CN111004856B - Rapid constant-temperature detection method, primer group and kit for vibrio vulnificus - Google Patents
Rapid constant-temperature detection method, primer group and kit for vibrio vulnificus Download PDFInfo
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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
The application is filed on 2016, 8, 30, and has the application number of 201610780407.X and the name of the invention as follows: the invention relates to a method, a primer and a kit for rapidly detecting vibrio vulnificus at constant temperature, belonging to divisional application of Chinese patent application.
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, traumatic infection and acute gastroenteritis, with septic shock 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 patents CN 103160604A and ZL201310556940.4 are taken as examples, and the invention patents adopt LAMP technology to detect Vibrio vulnificus by aiming at the sequences of vvhA gene and TolC gene which are specific genes of the Vibrio vulnificus reported in the literature respectively. 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 Vibrio vulnificus is a sequence of 2509-3725 bp bits of Vibrio vulnificus with GI number 320157827.
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 of 2509 to 3725bp of the genome (GI No. 320157827) 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 F, 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:
upstream outer primer F3_ a: 5'-GCAGAGAAGATGATCGTTTT-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-CTTACCATTTGGCGATCTG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3' (SEQ ID NO: 4);
primer set B:
upstream outer primer F3_ B: 5'-CGACTAGTGCCAAATCAC-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-CGTGCGATCATAATACCG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-TCTGATTGAAGGGCATGTCAAATGATCATGCTTTCGAGGT-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3' (SEQ ID NO: 8);
primer set C:
the upstream outer primer F3_ C: 5'-GTTGAGAAACTCGAACGC-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-GGTACCAAGATCGAAAGC-3' (SEQ ID NO: 10);
upstream inner primer FIP _ C: 5'-AGCGTGTTGGCTAGATGGATTCAGGATTGAGATAGCGG-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-CTTGCTTTGATCGTCGCTGAGGTGATACGCTGAAAACG-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-TGGTACCAAGTCGAAATGT-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-CCTGATCCAGACTTACCATT-3' (SEQ ID NO: 14);
upstream inner primer FIP _ D: 5'-GGATGAATGTGAGCAAGTTGGGAAAGATCCATTACGCCG-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D: 5'-ACCACAATGCTGCTTTGCTGTGAAAAGTGAACCAGACT-3' (SEQ ID NO: 16);
primer set E:
upstream outer primer F3_ E: 5'-GGCGGAGATCAGCAACTTG-3' (SEQ ID NO: 17);
downstream outer primer B3_ E: 5'-GAACCCACATCCATGTTAGACG-3' (SEQ ID NO: 18);
upstream inner primer FIP _ E: 5'-ACTACATCGCAGAAGAGACCGCGGATTGCTCAAAATGGCGC-3' (SEQ ID NO: 19);
the downstream inner primer BIP _ E: 5'-GGTAGCGAGATCGTGCGTGATGGGGGTATTGAACCTTATGCAG-3' (SEQ ID NO: 20);
a primer set F:
upstream outer primer F3 — F: 5'-ATGATTTGCGAACCCACCG-3' (SEQ ID NO: 21);
downstream outer primer B3 — F: 5'-TCCAACACACACCATTGAGC-3' (SEQ ID NO: 22);
upstream inner primer FIP _ F: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3' (SEQ ID NO: 23);
the downstream inner primer BIP _ F: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3' (SEQ ID NO: 24).
