CN111057779A

CN111057779A - Rapid constant-temperature detection method for vibrio vulnificus and application

Info

Publication number: CN111057779A
Application number: CN202010042444.7A
Authority: CN
Inventors: 曹永梅; 李雪玲; 李园园; 韦朝春; 刘伟; 贾犇; 陆长德; 李亦学
Original assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Shanghai Wangwang Food Group Co ltd
Current assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Shanghai Wangwang Food Group Co ltd
Priority date: 2015-09-02
Filing date: 2016-08-30
Publication date: 2020-04-24
Anticipated expiration: 2036-08-30
Also published as: CN106434884B; CN110938678B; CN106434897B; CN111020009B; CN111073989A; CN106434883B; CN106434897A; CN106434899B; CN111073956A; CN111073989B; CN111020008A; CN110964788B; CN111020045B; CN110951841A; CN111041116B; CN111020010B; CN106434886B; CN106367492A; CN106367500A; CN106434891A

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 for vibrio vulnificus and application

The application is filed on 2016, 8, 30, and has the application number of 201610767402.3 and the name of the invention: the divisional application of the Chinese patent application of 'method, primer and kit for rapid isothermal detection of Vibrio vulnificus and application'; 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, OkayamaH, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification reaction (2000, J8512; 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 patents CN 103160604A and ZL201310556940.4 are taken as examples, and the LAMP technology is adopted to detect Vibrio vulnificus by aiming at the specific genes of Vibrio vulnificus, namely vvhA gene and TolC gene sequences reported in the literature. 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, i.e., there is a possibility that non-vibrio vulnificus is 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 112393-113296 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 112393-113296 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, any one selected from the following primer sets A to C, or any one selected from the primer sets having a homology of 55% or more with a single sequence in the sequence of the primer set or the complementary strand sequence thereof.

Primer set a:

upstream outer primer F3_ a: 5'-GAAGTGTATCACCAGTTTAGC-3' (SEQ ID NO: 1);

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

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

the downstream inner primer BIP _ A:

5’-CAGTATTACCAAATCATTCATCCGCTTGAGTACAGGCATCGTTA-3’(SEQ ID NO：4)；

primer set B:

upstream outer primer F3_ B: 5'-TGGCTTAACGATAGCTACGC-3' (SEQ ID NO: 5);

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

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

the downstream inner primer BIP _ B:

5’-CGTTGGTTTAAGCCCAATCGATCGCGTTGAGTTTGCACACATGG-3’(SEQ ID NO：8)；

primer set C:

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3' (SEQ ID NO: 9);

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3' (SEQ ID NO: 10);

upstream inner primer FIP _ C: 5'-AGCGATTGATGAATCAGGCGTGCGGTCATCAAATTCTAAC-3' (SEQ ID NO: 11);

the downstream inner primer BIP _ C: 5'-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3' (SEQ ID NO: 12).

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 55% or more with a single sequence in the sequences of the aforementioned primer sets or the complementary strand sequences thereof, and the primer set includes, but is not limited to, any one of the following primer sets D to F:

primer set D:

upstream outer primer F3_ D: 5'-CAAGTGGAAGTGTATCACC-3' (SEQ ID NO: 13) (61.9% homology to primer F3_ A5'-GAAGTGTATCACCAGTTTAGC-3');

downstream outer primer B3_ D: 5'-GGAATAATGTACGACTCCTG-3' (SEQ ID NO: 14);

upstream inner primer FIP _ D: 5'-ATGCTTGTCGTCTTTCACCGCTCTAAAGATGAGATGATCGC-3' (SEQ ID NO: 15);

the downstream inner primer BIP _ D: 5'-GTTGCCTCGAAATGCATTAACTCTCTTGCGGATGAATGATT-3' (SEQ ID NO: 16);

primer set E:

upstream outer primer F3_ E: 5'-CAACGTATAGTTACCTAGCCGC-3' (SEQ ID NO: 17);

downstream outer primer B3_ E: 5'-CGGGTCAGTGGATTTGCTC-3' (SEQ ID NO: 18) (55% homology to primer B3_ B5'-TGGATTTGCTCCAAGACTGG-3');

upstream inner primer FIP _ E: 5'-CGCACAAACACAAAGAGCACCTTGGCTTAACGATAGCTACGC-3' (SEQ ID NO: 19);

the downstream inner primer BIP _ E:

