CN106434896B - Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application - Google Patents
Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application Download PDFInfo
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Abstract
The invention discloses a method, a primer group and a kit for rapidly detecting vibrio parahaemolyticus at constant temperature. 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 parahaemolyticus as a primer; and determining whether the vibrio parahaemolyticus exists in the sample to be detected 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
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 parahaemolyticus at a constant temperature.
Background
Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a gram-negative halophilic bacterium, widely distributed in rivers, oceans, tropical zones and coastal areas of temperate zones, and mainly parasitizes in aquatic products such as plankton, fish, shrimp, crab, shellfish and the like, and when directly or indirectly eating food polluted by the bacterium, the typical gastroenteritis reactions such as diarrhea, intestinal spasm, nausea, vomiting, fever and the like can be caused, and septicemia can be caused by severe cases. The incidence of vibrio parahaemolyticus is distributed worldwide, particularly, the incidence rate in coastal areas is high, and the food poisoning caused by vibrio parahaemolyticus in coastal cities in China accounts for a higher proportion than salmonella, escherichia coli O157: H7, staphylococcus aureus and the like in bacterial food poisoning events and is the top. Therefore, the method has important significance for the detection and prevention of the bacteria.
At present, the detection method for vibrio parahaemolyticus at home and abroad still takes a conventional culture method as a standard, the detection period is long (reaching 5-7 days), the operation is relatively complex, the detection efficiency is low, and the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society are difficult to meet. With the development of nucleic acid molecule detection technology in recent years, PCR technology established by taking specific genes as targets has been successfully applied to the detection of Vibrio parahaemolyticus, 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 parahaemolyticus in food is urgently needed.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min at an isothermal condition, and generates a visible reaction by-product, white magnesium pyrophosphate precipitate (see Notomi T, OkayamaH, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification reaction (2000, J8512; 63). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.
Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. That is, the user must first ensure that the target gene is a specific sequence of the species to be tested. The invention discloses a method for detecting vibrio parahaemolyticus by LAMP technology, which takes patents ZL201210062791.1 and CN 104513857A as examples, and respectively aims at the specific genes-tlh and irgB sequences of vibrio parahaemolyticus reported in the literature. However, the so-called "recognized specific genes" are often based on a delayed knowledge and are not necessarily updated based on the ever-increasing genome data of microorganisms, so that primers obtained based on the target gene sequences are not necessarily able to ensure their versatility and/or specificity in practical applications. The present invention is shown in Table 1, which shows the problem of the prior art that the primer versatility cannot be ensured. That is, the Vibrio parahaemolyticus detection sequence used in the prior art method is not actually common to Vibrio parahaemolyticus, i.e., there is a possibility that a partial strain of Vibrio parahaemolyticus may be missed. A similar problem exists in the confirmation of specificity, i.e., there is a possibility that non-vibrio parahaemolyticus is erroneously recognized as vibrio parahaemolyticus. Therefore, there is a need in the industry for a vibrio parahaemolyticus 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 in the production line of enterprises.
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 parahaemolyticus, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a vibrio parahaemolyticus LAMP primer based on microbial genome data resources (data up to 2013, 8, 5) in a GenBank database, and provides a method, a primer group and a kit for detecting vibrio parahaemolyticus by rapid isothermal amplification. The detection method for detecting the vibrio parahaemolyticus 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 a vibrio parahaemolyticus strain, 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 parahaemolyticus genome as a primer;
(3) and determining whether the vibrio parahaemolyticus exists in the sample to be detected by judging whether the reaction result is positive or not.
The method for detecting the vibrio parahaemolyticus strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and a vibrio parahaemolyticus specific amplification primer group as a primer to carry out constant temperature amplification reaction, and then determines whether the vibrio parahaemolyticus 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 Vibrio parahaemolyticus is the sequence of 22268-23012 bp bit of Vibrio parahaemolyticus with GI number 28899855.
