CN111020008B

CN111020008B - Rapid isothermal detection method and kit for nucleic acid of vibrio cholerae O1 group

Info

Publication number: CN111020008B
Application number: CN202010036105.8A
Authority: CN
Inventors: 李雪玲; 刘伟; 李园园; 贾犇; 韦朝春; 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: 2022-10-28
Anticipated expiration: 2036-08-30
Also published as: CN111041071B; CN106434885B; CN110951837A; CN106434888B; CN111020010B; CN110964786B; CN106434885A; CN106434884A; CN106434899A; CN110938676A; CN111304348A; CN110964788A; CN106434886B; CN111020049A; CN111100939A; CN106244706A; CN106367500B; CN111057778A; CN110760570A; CN106367492B

Abstract

The invention discloses a rapid constant temperature detection method, a primer group and a kit for vibrio cholerae O1 group. 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 a specific sequence of the vibrio cholerae O1 group as a primer; and determining whether the vibrio cholerae O1 group exists in the sample to be detected 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 isothermal detection method and kit for nucleic acid of vibrio cholerae O1 group

The application is filed on 2016, 8, 30, and has the application number of 201610780456.3 and the name of the invention: the divisional application of the Chinese patent application of 'a method, primers and a kit for rapidly detecting the vibrio cholerae O1 group at constant temperature'; the parent application claims the priority of Chinese patent application with the application date of 2015, 9/2, the application number of 201510556917.4 and the name of 'method, primer and kit for rapidly detecting cronobacter sakazakii at constant temperature'.

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 cholerae O1 group at constant temperature.

Background

Vibrio cholerae (Vibrio cholerae) is widely distributed in marine and freshwater environments and is an important disease inspection and quarantine object and food-borne pathogenic bacteria for aquaculture. Vibrio cholerae can be classified into many serogroups depending on the bacterial (O) antigen, but only the O1 and O139 groups were found to be able to induce cholera. Among them, the O1 group Vibrio cholerae has caused seven pandemics of cholera worldwide since 1817. Therefore, early detection and diagnosis of the Vibrio cholerae O1 group is important for the treatment and prevention of the occurrence and spread of cholera.

The traditional vibrio cholerae detection method has the defects of long detection period, relatively complex operation and low detection efficiency, and is difficult to meet the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means of PCR and fluorescence PCR technologies, but both methods require special detection instruments, and are not suitable for real-time field detection in basic detection departments, especially in enterprise production lines. In order to ensure the safety of food, a rapid, simple and accurate method for detecting the vibrio cholerae O1 group 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, BIP is composed of B1C and B2) for 6 regions of a target sequence, and performs a Nucleic acid amplification reaction by incubating for about 60min under isothermal conditions using a DNA polymerase having a strand displacement activity, to generate macroscopic reaction byproducts, namely white magnesium pyrophosphate precipitate (see Notomi T, okayama H, masubuchi H, yonekawa T, watane K, amino N, hase-mediated isothermal amplification of DNA, nucleic Acids Research, 20012E 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 discloses a method for detecting vibrio cholerae by LAMP technology, which takes patents ZL201010584749.7 and ZL201310119610.9 as examples, and respectively aims at specific genes of vibrio cholerae, namely ctxA gene and dnaA gene, reported in literatures. 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 versatility and/or specificity in practical applications. Table 1 of the present invention shows the problem that the versatility is not ensured in the prior art. That is, the detection sequence of the group of Vibrio cholerae O1 used in the prior art method is not actually common to the group of Vibrio cholerae O1, i.e., there is a possibility that some strains of the group of Vibrio cholerae O1 may be missed. A similar problem exists in the confirmation of specificity, that is, there is a possibility that the group of non-Vibrio cholerae O1 is erroneously identified as the group of Vibrio cholerae O1. Therefore, a vibrio cholerae O1 group detection method capable of ensuring specificity and universality is urgently needed in the industry, and meanwhile, the requirements of basic detection departments on rapidness and convenience are met, and real-time on-site detection can be conveniently developed in an enterprise production line.

Disclosure of Invention

The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the prior 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 the vibrio cholerae O1 group, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a vibrio cholerae O1 group LAMP primer based on microbial genome data resources (data up to 2013, 8 and 5 days) in a GenBank database, and provides a method, a primer group and a kit for rapidly detecting the vibrio cholerae O1 group by constant-temperature amplification. The detection method for detecting the vibrio cholerae O1 group has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenient operation and low cost.