5’-GAGACGACGTTGGTTTAAGCCCAACGTTGAGTTTGCACACATGG-3’(SEQ ID NO：20)；

a primer set F:

upstream outer primer F3 — F: 5'-TCGAAACACAAAGAGCACCG-3' (SEQ ID NO: 21);

downstream outer primer B3 — F: 5'-AATCGCCAAGAGCCGATG-3' (SEQ ID NO: 22) (57.9% homology of the complementary strand of this primer to primer F3_ C5'-TCTTGGCGATTGATGATGA-3');

upstream inner primer FIP _ F: 5'-GGGTGAATCGATCGATTGGGCTTAGGTGCTCTTTGTGTTTGTGC-3' (SEQ ID NO: 23);

the downstream inner primer BIP _ F: 5'-GACCATGTGTGCAAACTCAACGCGAAATCCGGCGGTTTCCA-3' (SEQ ID NO: 24).

In the method of the present invention, the primer set capable of amplifying a base sequence specific to the Vibrio vulnificus genome may or may not comprise a loop primer. The loop primer may be one or more, including primers LF and/or LB. The primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome is selected from any one of the following primer groups C ', E ' and F '; or any one selected from the group consisting of primers having a single sequence homology of 55% or more with the sequences of said primer groups C ', E ', F ' or the complementary strand sequences thereof:

a primer set C':

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3', respectively;

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3', respectively;

upstream inner primer FIP _ C: 5'-AGCGATTGATGAATCAGGCGTGCGGTCATCAAATTCTAAC-3', respectively;

the downstream inner primer BIP _ C:

5’-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3’；

upstream loop primer LF _ C: 5'-ATTACGAAACCACGGGCAAC-3' (SEQ ID NO: 25);

a primer set E':

upstream outer primer F3_ E: 5'-CAACGTATAGTTACCTAGCCGC-3', respectively;

downstream outer primer B3_ E: 5'-CGGGTCAGTGGATTTGCTC-3', respectively;

upstream inner primer FIP _ E: 5'-CGCACAAACACAAAGAGCACCTTGGCTTAACGATAGCTACGC-3', respectively;

the downstream inner primer BIP _ E:

5’-GAGACGACGTTGGTTTAAGCCCAACGTTGAGTTTGCACACATGG-3’；

downstream loop primer LB _ E: 5'-GATCGATTCACCCGCGTGCT-3' (SEQ ID NO: 26);

a primer set F':

upstream outer primer F3 — F: 5'-TCGAAACACAAAGAGCACCG-3', respectively;

downstream outer primer B3 — F: 5'-AATCGCCAAGAGCCGATG-3', respectively;

upstream inner primer FIP _ F:

5’-GGGTGAATCGATCGATTGGGCTTAGGTGCTCTTTGTGTTTGTGC-3’；

the downstream inner primer BIP _ F: 5'-GACCATGTGTGCAAACTCAACGCGAAATCCGGCGGTTTCCA-3', respectively;

upstream loop primer LF _ F: 5'-AACGTCGTCTCACACCAGCAA-3' (SEQ ID NO: 27);

and/or, the downstream loop primer LB _ F: 5'-GAGCAAATCCACTGACCCGCT-3' (SEQ ID NO: 28).

In specific embodiments, for example, the primer set F' may include only one forward loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer.