In the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio parahaemolyticus genome is a part of the nucleic acid sequence at position 22268-23012 bp of the genome (GI No. 28899855) or a part of the complementary strand thereof. Wherein the Vibrio parahaemolyticus genome-specific base sequence refers to a base sequence that is unique to the Vibrio parahaemolyticus 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 parahaemolyticus genome includes, but is not limited to, any one selected from the following primer sets A to 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:
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3' (SEQ ID NO: 4);
primer set B:
upstream outer primer F3_ B: 5'-TGGAAAGAATATACAGTCGC-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GGTAATTGTGATCACGCTT-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3' (SEQ ID NO: 8).
In the present invention, the primer set capable of amplifying the specific base sequence of the Vibrio parahaemolyticus genome may further include a primer set having a homology of 50% or more with respect to 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, any one of the following primer sets C to D:
primer set C:
upstream outer primer F3_ C: 5'-TCTTGAGTAGCGGTTCAA-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-GGAAGTCTCTATGAATCTAACC-3' (SEQ ID NO: 10) (50% homology to primer B3_ A5 '-CGAGAAGTAAGGAAGTCTCT-3');
upstream inner primer FIP _ C: 5'-ATTGATCAGACCCACACCACGAACATAGTTTTTCAGCTCG-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-TAGGTGAACCACATCATACCTAGTGCGACTGTATATTCTTTCCA-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-TGGAAAGAATATACAGTCGC-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-CTGCTCTCGGTAATTGTG-3' (SEQ ID NO: 14) (homology 52.6% to primer B3_ B5'-GGTAATTGTGATCACGCTT-3');
upstream inner primer FIP _ D: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3' (SEQ ID NO: 16).
In the method of the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio parahaemolyticus genome may include, but is not limited to, a loop primer. Preferably, the loop primer is one, and comprises loop primers LF or LB. The primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome is selected from any one of the following primer groups A ', B ' and D '; or any one selected from the group consisting of primers having a homology of 50% or more with respect to a single sequence of the sequences of the primer groups A ', B ', D ' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively; the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
upstream loop primer LF _ a: 5'-ATTGATCAGACCCACACCAC-3' (SEQ ID NO: 17);
a primer set B':
upstream outer primer F3_ B: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ B: 5'-GGTAATTGTGATCACGCTT-3', respectively;
upstream inner primer FIP _ B: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ B: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3', respectively; downstream loop primer LB _ B: 5'-AGAAGTACTGGAAATGCACGAT-3' (SEQ ID NO: 18);
a primer set D':
upstream outer primer F3_ D: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ D: 5'-CTGCTCTCGGTAATTGTG-3', respectively;
upstream inner primer FIP _ D: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ D: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3', respectively; downstream loop primer LB _ D: 5'-AGAAGTACTGGAAATGCACGAT-3' (SEQ ID NO: 19).
In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.4-1.0 mu mol/L LF 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 polymeraseSynthase reaction buffer, 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to the improvement of the reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mMMgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis chart shows a characteristic step-shaped strip, the sample to be detected is positive to the vibrio parahaemolyticus and contains the vibrio parahaemolyticus; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative to vibrio parahaemolyticus. 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 parahaemolyticus and contains the vibrio parahaemolyticus; if no turbidity is found, the sample to be tested is negative to vibrio parahaemolyticus. 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 parahaemolyticus and contains the vibrio parahaemolyticus; if no precipitate is formed at the bottom of the reaction tube, the sample to be detected is negative to vibrio parahaemolyticus.
The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or SYBRGreen I (30-50X), or hydroxynaphthol blue (i.e. HNB, 120-. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to vibrio parahaemolyticus; if the color after the reaction is green, the sample to be tested is positive for vibrio parahaemolyticus and contains vibrio parahaemolyticus. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to vibrio parahaemolyticus; if the color after the reaction is sky blue, the sample to be detected is positive for vibrio parahaemolyticus. The chromogenic detection can be used for detecting the reaction result in real time or at the end point through a detection instrument besides observing the reaction result through 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 vibrio parahaemolyticus; 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 parahaemolyticus. 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, 50 μ M calcein is added into enzyme reaction system, and 0.6-1mM [ Mn ] is added2+]For example, 0.6-1mM MnCl2。
The invention also provides a primer used in the method for detecting the vibrio parahaemolyticus strain at constant temperature. The primer includes a primer group capable of amplifying a specific base sequence of the Vibrio parahaemolyticus genome, including, but not limited to, a portion of the nucleic acid sequence of 22268-23012 bp site of the Vibrio parahaemolyticus genome with GI number 28899855 or a portion of the complementary strand thereof.