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 homology of 50% or more with each of the aforementioned sequences of the primer sets or their complementary strand sequences, and the primer set includes, but is not limited to, any one of the following primer sets G to L:
primer set G:
upstream outer primer F3_ G: 5'-TGATCGTTTTCAGCGTATC-3' (SEQ ID NO: 25) (homology 50% to primer F3_ A5'-GCAGAGAAGATGATCGTTTT-3');
downstream outer primer B3_ G: 5'-GACTATTCATTAGATGCCGAC-3' (SEQ ID NO: 26);
upstream inner primer FIP _ G: 5'-CGTAATGGATCTTTCTATCTGGCTAACCTTGATCATGCTTTCG-3' (SEQ ID NO: 27);
the downstream inner primer BIP _ G: 5'-CCGCCTTAGTCCATTGTTTTACCATTGTGGTGGATGAATGT-3' (SEQ ID NO: 28);
a primer set H:
upstream outer primer F3 — H: 5'-CCAAATCACTTGATCATGCT-3' (SEQ ID NO: 29) (50% homology to primer F3_ B5 '-CGACTAGTGCCAAATCAC-3');
downstream outer primer B3 — H: 5'-CGTGCGATCATAATACCG-3' (SEQ ID NO: 30);
upstream inner primer FIP _ H: 5'-GATCAACAAAACTGGCTCACTAGCTAACGCTTCCATCAATTTG-3' (SEQ ID NO: 31);
the downstream inner primer BIP _ H: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3' (SEQ ID NO: 32);
primer set I:
upstream outer primer F3_ I: 5'-CCAAATCACTTGATCATGCT-3' (SEQ ID NO: 33);
downstream outer primer B3 — I: 5'-TTTCTCAACTACACGTCAAG-3' (SEQ ID NO: 34) (50% homology to the complementary strand 5'-GCGTTCGAGTTTCTCAAC-3' of primer F3_ C);
upstream inner primer FIP _ I: 5'-GATCAACAAAACTGGCTCACTAGCTAACGCTTCCATCAATTTG-3' (SEQ ID NO: 35);
the downstream inner primer BIP _ I: 5'-CTCTAAGCTCAGTACCGCCATAGTGCGATCATAATACCGATA-3' (SEQ ID NO: 36);
primer set J:
upstream outer primer F3_ J: 5'-CGCAGAGAAGATGATCGT-3' (SEQ ID NO: 37);
downstream outer primer B3_ J: 5'-ACTTACCATTTGGCGATCT-3' (SEQ ID NO: 38) (50% homology to primer B3_ D5'-CCTGATCCAGACTTACCATT-3');
upstream inner primer FIP _ J: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3' (SEQ ID NO: 39);
the downstream inner primer BIP _ J: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3' (SEQ ID NO: 40);
primer set K:
upstream outer primer F3_ K: 5'-CTTGATCATGCTTTCGATCT-3' (SEQ ID NO: 41);
downstream outer primer B3_ K: 5'-CCTTACACCAAGTTGCTG-3' (SEQ ID NO: 42) (52.6% homology to the complementary strand 5'-CAAGTTGCTGATCTCCGCC-3' of primer F3_ E);
upstream inner primer FIP _ K: 5'-TGAGCAAGTTGGGCCAAATGTTTAGCCAGATAGAAAGATCC-3' (SEQ ID NO: 43);
the downstream inner primer BIP _ K: 5'-GTCGGCATCTAATGAATAGTCTGGCCTGATCCAGACTTACCATT-3' (SEQ ID NO: 44);
a primer set L:
upstream outer primer F3_ L: 5'-ATGATTTGCGAACCCACCG-3' (SEQ ID NO: 45);
downstream outer primer B3_ L: 5'-GCTCCCTCAATCCAACACAC-3' (SEQ ID NO: 46) (50% homology to primer B3_ F5'-TCCAACACACACCATTGAGC-3');
upstream inner primer FIP _ L: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3' (SEQ ID NO: 47);
the downstream inner primer BIP _ L: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3' (SEQ ID NO: 48).
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 A ', C', E ', F', G ', J', K 'and L'; or any one selected from the group consisting of primer groups having a homology of 50% or more with respect to a single sequence in the sequences of the primer groups A ', C', E ', F', G ', J', K ', L' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GCAGAGAAGATGATCGTTTT-3';
downstream outer primer B3_ a: 5'-CTTACCATTTGGCGATCTG-3', respectively;
upstream inner primer FIP _ A: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3', respectively;
the downstream inner primer BIP _ A: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3', respectively;
upstream loop primer LF _ a: 5'-TTCGACTTGGTACCAAGATCG-3' (SEQ ID NO: 49);
and/or, the downstream loop primer LB _ A: 5'-ACATTCATCCACCACAATGCT-3' (SEQ ID NO: 50);
a primer set C':
upstream outer primer F3_ C: 5'-GTTGAGAAACTCGAACGC-3', respectively;
downstream outer primer B3 — C: 5'-GGTACCAAGATCGAAAGC-3', respectively;
upstream inner primer FIP _ C: 5'-AGCGTGTTGGCTAGATGGATTCAGGATTGAGATAGCGG-3';
the downstream inner primer BIP _ C: 5'-CTTGCTTTGATCGTCGCTGAGGTGATACGCTGAAAACG-3', respectively;
downstream loop primer LB _ C: 5'-CTCAGGCCCAAACACCGAGT-3' (SEQ ID NO: 51);
a primer set E':
upstream outer primer F3_ E: 5'-GGCGGAGATCAGCAACTTG-3', respectively;
downstream outer primer B3_ E: 5'-GAACCCACATCCATGTTAGACG-3', respectively;
