CN111041112B

CN111041112B - Rapid constant-temperature detection method of vibrio vulnificus, primer set and application

Info

Publication number: CN111041112B
Application number: CN202010035775.8A
Authority: CN
Inventors: 李雪玲; 李园园; 刘伟; 韦朝春; 贾犇; 陆长德; 李亦学; 曹永梅
Original assignee: Shanghai Wangwang Food Group Co ltd; SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE
Current assignee: Shanghai Wangwang Food Group Co ltd; SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE
Priority date: 2015-09-02
Filing date: 2016-08-30
Publication date: 2022-09-20
Anticipated expiration: 2036-08-30
Also published as: CN106434897B; CN111057781A; CN106434883B; CN111073955B; CN111304348A; CN110938676B; CN111057780B; CN106434886A; CN111041113A; CN106434898A; CN106434896B; CN110951837B; CN111020045B; CN111304348B; CN111041115B; CN106434888B; CN111020046A; CN110760570A; CN111041071B; CN106434891B

Abstract

The invention discloses a rapid constant temperature detection method, a primer group and a kit for vibrio vulnificus. The method comprises the following steps: extracting genome DNA from a sample to be detected; performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying the specific sequence of the vibrio vulnificus as a primer; and determining whether the sample to be detected has vibrio vulnificus or not by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Rapid constant-temperature detection method of vibrio vulnificus, primer set and application

The application is filed in 2016, 8, month and 30, and has the application number of 201610780421.X and the name of the invention: the divisional application of the Chinese invention patent application of 'method, primer and application for rapid constant temperature detection of Vibrio vulnificus'; the parent application claims the priority of the Chinese patent application with the application date of 2015, 9 and 2, the application number of 201510556917.4, named as 'method, primer and kit for rapid isothermal detection of cronobacter sakazakii'.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting vibrio vulnificus at a constant temperature.

Background

Vibrio vulnificus (Vibrio vulgaris), also known as Vibrio maritima, is a halophilic gram-negative pathogenic bacterium found in sea water and some marine foods. Infection of humans by living contaminated marine products, or by exposure of wounds to contaminated sea water or marine animals, often causes symptoms such as primary sepsis, wound infections and acute gastroenteritis, with septic shock causing mortality rates of up to 50% or more. In China, Vibrio vulnificus infection mostly occurs in coastal areas and is listed as one of eight high-risk microorganisms in food pollution sources. In addition, the initial symptoms caused by Vibrio vulnificus are not significantly specific, and therefore prevention and detection of the bacteria are particularly important.

At present, detection of vibrio vulnificus is mainly completed through pathogen separation and biochemical identification, but the defects of long detection period, complex operation, difficulty in identifying similar species and the like exist. With the development of nucleic acid molecule detection technology in recent years, the conventional PCR or real-time PCR technology established by using a specific gene as a target has been successfully applied to the laboratory diagnosis of Vibrio vulnificus, and has the advantages of high sensitivity, short detection time and the like. Therefore, the method is not suitable for real-time on-site detection widely applied to basic detection departments, especially in enterprise production lines. In order to ensure the safety of food, a rapid, simple and accurate method for detecting vibrio vulnificus in food is urgently needed.

Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min at an isothermal condition, and generates a visible reaction by-product, white magnesium pyrophosphate precipitate (see Notomi T, Oyayaama H, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification (8512, 2000, 63). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.

Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. That is, the user must first ensure that the target gene is a specific sequence of the species to be tested. The invention uses patent CN 103160604A and ZL201310556940.4 as examples, and respectively aims at the specific genes of vibrio vulnificus reported in literature, namely vvhA gene and TolC gene sequences, and the LAMP technology is adopted to detect the vibrio vulnificus. However, the so-called "recognized specific genes" are often based on a delayed knowledge and are not necessarily updated based on the ever-increasing genome data of microorganisms, so that primers obtained based on the target gene sequences are not necessarily able to ensure their specificity and/or versatility in practical use. The invention presents the problem of insufficient primer versatility in the prior art as shown in Table 1. That is, the Vibrio vulnificus detection sequence used in the prior art method is not actually common to all of the Vibrio vulnificus strains, i.e., there is a possibility that a part of the strains of Vibrio vulnificus may be overlooked. A similar problem also exists in the confirmation of specificity, that is, there is a possibility that Vibrio non-vulnificus may be erroneously identified as Vibrio vulnificus. Therefore, there is a need in the industry for a vibrio vulnificus detection method that can ensure specificity and versatility, and at the same time, meet the needs of the primary detection department for rapidness and convenience, and can conveniently carry out real-time on-site detection inside the production line of an enterprise.