The invention provides a method for rapidly detecting a vibrio cholerae O1 group strain, which comprises the following steps:

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

(2) Performing constant-temperature amplification reaction under 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 genome of the vibrio cholerae O1 group as a primer;

(3) And determining whether the vibrio cholerae O1 group exists in the sample to be detected or not by judging whether the reaction result is positive or not.

The invention relates to a method for detecting a vibrio cholerae O1 group strain at constant temperature, which comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by using the genome DNA as a template and using a vibrio cholerae O1 group specific amplification primer group as a primer, and then determining whether the vibrio cholerae O1 group 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 base sequence of the vibrio cholerae O1 group is a sequence 170082-171001 bp of the vibrio cholerae O1 group with GI number 15640032.

In the present invention, the primer set capable of amplifying the genome-specific nucleotide sequence of Vibrio cholerae O1 group is a part of the nucleic acid sequence of positions 170082-171001 bp of the genome (GI No. 15640032) or a part of the complementary strand thereof. Wherein the genome-specific base sequence of Vibrio cholerae O1 group is a base sequence that is unique to the genome of Vibrio cholerae O1 group and is not contained in the genome of other microorganisms.

Wherein the primer set capable of amplifying the genome specific base sequence of Vibrio cholerae O1 group includes, but is not limited to, any one selected from the following primer sets A-B, or any one selected from the primer sets having a homology of 50% 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'-CCACTTATTCGCTTTACACA-3' (SEQ ID NO: 1);

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

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

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

primer set B:

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

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

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

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

In the present invention, the primer set capable of amplifying the specific base sequence of the genome of vibrio cholerae O1 group may further include a primer set having a homology of 50% or more with a single sequence in the sequence of each of the aforementioned primer sets or the sequence of the complementary strand thereof, and the primer set includes, but is not limited to, primer set C:

primer set C

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

downstream outer primer B3_ C:5'-CCGTTTTGATTTCGATGGT-3' (SEQ ID NO: 10) (50% homology to primer B3_ A5'-TTTCGATGGTGTAATCATCC-3');

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

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

In the method of the present invention, the primer set capable of amplifying the genome-specific base sequence of Vibrio cholerae O1 group 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 genome of the vibrio cholerae O1 group is a primer group C'; or any one of the primer sets having a homology of 50% or more with respect to a single sequence in the sequence of the primer set C' or the sequence of the complementary strand thereof:

a primer set C':

the upstream outer primer F3_ C:5'-AATGGAATTTGAACCTCGTC-3';

downstream outer primer B3_ C:5'-CCGTTTTGATTTCGATGGT-3';

upstream inner primer FIP _ C:5'-ATCATTGCCAGAGTGGAACTTAGCGTGTGGATAACGAAA-3';

the downstream inner primer BIP _ C:5'-GTGGTATGGACGTTCAATCGTTAAAGAGACCAGAGGTTCAA-3'; upstream loop primer LF _ C:5'-GCGCAAAGTGAAACGCAT-3' (SEQ ID NO: 13);

and/or, the downstream loop primer LB _ C:5'-AGAATCGGCGGATTTTAAAGGG-3' (SEQ ID NO: 14).

In specific embodiments, for example, the primer set C' may comprise 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 (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 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, 0.4-1.0 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 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. 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 (NH 4) 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 of the invention, the reaction procedure of the isothermal amplification reaction is (1) incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; (2) the reaction is stopped at 80 ℃ for 2-20 min. 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 to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative to the vibrio cholerae O1 group. The turbidity detection is to detect turbidity by visual observation or a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group; if no turbidity is found, the sample to be detected is negative to the vibrio cholerae O1 group. Or the reaction tube bottom can be visually observed whether the sediment exists or not after the centrifugation, if the sediment exists at the reaction tube bottom, the sample to be detected is positive to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group; if no precipitate is left at the bottom of the reaction tube, the sample to be detected is negative to the vibrio cholerae O1 group.