In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg²⁺(MgSO₄Or MgCl₂) 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, 0.4-1.0 mu mol/L LF and/or LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg²⁺(MgSO₄Or MgCl₂) 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. The loop primer contributes to the improvement of the reaction efficiency. 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)₂SO4，0.1％Triton X-100，2mM MgSO₄. MgSO in 1 XBst DNA polymerase reaction buffer₄And magnesium ion Mg in enzyme reaction system²⁺And (6) merging.

In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.

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 chart shows a characteristic step-shaped strip, the sample to be detected is positive to the vibrio vulnificus and contains the vibrio vulnificus; and if the electrophoretogram does not present a characteristic step-shaped strip, the sample to be detected is negative to the 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 SYBRGreen I (30-50X), or hydroxynaphthol blue (i.e. HNB, 120-. 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 positive to the vibrio vulnificus. 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 the color development agent, the color development agent can be added before the isothermal amplification reaction, or can be added after the isothermal amplification reaction is finished, preferably before the isothermal amplification reaction, or can be added before the isothermal amplification reactionEffectively reducing the possibility of reaction pollution. If SYBR Green I is adopted as a 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 calcein is added into enzyme reaction system, and 0.6-1mM [ Mn ] is added²⁺]For example, 0.6-1mM MnCl₂。

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 112393-113296 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 is selected from any one of the primer groups having a single sequence homology of 55% or more with the sequences of the primer groups or the complementary strand sequences thereof. Wherein the primer set includes, but is not limited to, any one of the following primer sets A to C. The primer set having a homology of 55% or more with a single sequence in the sequence of the aforementioned primer set or the sequence of the complementary strand thereof includes, but is not limited to, any one of the following primer sets D to F.

Primer set a:

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

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

Upstream inner primer FIP _ A: 5'-ACATGCTTGTCGTCTTTCACCTAAAGATGAGATGATCGCCA-3', respectively;

the downstream inner primer BIP _ A:

5’-CAGTATTACCAAATCATTCATCCGCTTGAGTACAGGCATCGTTA-3’；

primer set B:

upstream outer primer F3_ B: 5'-TGGCTTAACGATAGCTACGC-3', respectively;

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

upstream inner primer FIP _ B: 5'-CGTCTCACACCAGCAACGCATCGAAACACAAAGAGCACCG-3', respectively;

the downstream inner primer BIP _ B:

5’-CGTTGGTTTAAGCCCAATCGATCGCGTTGAGTTTGCACACATGG-3’；

primer set C:

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3', respectively;

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3', respectively;

the downstream inner primer BIP _ C:

5’-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3’；

primer set D:

upstream outer primer F3_ D: 5'-CAAGTGGAAGTGTATCACC-3', respectively;

downstream outer primer B3_ D: 5'-GGAATAATGTACGACTCCTG-3', respectively;

upstream inner primer FIP _ D: 5'-ATGCTTGTCGTCTTTCACCGCTCTAAAGATGAGATGATCGC-3', respectively;

the downstream inner primer BIP _ D: 5'-GTTGCCTCGAAATGCATTAACTCTCTTGCGGATGAATGATT-3', respectively;

primer set E:

upstream outer primer F3_ E: 5'-CAACGTATAGTTACCTAGCCGC-3', respectively;

downstream outer primer B3_ E: 5'-CGGGTCAGTGGATTTGCTC-3', respectively;

the downstream inner primer BIP _ E:

5’-GAGACGACGTTGGTTTAAGCCCAACGTTGAGTTTGCACACATGG-3’；

a primer set F:

upstream outer primer F3 — F: 5'-TCGAAACACAAAGAGCACCG-3', respectively;

downstream outer primer B3 — F: 5'-AATCGCCAAGAGCCGATG-3', respectively;

upstream inner primer FIP _ F:

5’-GGGTGAATCGATCGATTGGGCTTAGGTGCTCTTTGTGTTTGTGC-3’；

the downstream inner primer BIP _ F: 5'-GACCATGTGTGCAAACTCAACGCGAAATCCGGCGGTTTCCA-3' are provided.