Disclosure of Invention

The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying vibrio vulnificus, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs Vibrio vulnificus LAMP primers based on microbial genome data resources (data obtained by 8/5/2013) in a GenBank database, and provides a method, a primer group and a kit for rapid isothermal amplification detection of Vibrio vulnificus. The detection method for detecting the vibrio vulnificus has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenience in operation and low cost.

The invention provides a method for rapidly detecting vibrio vulnificus strains, which comprises the following steps:

(1) extracting genome DNA from a sample to be detected;

(2) carrying out constant-temperature amplification reaction under an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome as a primer;

(3) and determining whether the sample to be detected has vibrio vulnificus or not by judging whether the reaction result is positive or not.

The method for detecting the vibrio vulnificus strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and a vibrio vulnificus specific amplification primer group as a primer to carry out constant temperature amplification reaction, and then determines whether the vibrio vulnificus exists in the sample to be detected by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.

In the invention, the genome-specific alkali sequence of the vibrio vulnificus is a bit sequence of 62063-62415 bp of the vibrio vulnificus with the GI number of 320154846.

In the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio vulnificus genome is a part of the nucleic acid sequence at the 62063-62415 bp position of the genome (GI No. 320154846) or a part of the complementary strand thereof. Wherein the Vibrio vulnificus genome-specific base sequence refers to a base sequence that is unique to the Vibrio vulnificus genome only and is not contained in the genome of other microorganisms.

Wherein the primer set capable of amplifying the specific base sequence of the Vibrio vulnificus genome includes, but is not limited to, primer set A, or any one selected from the group consisting of primers having a homology of 60% or more with a single sequence in the sequence of the primer set or the complementary strand sequence thereof.

Primer set a:

the upstream outer primer F3_ A: 5'-GTCTCCAAACTTAGACCAAA-3' (SEQ ID NO: 1);

downstream outer primer B3_ a: 5'-TCTACTTTGAGCAACCGTAT-3' (SEQ ID NO: 2);

upstream inner primer FIP _ A: 5'-GTTGCTTTAATGTTCGATATGGGCCAAAATTTCGTTATCCAGCG-3' (SEQ ID NO: 3);

the downstream inner primer BIP _ A: 5'-ACATTTTCTTTTCTAGCTCGCGTCAGTACGAATCACTACCTT-3' (SEQ ID NO: 4).

In the present invention, the primer set capable of amplifying the specific base sequence of the vibrio vulnificus genome may further include a primer set having a homology of 60% or more with each of the sequences of the aforementioned primer sets or their complementary strand sequences, and the primer set includes, but is not limited to, the following primer set B:

primer set B:

upstream outer primer F3_ B: 5'-GTACTGAATACGGTTGCTC-3' (SEQ ID NO: 5) (60% homology to the complementary strand 5'-ATACGGTTGCTCAAAGTAGA-3' of primer B3_ A);

downstream outer primer B3_ B: 5'-CTGGATGAACTCAGGCTA-3' (SEQ ID NO: 6);

upstream inner primer FIP _ B: 5'-CAACAATATCGCACCATGTGGAAGTAGACAAGTTTGAAGCC-3' (SEQ ID NO: 7);

a downstream inner primer BIP _ B: 5'-TTTCAAAGGCATATCACGGGTGTATTACTCAACGAGTTGCC-3' (SEQ ID NO: 8).

In the method of the present invention, in a specific embodiment, the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And magnesium ion Mg in enzyme reaction system ²⁺ And (6) merging.

In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; ② stopping the reaction for 2-20 min at 80 ℃. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.

In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic step-shaped strip, the sample to be detected is positive for vibrio vulnificus and contains vibrio vulnificus; if the electrophoretogram does not present the characteristic ladder-shaped strip, the sample to be detected is negative to vibrio vulnificus. The turbidity detection is carried out by observing with naked eyes or detecting turbidity by a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive to vibrio vulnificus and contains vibrio vulnificus; if no turbidity is found, the sample to be detected is negative to vibrio vulnificus. Or the reaction tube bottom can be observed by naked eyes after centrifugation to see whether the sediment exists or not, if the sediment exists at the reaction tube bottom, the sample to be detected is positive to the vibrio vulnificus and contains the vibrio vulnificus; if no sediment is left at the bottom of the reaction tube, the sample to be detected is negative to vibrio vulnificus.