Wherein the primer group capable of amplifying the base sequence specific to the Vibrio parahaemolyticus 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 B. 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 C to D.
Primer set a:
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively; the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ B: 5'-GGTAATTGTGATCACGCTT-3', respectively;
upstream inner primer FIP _ B: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ B: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3', respectively;
primer set C:
upstream outer primer F3_ C: 5'-TCTTGAGTAGCGGTTCAA-3', respectively;
downstream outer primer B3 — C: 5'-GGAAGTCTCTATGAATCTAACC-3', respectively;
upstream inner primer FIP _ C: 5'-ATTGATCAGACCCACACCACGAACATAGTTTTTCAGCTCG-3', respectively;
the downstream inner primer BIP _ C: 5'-TAGGTGAACCACATCATACCTAGTGCGACTGTATATTCTTTCCA-3', respectively;
primer set D:
upstream outer primer F3_ D: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ D: 5'-CTGCTCTCGGTAATTGTG-3', respectively;
upstream inner primer FIP _ D: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ D: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3' are provided.
In the primers used in the method for detecting vibrio parahaemolyticus at constant temperature, the primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome can also comprise but is not limited to a loop primer; preferably, the loop primer is one, including LF or LB. The primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome is selected from any one of the following primer groups A ', B ' and D '; or any one selected from the group consisting of primers having a homology of 50% or more with respect to a single sequence of the sequences of the primer groups A ', B ', D ' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively;
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
upstream loop primer LF _ a: 5'-ATTGATCAGACCCACACCAC-3', respectively;
a primer set B':
upstream outer primer F3_ B: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ B: 5'-GGTAATTGTGATCACGCTT-3', respectively;
upstream inner primer FIP _ B: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ B: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3', respectively; downstream loop primer LB _ B: 5'-AGAAGTACTGGAAATGCACGAT-3', respectively;
a primer set D':
upstream outer primer F3_ D: 5'-TGGAAAGAATATACAGTCGC-3', respectively;
downstream outer primer B3_ D: 5'-CTGCTCTCGGTAATTGTG-3', respectively;
upstream inner primer FIP _ D: 5'-GGCAAATCGATTGAATTTTGCCGGTTAGATTCATAGAGACTTCC-3', respectively;
the downstream inner primer BIP _ D: 5'-GCAGATTAACCGACTTAGTAGGATGTTAATACGCTCGCGATA-3', respectively; downstream loop primer LB _ D: 5'-AGAAGTACTGGAAATGCACGAT-3' are provided.
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 parahaemolyticus strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome. In the kit of the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio parahaemolyticus genome includes, but is not limited to, a portion of the nucleic acid sequence at position 22268-23012 bp of the genome (GI No. 28899855) 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 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 C, primer set D, etc.
In the kit of the present invention, the primer set capable of amplifying the base sequence specific to the Vibrio parahaemolyticus genome may include, but is not limited to, a loop primer; the loop primer serves as an optional component. Preferably, the loop primer is one, including LF or LB. The primer set comprising the loop primer LF or LB includes, but is not limited to, primer sets A ', B ', D ', etc. In a specific embodiment, the kit of the invention may comprise 0.4-1.0. mu. mol/L of LF or LB loop primer. In a specific embodiment, the sequences of the primer sets are FIP, BIP, F3, B3 and LF, or FIP, BIP, F3, B3 and LB, or primers with single primer homology of 50% or more with the aforementioned sequences or their complementary strand sequences, respectively.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg2+(MgSO4Or MgCl2) And betaine. In one embodiment, the inventionThe enzyme reaction system of the enzyme kit comprises 1 xBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/LdNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L BstDNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
The kit of the invention also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, the whole genomic DNA, a part of the genomic DNA of vibrio parahaemolyticus, or a vector comprising the whole genomic DNA or a part of the genomic DNA of vibrio parahaemolyticus.
The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.
The kit further comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn2+]For example, MnCl2。
The kit of the invention also comprises double distilled water.
The kit of the invention also comprises a nucleic acid extraction reagent.