upstream inner primer FIP _ E: 5'-ACTACATCGCAGAAGAGACCGCGGATTGCTCAAAATGGCGC-3', respectively; the downstream inner primer BIP _ E: 5'-GGTAGCGAGATCGTGCGTGATGGGGGTATTGAACCTTATGCAG-3', respectively;
upstream loop primer LF _ E: 5'-AGATTGTCGAATGGGGTGACACC-3' (SEQ ID NO: 52);
a primer set F':
upstream outer primer F3 — F: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3 — F: 5'-TCCAACACACACCATTGAGC-3', respectively;
upstream inner primer FIP _ F: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3', respectively;
the downstream inner primer BIP _ F: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3', respectively; downstream loop primer LB _ F: 5'-CCGGTACTTCTGCGTCTGAGGA-3' (SEQ ID NO: 53);
a primer group G':
the upstream outer primer F3_ G: 5'-TGATCGTTTTCAGCGTATC-3', respectively;
downstream outer primer B3_ G: 5'-GACTATTCATTAGATGCCGAC-3', respectively;
upstream inner primer FIP _ G: 5'-CGTAATGGATCTTTCTATCTGGCTAACCTTGATCATGCTTTCG-3';
the downstream inner primer BIP _ G: 5'-CCGCCTTAGTCCATTGTTTTACCATTGTGGTGGATGAATGT-3', respectively;
upstream loop primer LF _ G: 5'-ACAACATTTCGACTTGGTACCA-3' (SEQ ID NO: 54);
and/or, the downstream loop primer LB _ G: 5'-AAATGATTTGGCCCAACTTGC-3' (SEQ ID NO: 55);
primer set J':
upstream outer primer F3_ J: 5'-CGCAGAGAAGATGATCGT-3', respectively;
downstream outer primer B3_ J: 5'-ACTTACCATTTGGCGATCT-3', respectively;
upstream inner primer FIP _ J: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3', respectively;
the downstream inner primer BIP _ J: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3', respectively;
downstream loop primer LB _ J: 5'-ACATTCATCCACCACAATGCT-3' (SEQ ID NO: 56);
a primer set K':
upstream outer primer F3_ K: 5'-CTTGATCATGCTTTCGATCT-3', respectively;
downstream outer primer B3_ K: 5'-CCTTACACCAAGTTGCTG-3';
upstream inner primer FIP _ K: 5 '-TGAGCAAGTTGGGCCAAAT-GTTTAGCCAGATAGAAAGATCC-3';
the downstream inner primer BIP _ K: 5'-GTCGGCATCTAATGAATAGTCTGGCCTGATCCAGACTTACCATT-3'
Upstream loop primer LF _ K: 5'-AAACAATGGACTAAGGCGGC-3' (SEQ ID NO: 57);
and/or, the downstream loop primer LB _ K: 5'-TCACTTTTCACCAGATCGCC-3' (SEQ ID NO: 58);
a primer set L':
upstream outer primer F3_ L: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3_ L: 5'-GCTCCCTCAATCCAACACAC-3', respectively;
upstream inner primer FIP _ L: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3', respectively;
the downstream inner primer BIP _ L: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3' downstream loop primer LB _ L: 5'-CCGGTACTTCTGCGTCTGAGGA-3' (SEQ ID NO: 59).
In specific embodiments, for example, the primer sets a ', G ' and K ' may comprise 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 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, 0.4-1.0 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 (pH8.8), 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 terminating 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 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 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 hydroxynaphthol blue is used as a color developing agent, if the color after the 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 detect the reaction result in real time or at an end point through a detection instrument besides observing the reaction result through naked eyes, and the sample to be detected is vibrio vulnificus negative by reasonably setting a 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; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is vibrio vulnificus positive. 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 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, adding 50 μ M calcein into enzyme reaction system, and adding 0.6-1mM [ Mn ] 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 2509 to 3725bp of the Vibrio vulnificus genome with GI number 320157827 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 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 F. The primer set having a homology of 50% or more with respect to a single sequence in the aforementioned primer set sequence or the complementary strand sequence thereof includes, but is not limited to, any one of the following primer sets G to L.