In the primers used in the method for detecting vibrio vulnificus at constant temperature, the primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome may or may not comprise one or more loop primers; the loop primer is LF and/or LB. The primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome is selected from any one of the following primer groups C ', E ' and F '; or any one selected from the group consisting of primers having a single sequence homology of 55% or more with the sequences of said primer groups C ', E ', F ' or the complementary strand sequences thereof:

a primer set C':

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3', respectively;

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3', respectively;

the downstream inner primer BIP _ C:

5’-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3’；

upstream loop primer LF _ C: 5'-ATTACGAAACCACGGGCAAC-3', respectively;

a primer set E':

upstream outer primer F3_ E: 5'-CAACGTATAGTTACCTAGCCGC-3', respectively;

downstream outer primer B3_ E: 5'-CGGGTCAGTGGATTTGCTC-3', respectively;

the downstream inner primer BIP _ E:

5’-GAGACGACGTTGGTTTAAGCCCAACGTTGAGTTTGCACACATGG-3’；

downstream loop primer LB _ E: 5'-GATCGATTCACCCGCGTGCT-3', respectively;

a primer set F':

upstream outer primer F3 — F: 5'-TCGAAACACAAAGAGCACCG-3', respectively;

downstream outer primer B3 — F: 5'-AATCGCCAAGAGCCGATG-3', respectively;

upstream inner primer FIP _ F:

5’-GGGTGAATCGATCGATTGGGCTTAGGTGCTCTTTGTGTTTGTGC-3’；

upstream loop primer LF _ F: 5'-AACGTCGTCTCACACCAGCAA-3', respectively;

and/or, the downstream loop primer LB _ F: 5'-GAGCAAATCCACTGACCCGCT-3' are provided.

In a specific embodiment, the primer set F' may include only one forward loop primer, only one downstream loop primer, or both a forward loop primer and a downstream loop primer. In a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with 55% or more homology with single primer in the aforementioned primer sequence or its complementary strand sequence.

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 112393-113296 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, any one of the primer set A, the primer set B, the primer set C, and the like. But not limited to, a primer group having a homology of 55% or more with a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer set D, primer set E, primer set F, etc.

In the kit of the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio vulnificus genome may or may not comprise one or more loop primers; the loop primer serves as an optional component. The loop primer is LF and/or LB. The primer set comprising the loop primer LF and/or LB includes, but is not limited to, primer sets C ', E ', F ', etc. In a specific embodiment, the kit of the invention may comprise 0.4-1.0. mu. mol/L of LF and/or LB loop primers. In a specific embodiment, the sequences of the primer sets are respectively the primers shown by FIP, BIP, F3, B3, LF and LB, or the primers with 55% or more homology to the single primer of the aforementioned sequence or its complementary strand sequence.

The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg²⁺(MgSO₄Or MgCl₂) 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 Mg²⁺(MgSO₄Or MgCl₂) 1.0-1.6mmol/LdNTP, 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 BstDNA 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)₂SO4，0.1％Triton X-100，2mM MgSO₄. MgSO in 1 XBst DNA polymerase reaction buffer₄And magnesium ion Mg in enzyme reaction system²⁺And (6) merging.

The kit of the invention also 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 [ Mn²⁺]For example, MnCl₂。

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 carrier, which comprises any one primer selected from the group consisting of primer groups A-C, D-F, C ', E ' and F '. 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-D containing any one of the primers of primer set D in … …, vector pBR322-F 'containing any one of the primers of primer set F' in … …, and the like. Vector lambda phage-A containing any one primer of primer set A, … … vector lambda phage-D containing any one primer of primer set D, … … vector lambda phage-F 'containing any one primer of primer set F', etc.

