corresponding to O1, O4, O14 and O17 were added to the reaction mixture, and the mixture was placed in a water bath to carry out the reaction under the following conditions.
Example 4
Agarose gel electrophoresis detection
From each reaction system of example 3, 2. mu.L of the reaction product was aspirated, and subjected to agarose (2%) gel electrophoresis at a voltage of 120v for a period of 20 min. And then observing under an ultraviolet lamp.
The detection results are shown in fig. 1-4:
FIG. 1, LAMP reaction specificity detection of O1 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are respectively added into the LAMP system of O1, and no amplified band appears in electrophoresis detection except the genome of O1, which indicates that the LAMP system of Vibrio fluvialis O1 has good specificity;
FIG. 2, LAMP reaction specificity detection of O4 system: the O4 LAMP system is added with the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 respectively, and no amplified band appears in electrophoresis detection except the genome of O4, which shows that the LAMP system specificity of Vibrio fluvialis O4 is good.
FIG. 3, LAMP reaction specificity detection of O14 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are added into the LAMP system of O14 respectively, and no amplified band appears in electrophoresis detection except the genome of O14, which indicates that the LAMP system of Vibrio fluvialis O14 has good specificity.
FIG. 4, LAMP reaction specificity detection of O17 system: the genomes of Vibrio fluvialis O1, O4, O9, O10, O12, O14, O17 and O18 are added into the LAMP system of O17 respectively, and no amplified band appears in electrophoresis detection except the genome of O17, which indicates that the LAMP system of Vibrio fluvialis O17 has good specificity.
SEQUENCE LISTING
<110> university of southern kayak
<120> loop-mediated isothermal amplification detection method for 4O antigen serotypes of Vibrio fluvialis and application thereof
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Claims (4)
1. The LAMP primer for different O antigen serotypes of Vibrio fluvialis is characterized by comprising 4 DNA fragments respectively selected from wzy genes of Vibrio fluvialis O1, O4, O14 and O17, and the nucleotide sequence of the LAMP primer is shown as SEQ ID NO 1-SEQ ID NO 16.
2. A loop-mediated isothermal amplification LAMP reaction system, which contains LAMP primers of Vibrio fluvialis O1, O4, O14 and O17 of claim 1, respectively.
3. An application of the loop-mediated isothermal amplification LAMP reaction system of claim 2 in the aspect of detecting O antigen serotype of Vibrio fluvialis strain.
4. The use as claimed in claim 3, wherein the Vibrio fluvialis refers to a crude extract of DNA isolated from a pure culture of a sample to be tested in an environment suitable for Vibrio fluvialis.
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Citations (4)
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|---|---|---|---|---|
| CN103614455A (en) * | 2013-07-03 | 2014-03-05 | 淮海工学院 | Kit for rapid detection of Vibrio aestuarianus through loop-mediated isothermal amplification and detection method |
| CN103898108A (en) * | 2014-04-21 | 2014-07-02 | 南开大学 | Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof |
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2020
- 2020-11-30 CN CN202011368066.8A patent/CN112375835A/en active Pending
Patent Citations (4)
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|---|---|---|---|---|
| CN103614455A (en) * | 2013-07-03 | 2014-03-05 | 淮海工学院 | Kit for rapid detection of Vibrio aestuarianus through loop-mediated isothermal amplification and detection method |
| CN103898108A (en) * | 2014-04-21 | 2014-07-02 | 南开大学 | Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof |
| CN105200044A (en) * | 2015-08-03 | 2015-12-30 | 南开大学 | Nucleotides specific to vibrio fluvialis O1, O6, O7, O8 and O9 as well as application of nucleotides |
| CN111850145A (en) * | 2020-07-08 | 2020-10-30 | 南开大学 | Detection method for serotype O antigen molecule parting of aeromonas hydrophila O7, O16, O19, O24 and the like |
Non-Patent Citations (2)
| Title |
|---|
| QIAN-JINZHOU；LEIWANG；JIONGCHEN；等: "Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| 胡少辉: "18种河流弧菌O血清型的分子分型系统的建立", 《万方学位论文》 * |
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