Primer set a:
upstream outer primer F3_ a: 5'-GCAGAGAAGATGATCGTTTT-3', respectively;
downstream outer primer B3_ a: 5'-CTTACCATTTGGCGATCTG-3', respectively;
upstream inner primer FIP _ A:
5’-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3’；
the downstream inner primer BIP _ A: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3', respectively;
primer set B:
the upstream outer primer F3_ B: 5'-CGACTAGTGCCAAATCAC-3', respectively;
downstream outer primer B3_ B: 5'-CGTGCGATCATAATACCG-3', respectively;
upstream inner primer FIP _ B: 5'-TCTGATTGAAGGGCATGTCAAATGATCATGCTTTCGAGGT-3', respectively;
the downstream inner primer BIP _ B: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3', respectively;
primer set C:
upstream outer primer F3_ C: 5'-GTTGAGAAACTCGAACGC-3', respectively;
downstream outer primer B3 — C: 5'-GGTACCAAGATCGAAAGC-3', respectively;
upstream inner primer FIP _ C: 5'-AGCGTGTTGGCTAGATGGATTCAGGATTGAGATAGCGG-3', respectively;
the downstream inner primer BIP _ C: 5'-CTTGCTTTGATCGTCGCTGAGGTGATACGCTGAAAACG-3', respectively;
primer set D:
upstream outer primer F3_ D: 5'-TGGTACCAAGTCGAAATGT-3', respectively;
downstream outer primer B3_ D: 5'-CCTGATCCAGACTTACCATT-3', respectively;
upstream inner primer FIP _ D: 5'-GGATGAATGTGAGCAAGTTGGGAAAGATCCATTACGCCG-3', respectively;
the downstream inner primer BIP _ D: 5'-ACCACAATGCTGCTTTGCTGTGAAAAGTGAACCAGACT-3', respectively;
primer set E:
upstream outer primer F3_ E: 5'-GGCGGAGATCAGCAACTTG-3';
downstream outer primer B3_ E: 5'-GAACCCACATCCATGTTAGACG-3', respectively;
an upstream inner primer FIP _ E: 5'-ACTACATCGCAGAAGAGACCGCGGATTGCTCAAAATGGCGC-3', respectively;
the downstream inner primer BIP _ E: 5'-GGTAGCGAGATCGTGCGTGATGGGGGTATTGAACCTTATGCAG-3', respectively;
a primer set F:
upstream outer primer F3 — F: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3 — F: 5'-TCCAACACACACCATTGAGC-3', respectively;
upstream inner primer FIP _ F: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3';
the downstream inner primer BIP _ F: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3', respectively;
primer set G:
upstream outer primer F3_ G: 5'-TGATCGTTTTCAGCGTATC-3', respectively;
downstream outer primer B3_ G: 5'-GACTATTCATTAGATGCCGAC-3', respectively;
upstream inner primer FIP _ G: 5'-CGTAATGGATCTTTCTATCTGGCTAACCTTGATCATGCTTTCG-3', respectively;
the downstream inner primer BIP _ G: 5'-CCGCCTTAGTCCATTGTTTTACCATTGTGGTGGATGAATGT-3', respectively;
a primer set H:
upstream outer primer F3 — H: 5'-CCAAATCACTTGATCATGCT-3', respectively;
downstream outer primer B3 — H: 5'-CGTGCGATCATAATACCG-3', respectively;
upstream inner primer FIP _ H: 5'-GATCAACAAAACTGGCTCACTAGCTAACGCTTCCATCAATTTG-3', respectively;
the downstream inner primer BIP _ H: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3', respectively;
primer set I:
upstream outer primer F3_ I: 5'-CCAAATCACTTGATCATGCT-3', respectively;
downstream outer primer B3 — I: 5'-TTTCTCAACTACACGTCAAG-3', respectively;
upstream inner primer FIP _ I: 5'-GATCAACAAAACTGGCTCACTAGCTAACGCTTCCATCAATTTG-3', respectively;
the downstream inner primer BIP _ I: 5'-CTCTAAGCTCAGTACCGCCATAGTGCGATCATAATACCGATA-3', respectively;
primer set J:
upstream outer primer F3_ J: 5'-CGCAGAGAAGATGATCGT-3', respectively;
downstream outer primer B3_ J: 5'-ACTTACCATTTGGCGATCT-3';
upstream inner primer FIP _ J: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3', respectively;
the downstream inner primer BIP _ J: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3', respectively;
primer set K:
upstream outer primer F3_ K: 5'-CTTGATCATGCTTTCGATCT-3', respectively;
downstream outer primer B3 — K: 5'-CCTTACACCAAGTTGCTG-3', respectively;
upstream inner primer FIP _ K: 5'-TGAGCAAGTTGGGCCAAATGTTTAGCCAGATAGAAAGATCC-3', respectively;
a downstream inner primer BIP _ K: 5'-GTCGGCATCTAATGAATAGTCTGGCCTGATCCAGACTTACCATT-3';
a primer set L:
upstream outer primer F3_ L: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3_ L: 5'-GCTCCCTCAATCCAACACAC-3';