The invention also provides a carrier, which comprises any one primer selected from the primer groups A-D, A ', B ' and D '. The vector contains a DNA sequence with vibrio parahaemolyticus specificity, so that the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application fields of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one of the primers of primer set A, vector pBR322-B containing any one of the primers of primer set B, and vector pBR322-D 'containing any one of the primers of primer set D' … …. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, … … a vector lambda phage-D 'containing any one of the primers of the primer set D', and the like.
The invention also provides application of any one primer selected from the primer groups A-D, A ', B ' and D ' in constant temperature detection of vibrio parahaemolyticus.
The invention also provides application of the kit in constant temperature detection of vibrio parahaemolyticus.
The invention also provides application of the vector in constant temperature detection of vibrio parahaemolyticus.
The invention provides a simple, rapid and sensitive method for detecting vibrio parahaemolyticus, 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 parahaemolyticus 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 detection method of Vibrio parahaemolyticus of example 7 of the present invention.
FIG. 2 shows the sensitivity of the method for detecting Vibrio parahaemolyticus 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 parahaemolyticus 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 parahaemolyticus strain for detection is from China general microbiological culture collection center with the number of CGMCC1.1997 (ATCC 17802). 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co260/OD280At a concentration of 1.8, 216 ng/. mu.L.
(2) Taking the genomic DNA of vibrio parahaemolyticus to be detected as a template, respectively adopting self-prepared kits (shown in table 2 and table 3), preparing a reaction system according to the conditions in table 3, and taking a specific amplification primer group of vibrio parahaemolyticus as a primer to carry out isothermal amplification reaction. The primers in examples 1 to 6 were primer sets A, A ', B, C, D', 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 the vibrio parahaemolyticus 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 parahaemolyticus.
According to the method of the above embodiment, the specific fragment of Vibrio parahaemolyticus can be amplified and the detection result can be obtained by using the primer set D and the primer set B', respectively.
Example 7 Vibrio parahaemolyticus-specific assay
28 strains of non-Vibrio parahaemolyticus (1 to 26, 28 to 29 in Table 4 and FIG. 1) were collected, these strains and the Vibrio parahaemolyticus strain (27 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 carried out 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, 1-26 are Staphylococcus aureus, Staphylococcus aureus Chryseozoea, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria Ennok, Listeria Israeli, Salmonella enterica, 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 enterohemorrhagic, Cronobacter sakazakii, Yersinia enterocolitica, Escherichia pseudotuberculosis, Vibrio vulnificus, Vibrio paravibrio haemolyticus, 28-29 are Vibrio freundii and Vibrio cholerae, NTC: negative control, 27: vibrio parahaemolyticus. In FIG. 1, the product obtained after the amplification reaction of only the Vibrio parahaemolyticus strain appeared bright green and was a positive result, as shown in tube No. 27. The products of the other non-Vibrio parahaemolyticus strains and the negative control amplification reaction are orange, which is a negative result, as shown in tubes No. 1-26, No. 28-29 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 specificity for Vibrio parahaemolyticus strains, i.e., only Vibrio parahaemolyticus strains are amplified positively, and other non-Vibrio parahaemolyticus strains are negative.
Preparing a detection kit, wherein the primers adopted in the kit are respectively primer groups B-D, and the primer groups A ', B ' and D ' respectively obtain the same detection results according to the specific detection method, namely, the products after the amplification reaction of the non-vibrio parahaemolyticus strains and the negative control are negative results, and the products after the amplification reaction of the vibrio parahaemolyticus strains are positive results.
In addition, theoretical analysis was performed on the specificity of the primer sets A to D and the primer sets A ', B ', D ' according to the method described in Table 1, and as a result, it was found that, when at most three mismatches were allowed for each primer, at most two primers were simultaneously aligned to Vibrio parahaemolyticus in each primer set, indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNA of the bacterium CGMCC1.1997 was extracted by the method of example 1, and the DNA was added to the reaction system using kit IB in a gradient of 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg DNA, and LAMP amplification (primer set A) and visualization by adding color developing agent were carried out respectively under the other reaction conditions according to the method of example 1 of Table 3. As shown in fig. 2, 1 to 7 are 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 50ng, 5ng, 500pg, 50pg and 5pg treatments appeared bright green and as positive results, the reaction products of 500fg, 50fg treatments and negative control appeared orange and as negative results. The results of the tests showed that a minimum of 5pg (corresponding to about 900 bacteria) of DNA was still detected in each reaction tube.