The color development detection is to add color development reagent including but not limited to calcein (50 μ M) or SYBR Green I (30-50 ×), or hydroxynaphthol blue (i.e. HNB,120-150 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 cholerae O1 group; if the color after the reaction is green, the sample to be detected is positive to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group. When hydroxyl naphthol blue is used as a color developing agent, if the color after the reaction is violet, the sample to be detected is negative to vibrio cholerae O1 group; if the color after the reaction is sky blue, the sample to be detected is positive to the vibrio cholerae O1 group. The color development detection can be carried out in real time or at the end point by a detection instrument besides observing the reaction result by naked eyes, and by reasonably setting the threshold value of the 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 negative to the vibrio cholerae O1 group; 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 cholerae O1 group. 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 adopted as the color development agent, the color development agent can be added before the constant-temperature amplification reaction, and can also be added before the constant-temperature amplification reactionSo as to be added after the isothermal amplification reaction is completed, preferably before the isothermal amplification reaction, the possibility of reaction contamination 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 the color developing agent, 0.6 to 1mM of Mn is added while adding 50. Mu.M of calcein to the enzyme reaction system ²⁺ ]For example, 0.6-1mM MnCl ₂ 。

The invention also provides a primer used in the method for detecting the vibrio cholerae O1 group strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of the genome of the Vibrio cholerae O1 group, and the sequence of the primer is part of the nucleic acid sequence of 170082-171001 bp of the Vibrio cholerae O1 group genome with the GI number of 15640032 or part of the complementary strand thereof.

Wherein the primer group capable of amplifying the genome-specific base sequence of Vibrio cholerae O1 group is selected from any one of the following primer groups, or is selected from any one of the primer groups having a homology of 50% or more with a single sequence in the sequence of each of the primer groups or the complementary strand sequence thereof. Wherein the primer set includes, but is not limited to, any one of the following primer sets A to B. The primer set having a homology of 50% or more with respect to a single sequence in the aforementioned primer set sequence or its complementary strand sequence includes, but is not limited to, primer set C.

Primer set a:

the upstream outer primer F3_ A:5'-CCACTTATTCGCTTTACACA-3';

downstream outer primer B3_ A:5'-TTTCGATGGTGTAATCATCC-3';

upstream inner primer FIP _ A:5'-GCAAAGTGAAACGCATGGTTGGAATTTGAACCTCGTCTG-3';

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

primer set B:

the upstream outer primer F3_ B:5'-ATCCGCCAGATGTGCGAT-3';

downstream outer primer B3_ B:5'-GGGAATCCAGATCCCTAGTTTG-3';

upstream inner primer FIP _ B:5'-GCGGATCGGTAAACAGCTGCTCCGTTGGGGTCATGCAGATG-3'; the downstream inner primer BIP _ B:5'-TGCCTGAATTTACAGGCTTACGCGATCGCCAACCCAAGTTGG-3';

primer set C:

an upstream outer primer F3_ C:5'-AATGGAATTTGAACCTCGTC-3';

downstream outer primer B3_ C:5'-CCGTTTTGATTTCGATGGT-3';

upstream inner primer FIP _ C:5'-ATCATTGCCAGAGTGGAACTTAGCGTGTGGATAACGAAA-3';

the downstream inner primer BIP _ C:5'-GTGGTATGGACGTTCAATCGTTAAAGAGACCAGAGGTTCAA-3'.

In the primers used in the method for detecting the vibrio cholerae O1 group at the constant temperature, the primer group capable of amplifying the specific base sequence of the vibrio cholerae O1 group genome can also comprise or 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 genome of the vibrio cholerae O1 group is a primer group C'; or any one selected from the group consisting of primer groups having a homology of 50% or more with the individual sequences in the C' sequences of the primer group or the complementary strand sequences thereof:

a primer set C':

the upstream outer primer F3_ C:5'-AATGGAATTTGAACCTCGTC-3';

downstream outer primer B3_ C:5'-CCGTTTTGATTTCGATGGT-3';

upstream inner primer FIP _ C:5'-ATCATTGCCAGAGTGGAACTTAGCGTGTGGATAACGAAA-3';

the downstream inner primer BIP _ C:5'-GTGGTATGGACGTTCAATCGTTAAAGAGACCAGAGGTTCAA-3'; upstream loop primer LF _ C:5'-GCGCAAAGTGAAACGCAT-3';

and/or, the downstream loop primer LB _ C:5'-AGAATCGGCGGATTTTAAAGGG-3'.

In a specific embodiment, the primer set C' may include only one upstream loop primer, only one downstream loop primer, or both of the upstream loop primer and the downstream loop primer. In a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with homology of 50% or more with the aforementioned primer sequence or single primer in the complementary strand sequence thereof.

The invention also provides a kit used in the method for detecting the vibrio cholerae O1 group strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the genome of the vibrio cholerae O1 group. In the kit of the present invention, the primer set capable of amplifying the genome-specific nucleotide sequence of Vibrio cholerae O1 group includes, but is not limited to, a portion of the nucleic acid sequence at position 170082-171001 bp of the genome (GI No.: 15640032) or a portion of the complementary strand thereof as the primer sequence; the primer includes, but is not limited to, any one of the primer set A and the primer set B. But not limited to, a primer group having 50% or more homology with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set C, etc.