upstream inner primer FIP _ L: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3', respectively;
the downstream inner primer BIP _ L: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-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 A ', C', E ', F', G ', J', K 'and L'; or any one selected from the group consisting of primer groups having a homology of 50% or more with respect to a single sequence in the sequences of the primer groups A ', C', E ', F', G ', J', K ', L' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GCAGAGAAGATGATCGTTTT-3', respectively;
downstream outer primer B3_ a: 5'-CTTACCATTTGGCGATCTG-3', respectively;
upstream inner primer FIP _ A:
5’-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3’；
the downstream inner primer BIP _ A: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3';
upstream loop primer LF _ a: 5'-TTCGACTTGGTACCAAGATCG-3', respectively;
and/or, the downstream loop primer LB _ A: 5'-ACATTCATCCACCACAATGCT-3', respectively;
a primer set C':
upstream outer primer F3_ C: 5'-GTTGAGAAACTCGAACGC-3', respectively;
downstream outer primer B3 — C: 5'-GGTACCAAGATCGAAAGC-3', respectively;
upstream inner primer FIP _ C: 5'-AGCGTGTTGGCTAGATGGATTCAGGATTGAGATAGCGG-3', respectively;
the downstream inner primer BIP _ C: 5'-CTTGCTTTGATCGTCGCTGAGGTGATACGCTGAAAACG-3', respectively;
downstream loop primer LB _ C: 5'-CTCAGGCCCAAACACCGAGT-3', respectively;
a primer set E':
upstream outer primer F3_ E: 5'-GGCGGAGATCAGCAACTTG-3', respectively;
downstream outer primer B3_ E: 5'-GAACCCACATCCATGTTAGACG-3', respectively;
upstream inner primer FIP _ E: 5'-ACTACATCGCAGAAGAGACCGCGGATTGCTCAAAATGGCGC-3', respectively;
the downstream inner primer BIP _ E: 5'-GGTAGCGAGATCGTGCGTGATGGGGGTATTGAACCTTATGCAG-3', respectively;
upstream loop primer LF _ E: 5'-AGATTGTCGAATGGGGTGACACC-3', respectively;
a primer set F':
upstream outer primer F3 — F: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3 — F: 5'-TCCAACACACACCATTGAGC-3', respectively;
upstream inner primer FIP _ F: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3', respectively;
the downstream inner primer BIP _ F: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3', respectively;
downstream loop primer LB _ F: 5'-CCGGTACTTCTGCGTCTGAGGA-3';
a primer group G':
upstream outer primer F3_ G: 5'-TGATCGTTTTCAGCGTATC-3', respectively;
downstream outer primer B3_ G: 5'-GACTATTCATTAGATGCCGAC-3', respectively;
upstream inner primer FIP _ G: 5'-CGTAATGGATCTTTCTATCTGGCTAACCTTGATCATGCTTTCG-3', respectively;
a downstream inner primer BIP _ G: 5'-CCGCCTTAGTCCATTGTTTTACCATTGTGGTGGATGAATGT-3', respectively;
upstream loop primer LF _ G: 5'-ACAACATTTCGACTTGGTACCA-3', respectively;
and/or, the downstream loop primer LB _ G: 5'-AAATGATTTGGCCCAACTTGC-3';
primer set J':
upstream outer primer F3_ J: 5'-CGCAGAGAAGATGATCGT-3';
downstream outer primer B3_ J: 5'-ACTTACCATTTGGCGATCT-3', respectively;
upstream inner primer FIP _ J: 5'-GGATCTTTCTATCTGGCTAAACAACGTATCACCTTGATCATGCTT-3', respectively;
the downstream inner primer BIP _ J: 5'-AATGATTTGGCCCAACTTGCTACTATTCATTAGATGCCGAC-3', respectively;
downstream loop primer LB _ J: 5'-ACATTCATCCACCACAATGCT-3';
a primer set K':
upstream outer primer F3_ K: 5'-CTTGATCATGCTTTCGATCT-3', respectively;
downstream outer primer B3_ K: 5'-CCTTACACCAAGTTGCTG-3', respectively;
upstream inner primer FIP _ K: 5 '-TGAGCAAGTTGGGCCAAAT-GTTTAGCCAGATAGAAAGATCC-3';
the downstream inner primer BIP _ K: 5'-GTCGGCATCTAATGAATAGTCTGGCCTGATCCAGACTTACCATT-3'
Upstream loop primer LF _ K: 5'-AAACAATGGACTAAGGCGGC-3';
and/or, the downstream loop primer LB _ K: 5'-TCACTTTTCACCAGATCGCC-3', respectively;
a primer set L':
upstream outer primer F3_ L: 5'-ATGATTTGCGAACCCACCG-3', respectively;
downstream outer primer B3_ L: 5'-GCTCCCTCAATCCAACACAC-3', respectively;
upstream inner primer FIP _ L: 5'-GACGTTAACCAAGTTTCCCCACCACGCTCGGGCTAAATTGACA-3', respectively;
the downstream inner primer BIP _ L: 5'-GCGCCAATAACTCGGCCAATTTGCATTTGACTCGCCCTCTGAA-3'
Downstream loop primer LB _ L: 5'-CCGGTACTTCTGCGTCTGAGGA-3' are provided.