The color development detection is to add color development reagent including but not limited to calcein (50 μ M) or SYBR Green I (30-50X), or hydroxyl naphthol blue (i.e. HNB, 120-ion 150 μ M) into the reaction tube. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to vibrio vulnificus; if the color after the reaction is green, the sample to be detected is positive to the vibrio vulnificus and contains the vibrio vulnificus. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is vibrio vulnificus negative; if the color after the reaction is sky blue, the sample to be detected is vibrio vulnificus positive. The color development detection can be carried out in real time or end point detection reaction results through a detection instrument besides the reaction results observed by naked eyes, and by reasonably setting a threshold value of negative reaction, when the reaction result of the sample to be detected is lower than or equal to the threshold value, the sample to be detected is vibrio vulnificus negative; and when the reaction result of the sample to be detected is greater than the threshold value, the sample to be detected is positive for the vibrio vulnificus. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.

In the color development detection, if calcein or hydroxynaphthol blue is used as a color developing agent, the color developing agent can be added before the constant-temperature amplification reaction, or can be added after the constant-temperature amplification reaction is completed, preferably before the constant-temperature amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is adopted as the color developing agent, the SYBR Green I is added after the isothermal amplification reaction is finished. If calcein is used as color-developing agent, 50 μ M of calcein is added into enzyme reaction systemSimultaneously, 0.6-1mM [ Mn ] is added ²⁺ ]For example, 0.6-1mM MnCl ₂ 。

The invention also provides a primer used in the method for detecting the vibrio vulnificus strain at constant temperature. The primer comprises a primer group capable of amplifying a specific base sequence of the Vibrio vulnificus genome, including but not limited to, a part of a nucleic acid sequence of 62063-62415 bp of the Vibrio vulnificus genome with GI number 320154846 or a part of a complementary strand thereof.

Wherein the primer group capable of amplifying the base sequence specific to the Vibrio vulnificus genome is selected from any one of the following primer groups, or from any one of the primer groups having a homology of 60% or more with a single sequence in the sequence of each of the primer groups or the complementary strand sequence thereof. Wherein, the primer group includes but is not limited to the following primer group A. The primer set having a homology of 60% or more with a single sequence in the aforementioned primer set sequence or its complementary strand sequence includes, but is not limited to, the following primer set B.

Primer set a:

upstream outer primer F3_ a: 5'-GTCTCCAAACTTAGACCAAA-3'

Downstream outer primer B3_ a: 5'-TCTACTTTGAGCAACCGTAT-3'

Upstream inner primer FIP _ A:

5’-GTTGCTTTAATGTTCGATATGGGCCAAAATTTCGTTATCCAGCG-3’；

the downstream inner primer BIP _ A: 5'-ACATTTTCTTTTCTAGCTCGCGTCAGTACGAATCACTACCTT-3';

primer set B:

upstream outer primer F3_ B: 5'-GTACTGAATACGGTTGCTC-3';

downstream outer primer B3_ B: 5'-CTGGATGAACTCAGGCTA-3', respectively;

upstream inner primer FIP _ B: 5'-CAACAATATCGCACCATGTGGAAGTAGACAAGTTTGAAGCC-3', respectively; the downstream inner primer BIP _ B: 5'-TTTCAAAGGCATATCACGGGTGTATTACTCAACGAGTTGCC-3', respectively;

the invention also provides a kit used in the method for detecting the vibrio vulnificus strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome. In the kit of the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio vulnificus genome includes, but is not limited to, a part of the nucleic acid sequence at position 62063-62415 bp of the genome (GI No. 320154846) or a part of the complementary strand thereof as the primer sequence; the primer includes but is not limited to the primer set a. But not limited to, a primer set having a homology of 60% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set B.

The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And magnesium ion Mg in enzyme reaction system ²⁺ And (6) merging.

The kit of the invention further comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, the whole genomic DNA, a portion of the genomic DNA of vibrio vulnificus, or a vector comprising the whole genomic DNA or a portion of the genomic DNA of vibrio vulnificus.

The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.

The kit further comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn ²⁺ ]For example, MnCl ₂ 。

The kit of the invention also comprises double distilled water.

The kit of the invention also comprises a nucleic acid extraction reagent.

The invention also provides a vector, which comprises any one primer selected from the primer group A, B. The vector contains a DNA sequence with vibrio vulnificus specificity, so that the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application fields of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one of the primers of primer set A, vector pBR322-B containing any one of the primers of primer set B, and the like. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, and the like.

The invention also provides application of the primers selected from any one of the primer groups A, B in constant temperature detection of Vibrio vulnificus.