In the kit of the present invention, the primer set capable of amplifying the specific base sequence of the genome of vibrio cholerae O1 group 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, the primer set C' and the like. In a specific embodiment, the kit of the present 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 50% 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 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,10mmoll/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 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 partial genomic DNA of the vibrio cholerae O1 group, or a vector comprising the whole genomic DNA or a partial genomic DNA of the vibrio cholerae O1 group.

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 carrier, which comprises any one group of primers selected from the group A-B, C, C'. The vector contains a DNA sequence with the specificity of the vibrio cholerae O1 group, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application field 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 primer of primer set A, vector pBR322-B containing any one primer of primer set B, vector pBR322-C' containing any one primer of primer set C of … …, and the like. A vector lambda phage-A containing any one primer of the primer set A, a vector lambda phage-B containing any one primer of the primer set B, a vector lambda phage-C' containing any one primer of the primer set C in … …, and the like.

The invention also provides the application of any one primer selected from the primer groups A-B, C, C' in constant temperature detection of the vibrio cholerae O1 group.

The invention also provides application of the kit in constant temperature detection of the vibrio cholerae O1 group.

The invention also provides application of the carrier in constant temperature detection of vibrio cholerae O1 group.

The invention provides a simple, rapid and sensitive method for detecting vibrio cholerae O1 group, 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 detection method for the vibrio cholerae O1 group 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 detection method for Vibrio cholerae O1 group in example 7 of the present invention.

FIG. 2 shows the sensitivity of the detection method of Vibrio cholerae O1 group in 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 are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims. 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.

Example 1-6 Vibrio cholerae O1 group constant temperature 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 cholerae O1 group strain for detection is derived from China general microbiological culture collection center with the number of CGMCC1.8676. 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 At a concentration of 1.8, 30.4 ng/. Mu.L.

(2) Taking genome DNA of a vibrio cholerae O1 group to be detected as a template, respectively adopting self-prepared kits (shown in tables 2 and 3), preparing a reaction system according to the conditions in the table 3, and taking a specific amplification primer group of the vibrio cholerae O1 group as a primer to carry out constant-temperature amplification reaction. The primers used in examples 1 to 6 were primer sets A, B, C, C '(2-loop primer), C' (1-loop primer), 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 fragment of the vibrio cholerae O1 group 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 invention can be applied to detecting whether the sample contains the vibrio cholerae O1 group.

Example 7 detection of Vibrio cholerae O1 group specificity

28 strains of the Vibrio cholerae O1 group (1 to 28 in Table 4 and FIG. 1) were collected, and these strains were cultured separately from the Vibrio cholerae O1 group (29 in Table 4 and FIG. 1), and 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 carried out separately with reference to the reaction system and conditions of example 1.

The results are shown in Table 4 and FIG. 1, and in FIG. 1, 1 to 28 are respectively Staphylococcus aureus, staphylococcus aureus subspecies aureoflavum, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inokei, 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, enterotoxigenic Escherichia coli, escherichia coli enterohemorrhagic, escherichia coli Cronobacter sakazakii, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus and Vibrio, vibrio NTC: negative control, 29: vibrio cholerae group O1. In FIG. 1, the amplification reaction product of only the Vibrio cholerae O1 group strain appeared bright green and was a positive result, as shown in tube 29. The products of the other strains of the Vibrio cholerae O1 group and the negative control after the amplification reaction are orange, which is a negative result, as shown in tubes No. 1-28 and NTC negative control tube.

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 cholerae O1 group strain specificity, i.e., only the Vibrio cholerae O1 group strain is positive in amplification, and the other Vibrio cholerae O1 group strains are negative.

Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B, a primer group C and a primer group C', and the same detection results are respectively obtained according to the specific detection method, namely, the products after the amplification reaction of the non-vibrio cholerae O1 group strain and the negative control are negative results, and the products after the amplification reaction of the vibrio cholerae O1 group strain are positive results.

In addition, theoretical analysis was performed on the specificities of the primer groups a to B, the primer group C, and the primer group C' according to the method described in table 1, and as a result, it was found that, in the case where three mismatches were allowed at most for each primer, at most two primers were simultaneously compared to vibrio cholerae non-O1 group bacteria, indicating that the specificity of each primer group was better.