In a specific embodiment, the primer sets a ', G ' and K ' 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, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with homology of 50% or more with the above primer sequence or single primer in the 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 primer sequence including a part of a nucleic acid sequence of 2509 to 3725bp of the genome (GI No. 320157827) or a part of the complementary strand thereof; the primer includes, but is not limited to, any one of the primer group A, the primer group B, the primer group C, the primer group D, the primer group E, the primer group F, and the like. But not limited to, a primer set having a homology of 50% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set G, primer set H, primer set I, primer set J, primer set K, primer set L, 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, the primer sets A ', C', E ', F', G ', J', K ', L', 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 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 (pH8.8), 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 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 also 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 consisting of primer groups A-F, G-L, A ', C', E ', F', G ', J', K 'and L'. 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 primer of primer set A, vector pBR322-G containing any one primer of primer set G in … …, vector pBR322-L 'containing any one primer of primer set L' in … …, and the like. Vector lambda phage-A containing any one of the primers of primer set A, … … vector lambda phage-G containing any one of the primers of primer set G, … … vector lambda phage-L 'containing any one of the primers of primer set L', etc.
The invention also provides application of any one of the primers A-F, G-L, A ', C', E ', F', G ', J', K 'and L' 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 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 used in examples 1 to 6 were primer set A, C ', G' (2-loop primer), G '(1-loop primer), and L, L', 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 F, the primer groups G to K and the primer groups A ', E', F 'and J' can be used for well amplifying the specific segments of the vibrio vulnificus and obtaining the detection result.
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 products after the negative control amplification reaction are orange, and are negative results, such as tubes 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-F, primer groups G-L, primer groups A ', C', E ', F', G ', J', K ', L', and the same detection results are obtained according to the specific detection method, namely, the products after the amplification reaction of the 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 F, G to L, and a ', C', E ', F', G ', J', K ', L', respectively, according to the method described in table 1, and it was found that, when at most three mismatches were allowed for each primer, at most two primers were simultaneously aligned to vibrio vulnificus, indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNA of 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, 10fg, and other reaction conditions were LAMP amplification (primer set A) and visualization by adding color reagent according to the method of example 1 of Table 3, respectively. As shown in fig. 2, 1 to 6 are 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, NTC: and (5) negative control. In FIG. 2, the reaction products of 1ng, 100pg, 10pg, 1pg and 100fg treatments appeared bright green and as positive results, and the reaction products of 10fg treatments and the negative control appeared orange and as negative results. The detection results showed that DNA of 100fg (equivalent to about 20 bacteria) was detected in each reaction tube, and the sensitivity was high.
According to the detection method, other steps and conditions are the same, the primer groups B-F, the primer groups G-L and the primer groups A ', C', E ', F', G ', J', K ', L' are respectively used, DNA as low as 1 pg-100 fg in each reaction tube can still be detected, and the detection sensitivity is higher.