According to the detection method, the DNA of 5 pg-500 fg in each reaction tube can be detected by using the primer group B, the primer groups C-D and the primer groups A ', B ', D ' respectively according to the other steps and conditions.
Example 9 commonality testing
Theoretical analysis of the versatility of the primer sets A to B, C to D, and A ', B ', D ' was carried out according to the method described in Table 1, and it was found that the primer regions of the primer sets completely match the genomic sequences of the second chromosomes (GI Nos. 28899855, 433659170, 525847173, and 525852846, respectively) of four Vibrio parahaemolyticus strains, and that they could be theoretically used for the detection of the four Vibrio parahaemolyticus strains, indicating that the versatility of each primer set was good.
TABLE 1 analysis of the universality and specificity of primers in the existing detection method of Vibrio parahaemolyticus
Note: a) each Vibrio parahaemolyticus strain has two chromosomes, and the position of the detection region in the genome of GI No. 28896774#1/28899855#2 is determined by performing Bowtie alignment of the sequence between primers F3 and B3 in the patent with 8 genomic sequences of four strains of Vibrio parahaemolyticus, #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) The sequences of the detection regions are subjected to Blast comparison in public database resources, and primers cannot be simultaneously compared with the sequences of non-vibrio parahaemolyticus, thereby indicating that the specificity of the primers is good.
TABLE 2 types and major Components of kits for isothermal detection of Vibrio parahaemolyticus
TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal detection of Vibrio parahaemolyticus 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.
Claims (5)
1. A method for rapidly detecting vibrio parahaemolyticus at constant temperature for non-diagnostic purposes 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 parahaemolyticus genome as a primer;
(3) determining whether the vibrio parahaemolyticus exists in the sample to be detected by judging whether the reaction result is positive or not;
wherein the Vibrio parahaemolyticus genome-specific base sequence is a sequence of 22268-23012 bp of the Vibrio parahaemolyticus genome with GI number 28899855;
wherein the primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3' (SEQ ID NO: 4);
primer set a':
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively;
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
upstream loop primer LF _ a: 5'-ATTGATCAGACCCACACCAC-3' (SEQ ID NO: 17).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0. mu. mol/L FIP _ A and BIP _ A primers, 0.15-0.3. mu. mol/L F3_ A and B3_ A primers, 0.16-0.64U/. mu.L Bst DNA polymerase, 0-1.5mol/L betaine, with or without 0.4-1.0. mu. mol/L LF _ A primer.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by incubating at ① 60-65 ℃ for 10-90 min and terminating at ② 80 ℃ for 2-20 min.
4. The primer used in the method according to claim 1, which is a primer set capable of amplifying a base sequence specific to the Vibrio parahaemolyticus genome having a sequence which is a part of a nucleic acid sequence at positions 22268 to 23012bp of the Vibrio parahaemolyticus genome having GI number 28899855 or a part of a complementary strand thereof;
wherein the primer group capable of amplifying the specific base sequence of the vibrio parahaemolyticus genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively;
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
primer set a':
upstream outer primer F3_ a: 5'-TGAGTAGCGGTTCAATCG-3', respectively;
downstream outer primer B3_ a: 5'-CGAGAAGTAAGGAAGTCTCT-3', respectively;
upstream inner primer FIP _ A: 5'-CAAACTAACGCTTATAACCAACAGCATAGTTTTTCAGCTCGGC-3', respectively;
the downstream inner primer BIP _ A: 5'-ACATCATACCTAGTGCAATGGTGACCATAAGAAAGCGACTGTA-3', respectively;
upstream loop primer LF _ a: 5'-ATTGATCAGACCCACACCAC-3' (SEQ ID NO: 17).
5. Use of a primer for isothermal detection of Vibrio parahaemolyticus for non-diagnostic purposes, wherein the primer is according to claim 4.
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| CN202010042440.9A CN111020011A (en) | 2015-09-02 | 2016-08-30 | Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application |
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