Example 8 sensitivity detection

DNA of bacterium CGMCC1.8676 was extracted by the method of example 1, and LAMP amplification (primer set A) and visualization by adding a color developing agent were carried out by using kit IB and by gradient addition of 10ng, 2ng, 400pg, 80pg, 16pg, 320fg and 64fg DNA according to the method of example 1 of Table 3 under other reaction conditions. As shown in fig. 2, 1-7 are 10ng, 2ng, 400pg, 80pg, 16pg, 320fg and 64fg, respectively, ntc: and (5) negative control. In FIG. 2, the reaction products of 10ng, 2ng, 400pg, 80pg, and 16pg treatments appeared bright green and as positive results, the reaction products of 320fg and 64fg treatments and the negative control appeared orange and as negative results. As a result of the examination, DNA was detected in a minimum amount of 16pg (equivalent to about 3600 bacteria) per reaction tube.

According to the detection method, the DNA as low as 16 pg-320 fg in each reaction tube can be detected by using the primer group B, the primer group C and the primer group C' respectively according to other steps and conditions.

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 B, the primer set C and the primer set C', and as a result, it was found that the primer region of each primer set completely matched with 8 chromosomes (GI numbers 15640032, 147671401, 227080237, 227116370, 229606122, 360034408, 379739908 and 384423362) of the Vibrio cholerae O1 group strain 1, and that each primer set could be theoretically used for the detection of the above-mentioned 8 Vibrio cholerae O1 group strains, indicating that the universality of each primer set was good.

TABLE 1 analysis of the universality and specificity of primers in the existing detection methods for the Vibrio cholerae O1 group

Note: a) Each Vibrio cholerae strain has two chromosomes, and the sequence between the primers F3 and B3 in the patent is subjected to Bowtie alignment with 16 chromosome genome sequences of 8 strains of the Vibrio cholerae O1 group to determine the position of a detection region in a GI No. 15640032# -1/15600771 # -2 genome, #1 represents the genome sequence of the first chromosome of the strain, and #2 represents the genome 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 simultaneously aligned to the non-cholera arc strain, the specificity is good.

TABLE 2 kit for isothermal detection of Vibrio cholerae O1 group and its main components

TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal testing of Vibrio cholerae O1 group according to 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 center for preservation and management of bacterial strains. b) +: positive result, -: and (5) negative result.

<110> Shanghai Industrial & technical research institute, shanghai Wangwang food group Co., ltd

Rapid isothermal detection method and kit for nucleic acid of <120> vibrio cholerae O1 group

<160> 14

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

ccacttattc gctttacaca 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

tttcgatggt gtaatcatcc 20

<210> 3

<211> 39

<212> DNA

<213> Artificial sequence

<400> 3

gcaaagtgaa acgcatggtt ggaatttgaa cctcgtctg 39

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence

<400> 4

gtggtatgga cgttcaatcg tcagaggttc aaaaatccct t 41

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence

<400> 5

atccgccaga tgtgcgat 18

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

gggaatccag atccctagtt tg 22

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence

<400> 7

gcggatcggt aaacagctgc tccgttgggg tcatgcagat g 41

<210> 8

<211> 42

<212> DNA

<213> Artificial sequence

<400> 8

tgcctgaatt tacaggctta cgcgatcgcc aacccaagtt gg 42

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

aatggaattt gaacctcgtc 20

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence

<400> 10

ccgttttgat ttcgatggt 19

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence

<400> 11

atcattgcca gagtggaact tagcgtgtgg ataacgaaa 39

<210> 12

<211> 41

<212> DNA

<213> Artificial sequence

<400> 12

gtggtatgga cgttcaatcg ttaaagagac cagaggttca a 41

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<400> 13

gcgcaaagtg aaacgcat 18

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<400> 14

agaatcggcg gattttaaag gg 22

Claims

1. A rapid constant temperature detection kit for a vibrio cholerae O1 group is characterized by comprising a primer group capable of amplifying a specific base sequence of a vibrio cholerae O1 group genome; wherein the primer group capable of amplifying the genome-specific base sequence of the vibrio cholerae O1 group is selected from a primer group A or a primer group B;

primer set a:

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

the downstream outer primer B3_ A:5'-TTTCGATGGTGTAATCATCC-3' (SEQ ID NO: 2);

primer set B:

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

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

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

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

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 and BIP primers, 0.15-0.3 μmol/L F3 and B3 primers, 0.16-0.64U/μ L Bst DNA polymerase, and 0-1.5mol/L betaine.

4. Use of the kit of claim 1~3 in the isothermal detection of vibrio cholerae group O1 for non-diagnostic purposes.