Example 9 commonality testing
Theoretical analysis of the universality of the primer sets A to F, G to L, A ', C', E ', F', G ', J', K ', L' was carried out according to the method described in Table 1, and it was found that the primer regions of the primer sets perfectly matched with the chromosomes No. 2 of three Vibrio vulnificus strains (GI Nos. 320157827, 326424156 and 37675660, respectively), and could be theoretically used for the detection of the above three Vibrio vulnificus strains, indicating that the universality of the primer sets was 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 bioinformatics technology research center
Rapid constant-temperature detection method, primer group and kit for vibrio vulnificus
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ggatctttct atctggctaa acaacgtatc accttgatca tgctt 45
<210> 4
<211> 41
<212> DNA
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<400> 4
aatgatttgg cccaacttgc tactattcat tagatgccga c 41
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tctgattgaa gggcatgtca aatgatcatg ctttcgaggt 40
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<212> DNA
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<400> 8
gctgattaca aaactctaag ctcagatatg gccattcgcg att 43
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<210> 10
<211> 18
<212> DNA
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<400> 10
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<212> DNA
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<400> 11
agcgtgttgg ctagatggat tcaggattga gatagcgg 38
<210> 12
<211> 38
<212> DNA
<213> Artificial sequence
<400> 12
cttgctttga tcgtcgctga ggtgatacgc tgaaaacg 38
<210> 13
<211> 19
<212> DNA
<213> Artificial sequence
<400> 13
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
<210> 15
<211> 39
<212> DNA
<213> Artificial sequence
<400> 15
ggatgaatgt gagcaagttg ggaaagatcc attacgccg 39
<210> 16
<211> 38
<212> DNA
<213> Artificial sequence
<400> 16
accacaatgc tgctttgctg tgaaaagtga accagact 38
<210> 17
<211> 19
<212> DNA
<213> Artificial sequence
<400> 17
ggcggagatc agcaacttg 19
<210> 18
<211> 22
<212> DNA
<213> Artificial sequence
<400> 18
<210> 19
<211> 41
<212> DNA
<213> Artificial sequence
<400> 19
actacatcgc agaagagacc gcggattgct caaaatggcg c 41
<210> 20
<211> 43
<212> DNA
<213> Artificial sequence
<400> 20
ggtagcgaga tcgtgcgtga tgggggtatt gaaccttatg cag 43
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence
<400> 21
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence
<400> 22
<210> 23
<211> 43
<212> DNA
<213> Artificial sequence
<400> 23
gacgttaacc aagtttcccc accacgctcg ggctaaattg aca 43
<210> 24
<211> 43
<212> DNA
<213> Artificial sequence
<400> 24
gcgccaataa ctcggccaat ttgcatttga ctcgccctct gaa 43
<210> 25
<211> 19
<212> DNA
<213> Artificial sequence
<400> 25
tgatcgtttt cagcgtatc 19
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence
<400> 26
gactattcat tagatgccga c 21
<210> 27
<211> 43
<212> DNA
<213> Artificial sequence
<400> 27
cgtaatggat ctttctatct ggctaacctt gatcatgctt tcg 43
<210> 28
<211> 41
<212> DNA
<213> Artificial sequence
<400> 28
ccgccttagt ccattgtttt accattgtgg tggatgaatg t 41
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence
<400> 29
<210> 30
<211> 18
<212> DNA
<213> Artificial sequence
<400> 30
<210> 31
<211> 43
<212> DNA
<213> Artificial sequence
<400> 31
gatcaacaaa actggctcac tagctaacgc ttccatcaat ttg 43
<210> 32
<211> 43
<212> DNA
<213> Artificial sequence
<400> 32
gctgattaca aaactctaag ctcagatatg gccattcgcg att 43
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence
<400> 33
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
<210> 35
<211> 43
<212> DNA
<213> Artificial sequence
<400> 35
gatcaacaaa actggctcac tagctaacgc ttccatcaat ttg 43
<210> 36
<211> 42
<212> DNA
<213> Artificial sequence
<400> 36
ctctaagctc agtaccgcca tagtgcgatc ataataccga ta 42
<210> 37
<211> 18
<212> DNA
<213> Artificial sequence
<400> 37
<210> 38
<211> 19
<212> DNA
<213> Artificial sequence
<400> 38
<210> 39
<211> 45
<212> DNA
<213> Artificial sequence
<400> 39
ggatctttct atctggctaa acaacgtatc accttgatca tgctt 45
<210> 40
<211> 41
<212> DNA
<213> Artificial sequence
<400> 40
aatgatttgg cccaacttgc tactattcat tagatgccga c 41
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence
<400> 41
<210> 42
<211> 18
<212> DNA
<213> Artificial sequence
<400> 42
<210> 43
<211> 41
<212> DNA
<213> Artificial sequence
<400> 43
tgagcaagtt gggccaaatg tttagccaga tagaaagatc c 41