The invention also provides the application of any one primer selected from the primer groups A-C, D-F, C ', E ' and F ' 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 for detecting vibrio vulnificus, a primer/primer group and a detection reagent/kit for 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, convenience in 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 Co₂₆₀/OD₂₈₀It 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, C ', D, E ', F ', F, 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. In addition, when the detection is performed by using a detector, the detection effect is good when the reaction time is shortened to 10min (as in example 6). Therefore, the present invention can be applied to the detection of whether or not a sample contains Vibrio vulnificus.

According to the method of the embodiment, the primer groups B to C and the primer group E are respectively used, so that the specific fragment of the vibrio vulnificus can be well amplified and the detection result can be obtained.

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 bright green and was 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 primers adopted in the kit are respectively primer groups B-C, primer groups D-F, primer groups C ', E ' and F ', and the same detection results are obtained according to the specific detection method, namely, the products after the amplification reaction of the non-vibrio vulnificus strain and the negative control are negative results, and the products after the amplification reaction of the vibrio vulnificus strain are positive results.

In addition, theoretical analysis was performed on the specificity of the primer sets A to C, D to F, and C ', E ', F ' respectively according to the method described in Table 1, and the results found that, when at most three mismatches were allowed for each primer, at most two primers were simultaneously aligned to Vibrio vulnificus in each primer set, indicating that the specificity of each primer set was better.

Example 8 sensitivity detection

DNA of the bacterium CICC10383 was extracted by the method of example 1, and the DNA was added to the reaction system using kit IB in a gradient of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, and LAMP amplification (primer set A) and visualization by adding color reagent 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 7 are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 10ng and 1ng of the treatments showed bright green color and positive results, and the reaction products of 100pg, 10pg, 1pg, 100fg and 10fg of the treatments and the negative control showed orange color and negative results. The test results showed that a minimum of 1ng of DNA was detected in each reaction tube.

According to the detection method, other steps and conditions are the same as above, DNA as low as 1ng to 100fg in each reaction tube can still be detected by using the primer sets B to C, the primer sets D to F and the primer sets A ', C ' and D ', respectively.

Example 9 commonality testing

According to the method described in table 1, theoretical analysis was performed on the universality of the primer sets a to C, D to F, C ', E ', F ', respectively, and as a result, it was found that the primer regions of the primer sets completely match with chromosomes #1 of three vibrio vulnificus (GI nos. 320154846, 326423644, and 37678184, respectively), and could be theoretically used for the detection of the above three vibrio vulnificus strains, indicating that the universality of the primer sets is good.

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) And performing Blast comparison on the detection region sequences in public database resources, wherein the primer regions are completely matched and have good universality. 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 the 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 Wangwang food group Co., Ltd., Shanghai Marine industry and technology research institute

Rapid constant-temperature detection method for <120> vibrio vulnificus and application

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<213> Artificial sequence

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<213> Artificial sequence

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<213> Artificial sequence

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tggcttaacg atagctacgc 20

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<213> Artificial sequence

<400>6

tggatttgct ccaagactgg 20

<210>7

<211>40

<212>DNA

<213> Artificial sequence

<400>7

cgtctcacac cagcaacgca tcgaaacaca aagagcaccg 40

<210>8

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<213> Artificial sequence

<400>8

cgttggttta agcccaatcg atcgcgttga gtttgcacac atgg 44

<210>9

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<213> Artificial sequence

<400>9

tcttggcgat tgatgatga 19

<210>10

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<213> Artificial sequence

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tgattcaatg ccccctata 19

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<213> Artificial sequence

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<213> Artificial sequence

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<213> Artificial sequence

<400>13

caagtggaag tgtatcacc 19

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<212>DNA

<213> Artificial sequence

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ggaataatgt acgactcctg 20

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<213> Artificial sequence

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atgcttgtcg tctttcaccg ctctaaagat gagatgatcg c 41