The invention also provides application of the kit in constant temperature detection of vibrio vulnificus.

The invention also provides application of the vector in constant temperature detection of vibrio vulnificus.

The invention provides a simple, rapid and sensitive method, primer/primer group and detection reagent/kit for detecting vibrio vulnificus in the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the vibrio vulnificus detection method has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the constant temperature amplification method adopted by the invention can be carried out under the constant temperature condition, only a simple constant temperature device is needed, expensive instruments in PCR experiments are not needed, and the steps of carrying out electrophoresis detection on the amplified products and the like are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments for popularization and use, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base of molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.

Drawings

FIG. 1 shows the specificity of the isothermal Vibrio vulnificus detection method of example 7 of the present invention.

FIG. 2 shows the sensitivity of the Vibrio vulnificus detection method of example 8 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.

Examples 1-6 Vibrio vulnificus isothermal reaction System and detection method

The detection is carried out according to the following steps (1) to (3):

(1) extraction of genomic DNA

The vibrio vulnificus strain used for detection is from China center for the culture collection management of industrial microorganisms and is numbered CICC10383 (ATCC 27562). 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co ₂₆₀ /OD ₂₈₀ It was 1.8, and the concentration was 210.8 ng/. mu.L.

(2) The vibrio vulnificus genome DNA to be detected is taken as a template, self-prepared kits (shown in table 2 and table 3) are respectively adopted, a reaction system is prepared according to the conditions in table 3, and a vibrio vulnificus specific amplification primer group is taken as a primer to carry out constant-temperature amplification reaction. The primers in examples 1 to 6 were primer sets A, A, A, B, B, and B, respectively.

(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 3.

As can be seen from Table 3, the detection method and the primer set and the reaction system adopted by the detection method can well amplify the specific segment of Vibrio vulnificus and obtain the detection result. Therefore, the present invention can be applied to the detection of whether or not a sample contains Vibrio vulnificus.

Example 7 Vibrio vulnificus specific detection

28 strains of Vibrio vulnificus (1 to 25, 27 to 29 in Table 4 and FIG. 1) were collected, these strains and the Vibrio vulnificus strain (26 in Table 4 and FIG. 1) were cultured separately, 1mL of the bacterial solution was taken, and bacterial DNA was extracted using kit IA, and LAMP amplification (primer set A) and visualization by adding a color developing agent were performed separately with reference to the reaction system and conditions of example 1.

The detection results are shown in Table 4 and FIG. 1, in FIG. 1, 1 to 25 are respectively Staphylococcus aureus, Staphylococcus aureus subspecies aureoflavus, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria inoke, Listeria ehelii, Salmonella enterica subspecies enterica, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, Escherichia coli diarrheal, Escherichia coli producing enterotoxin, Escherichia coli enterotoxigenic Escherichia coli, Escherichia enterohemorrhagic Escherichia coli, Cronobacter sakazakii, Yersinia enterocolitica and Yersinia pseudotuberculosis, 27 to 29 are respectively haemolytica, Vibrio parahaemolyticus, Vibrio, Vibrio freundii and vibrio cholerae, NTC: negative control, 26: vibrio vulnificus. In FIG. 1, the product obtained after the amplification reaction of only Vibrio vulnificus strain appeared in bright green color as a positive result, as shown in tube No. 26. The products of other non-Vibrio vulnificus strains and the negative control amplification reaction are orange, which are negative results, as shown in tubes No. 1-25, 27-29 and NTC negative control tubes.

As can be seen from the results of FIG. 1 and Table 4, the detection kit and the detection method of the present invention have good Vibrio vulnificus strain specificity, that is, only Vibrio vulnificus strains are amplified positively, and other Vibrio vulnificus strains are negative.

Preparing a detection kit, wherein the primer adopted in the kit is a primer group B, and obtaining the same detection result according to the specific detection method, namely, the product after the amplification reaction of the non-vibrio vulnificus strain and the negative control is a negative result, and the product after the amplification reaction of the vibrio vulnificus strain is a positive result.

In addition, theoretical analysis was performed on the specificities of the primer group a and the primer group B, respectively, according to the method described in table 1, and the results found that, under the condition that at most three mismatches were allowed for each primer, at most one primer in each primer group was aligned to vibrio vulnificus, indicating that the specificity of each primer group was better.