<210> 44
<211> 44
<212> DNA
<213> Artificial sequence
<400> 44
gtcggcatct aatgaatagt ctggcctgat ccagacttac catt 44
<210> 45
<211> 19
<212> DNA
<213> Artificial sequence
<400> 45
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence
<400> 46
<210> 47
<211> 43
<212> DNA
<213> Artificial sequence
<400> 47
gacgttaacc aagtttcccc accacgctcg ggctaaattg aca 43
<210> 48
<211> 43
<212> DNA
<213> Artificial sequence
<400> 48
gcgccaataa ctcggccaat ttgcatttga ctcgccctct gaa 43
<210> 49
<211> 21
<212> DNA
<213> Artificial sequence
<400> 49
ttcgacttgg taccaagatc g 21
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence
<400> 50
acattcatcc accacaatgc t 21
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence
<400> 51
<210> 52
<211> 23
<212> DNA
<213> Artificial sequence
<400> 52
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence
<400> 53
ccggtacttc tgcgtctgag ga 22
<210> 54
<211> 22
<212> DNA
<213> Artificial sequence
<400> 54
acaacatttc gacttggtac ca 22
<210> 55
<211> 21
<212> DNA
<213> Artificial sequence
<400> 55
aaatgatttg gcccaacttg c 21
<210> 56
<211> 21
<212> DNA
<213> Artificial sequence
<400> 56
acattcatcc accacaatgc t 21
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence
<400> 57
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence
<400> 58
<210> 59
<211> 22
<212> DNA
<213> Artificial sequence
<400> 59
ccggtacttc tgcgtctgag ga 22
Claims (9)
1. A rapid constant temperature detection method aiming at vibrio vulnificus for non-diagnosis purpose 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 2509-3725 bp bit sequence of the Vibrio vulnificus genome with GI number 320157827;
wherein the primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome is selected from a primer group B;
primer set B:
upstream outer primer F3_ B: 5'-CGACTAGTGCCAAATCAC-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-CGTGCGATCATAATACCG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-TCTGATTGAAGGGCATGTCAAATGATCATGCTTTCGAGGT-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3' (SEQ ID NO: 8).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP _ B and BIP _ B primers, 0.15-0.3 mu mol/L F3_ B and B3_ B primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: incubating for 10-90 min at 60-65 ℃; ② terminating the reaction for 2-20 min at 80 ℃.
4. The primer for rapid isothermal detection of Vibrio vulnificus is characterized by comprising a primer group capable of amplifying a Vibrio vulnificus genome specific base sequence which is a part of a nucleic acid sequence of 2509-3725 bp of a Vibrio vulnificus genome with GI number 320157827 or a part of a complementary strand thereof;
wherein the primer group capable of amplifying the specific base sequence of the vibrio vulnificus genome is selected from a primer group B;
primer set B:
upstream outer primer F3_ B: 5'-CGACTAGTGCCAAATCAC-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-CGTGCGATCATAATACCG-3' (SEQ ID NO: 6);
an upstream inner primer FIP _ B: 5'-TCTGATTGAAGGGCATGTCAAATGATCATGCTTTCGAGGT-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3' (SEQ ID NO: 8).
5. 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 selected from a primer group B;
primer set B:
upstream outer primer F3_ B: 5'-CGACTAGTGCCAAATCAC-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-CGTGCGATCATAATACCG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-TCTGATTGAAGGGCATGTCAAATGATCATGCTTTCGAGGT-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCTGATTACAAAACTCTAAGCTCAGATATGGCCATTCGCGATT-3' (SEQ ID NO: 8).
6. The kit of claim 5, further comprising Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg 2+ And one or more of betaine.
7. The kit of claim 5, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer solution, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ B and BIP _ B primers, 0.15-0.3 μmol/L F3_ B and B3_ B primers, 0.16-0.64U/μ L Bst DNA polymerase, and 0-1.5mol/L betaine.
8. Use of a primer for isothermal detection of Vibrio vulnificus for non-diagnostic purposes, wherein the primer is according to claim 4.
9. Use of a kit according to any one of claims 5 to 7 for the isothermal detection of Vibrio vulnificus for non-diagnostic purposes.
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| CN101348835B (en) * | 2008-09-09 | 2011-08-17 | 南开大学 | Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology |
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