<210>16

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<213> Artificial sequence

<400>16

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<210>17

<211>22

<212>DNA

<213> Artificial sequence

<400>17

caacgtatag ttacctagcc gc 22

<210>18

<211>19

<212>DNA

<213> Artificial sequence

<400>18

cgggtcagtg gatttgctc 19

<210>19

<211>42

<212>DNA

<213> Artificial sequence

<400>19

cgcacaaaca caaagagcac cttggcttaa cgatagctac gc 42

<210>20

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<213> Artificial sequence

<400>20

gagacgacgt tggtttaagc ccaacgttga gtttgcacac atgg 44

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<213> Artificial sequence

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tcgaaacaca aagagcaccg 20

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<213> Artificial sequence

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aatcgccaag agccgatg 18

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<213> Artificial sequence

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<213> Artificial sequence

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<213> Artificial sequence

<400>25

attacgaaac cacgggcaac 20

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<213> Artificial sequence

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gatcgattca cccgcgtgct 20

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<213> Artificial sequence

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Claims

1. A rapid constant temperature detection method for Vibrio vulnificus is characterized by comprising the following steps:

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

(2) 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 base sequence of the vibrio vulnificus genome as a primer;

(3) determining whether the vibrio vulnificus exists in the sample to be detected by judging whether the reaction result is positive or not;

wherein the Vibrio vulnificus genome specific alkali sequence is a bit sequence of 112393-113296 bp of the Vibrio vulnificus genome with GI number 320154846;

wherein the primer set capable of amplifying the base sequence specific to the Vibrio vulnificus genome is selected from any one of the following primer sets B, C, C';

primer set B:

upstream outer primer F3_ B: 5'-TGGCTTAACGATAGCTACGC-3' (SEQ ID NO: 5);

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

the downstream inner primer BIP _ B:

5’-CGTTGGTTTAAGCCCAATCGATCGCGTTGAGTTTGCACACATGG-3’(SEQ ID NO：8)；

primer set C:

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3' (SEQ ID NO: 9);

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3' (SEQ ID NO: 10);

the downstream inner primer BIP _ C: 5'-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3' (SEQ ID NO: 12);

a primer set C':

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3', respectively;

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3', respectively;

the downstream inner primer BIP _ C:

5’-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3’；

upstream loop primer LF _ C: 5'-ATTACGAAACCACGGGCAAC-3' (SEQ ID NO: 25).

2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg²⁺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, 0-1.5mol/L betaine, including or not including 0.4-1.0. mu. mol/L LF and/or LB primers.

3. The method of claim 1, wherein the isothermal amplification reaction is performed by incubating at ① 60-65 ℃ for 10-90 min and terminating at ② 80 ℃ for 2-20 min.

4. The primer for rapid isothermal detection of the vibrio vulnificus is characterized by comprising a primer group capable of amplifying a specific base sequence of a vibrio vulnificus genome, wherein the sequence is a part of a nucleic acid sequence of 112393-113296 bp of the vibrio vulnificus genome with the GI number of 320154846 or a part of a complementary strand thereof;

primer set B:

upstream outer primer F3_ B: 5'-TGGCTTAACGATAGCTACGC-3' (SEQ ID NO: 5);

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

the downstream inner primer BIP _ B:

5’-CGTTGGTTTAAGCCCAATCGATCGCGTTGAGTTTGCACACATGG-3’(SEQ ID NO：8)；

primer set C:

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3' (SEQ ID NO: 9);

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3' (SEQ ID NO: 10);

a primer set C':

upstream outer primer F3_ C: 5'-TCTTGGCGATTGATGATGA-3', respectively;

downstream outer primer B3 — C: 5'-TGATTCAATGCCCCCTATA-3', respectively;

the downstream inner primer BIP _ C:

5’-AGCTCGTTATCCACTAGTTGATGCACACTCAGGAAAAGCAATA-3’；

upstream loop primer LF _ C: 5'-ATTACGAAACCACGGGCAAC-3' (SEQ ID NO: 25).

5. Use of a primer for isothermal detection of Vibrio vulnificus according to claim 4.