Example 8 sensitivity detection

DNA of the bacterium CICC10383 was extracted by the method of example 1, and added to the reaction system using kit IB in a gradient of 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, and LAMP amplification (primer set A) and visualization by adding color developing agent were carried out respectively under the other reaction conditions according to the method of example 1 of Table 3. As shown in fig. 2, 1 to 6 are 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 1ng, 100pg and 10pg treatments appeared bright green and as positive results, the reaction products of 1pg, 100fg and 10fg treatments and the negative control appeared orange and as negative results. The results of the tests showed that the reaction tube contained a minimum of 10pg (about 2X 10. sup. th equivalent) per reaction tube ³ Individual bacteria) can still be detected.

According to the above detection method, other steps and conditions are as above, with the primer set B, DNA as low as 100fg in each reaction tube can still be detected, and the detection sensitivity is high.

Example 9 commonality testing

According to the method described in table 1, theoretical analysis was performed on the universality of the primer set a and the primer set B, respectively, and as a result, it was found that the primer regions of the primer sets completely match with the chromosomes 1 of three vibrio vulnificus strains (GI numbers 320154846, 326423644 and 37678184, respectively), and theoretically, the primer sets can be used for the detection of the three vibrio vulnificus strains, indicating that the universality of the primer sets is better.

TABLE 1 analysis of the universality and specificity of primers in the existing detection method of Vibrio vulnificus

Note: a) each Vibrio vulnificus strain has two chromosomes, and the position of the detection region in the genome of GI No. 320154846#1/320157827#2 is determined by performing Bowtie alignment of the sequence between primers F3 and B3 in the patent with the 6 chromosomal genomic sequences of 3 strains of Vibrio vulnificus, #1 represents the genomic sequence of the first chromosome of the strain, and #2 represents the genomic sequence of the second chromosome of the strain. b) The sequences of the detection regions are subjected to Blast comparison in public database resources, and the primer regions are completely matched, so that the universality is good. c) Performing Blast comparison on the detection region sequence in public database resources, wherein the higher the matching degree of the primer region is, the worse the specificity is; if the primers can not be compared to the non-traumatic arc strain at the same time, the specificity is good.

TABLE 2 kit for isothermal detection of Vibrio vulnificus and its main components

TABLE 3 examples 1 to 6 reaction conditions and test results in the method for isothermal detection of Vibrio vulnificus of the present invention

TABLE 4 strains used in the test and test results

Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china medical bacteria strain preservation and management center. b) +: positive result, -: and (5) negative result.

<110> Shanghai technical research institute of seafood industry, Shanghai Wangwang food group Co., Ltd

<160> 8

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

gtctccaaac ttagaccaaa 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

tctactttga gcaaccgtat 20

<210> 3

<211> 44

<212> DNA

<213> Artificial sequence

<400> 3

gttgctttaa tgttcgatat gggccaaaat ttcgttatcc agcg 44

<210> 4

<211> 42

<212> DNA

<213> Artificial sequence

<400> 4

acattttctt ttctagctcg cgtcagtacg aatcactacc tt 42

<210> 5

<211> 19

<212> DNA

<213> Artificial sequence

<400> 5

gtactgaata cggttgctc 19

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence

<400> 6

ctggatgaac tcaggcta 18

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence

<400> 7

caacaatatc gcaccatgtg gaagtagaca agtttgaagc c 41

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence

<400> 8

tttcaaaggc atatcacggg tgtattactc aacgagttgc c 41

Claims

1. A rapid constant temperature detection kit for Vibrio vulnificus is characterized in that the kit comprises a primer group capable of amplifying a specific base sequence of a Vibrio vulnificus genome; wherein the primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome is a primer group A;

primer set a:

the upstream outer primer F3_ A: 5'-GTCTCCAAACTTAGACCAAA-3', respectively;

downstream outer primer B3_ a: 5'-TCTACTTTGAGCAACCGTAT-3', respectively;

upstream inner primer FIP _ A: 5'-GTTGCTTTAATGTTCGATATGGGCCAAAATTTCGTTATCCAGCG-3', respectively;

the downstream inner primer BIP _ A: 5'-ACATTTTCTTTTCTAGCTCGCGTCAGTACGAATCACTACCTT-3' are provided.

2. The kit of claim 1, further comprising a Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg ²⁺ And one or more of betaine.

3. The kit of claim 1, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ 1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ A and BIP _ A primers, 0.15-0.3 μmol/L F3_ A and B3_ A primers, 0.16-0.64U/μ L Bst DNA polymerase, and 0-1.5mol/L betaine.

4. Use of the kit according to any one of claims 1 to 3 for isothermal detection of Vibrio vulnificus for non-diagnostic purposes.