CN110951841A - Rapid constant-temperature detection method, primer group and kit for vibrio cholerae O1 group - Google Patents
Rapid constant-temperature detection method, primer group and kit for vibrio cholerae O1 group Download PDFInfo
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Abstract
The invention discloses a rapid constant temperature detection method, a primer group and a kit for vibrio cholerae O1. 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 cholerae O1 group as a primer; and determining whether the vibrio cholerae O1 group 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
The application is filed on 2016, 8, 30, and has the application number of 201610767491.1 and the name of the invention: the divisional application of the Chinese invention patent application of 'a method for rapidly detecting the vibrio cholerae O1 group at constant temperature, a primer and application'; the parent application claims the priority of the Chinese patent application with the application date of 2015, 9 and 2, the application number of 201510556917.4, named as 'method, primer and kit for rapid isothermal detection of cronobacter sakazakii'.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting vibrio cholerae O1 group at constant temperature.
Background
Vibrio cholerae (Vibrio cholerae) is widely distributed in marine and freshwater environments and is an important disease inspection and quarantine object and food-borne pathogenic bacteria for aquaculture. Vibrio cholerae can be classified into several serogroups depending on the bacterial (O) antigen, but only the O1 and O139 groups have been found to cause cholera. Among them, the O1 group Vibrio cholerae has caused seven pandemics of cholera worldwide since 1817. Therefore, early detection and diagnosis of the Vibrio cholerae O1 group is important for the treatment and prevention of the occurrence and spread of cholera.
The traditional vibrio cholerae detection method has the defects of long detection period, relatively complex operation and low detection efficiency, and is difficult to meet the requirements of high throughput, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means of PCR and fluorescence PCR technologies, but both methods require special detection instruments, and are not suitable for real-time field detection in basic detection departments, especially in enterprise production lines. In order to ensure the safety of food, a rapid, simple and accurate method for detecting the vibrio cholerae O1 group in food is urgently needed.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, 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 takes patents ZL201010584749.7 and ZL201310119610.9 as examples, and the patents respectively aim at specific genes of vibrio cholerae, namely ctxA gene and dnaA gene, and detect the vibrio cholerae O1 group by adopting LAMP technology. However, the so-called "recognized specific genes" are often based on a delayed knowledge and are not necessarily updated based on the ever-increasing genome data of microorganisms, so that primers obtained based on the target gene sequences are not necessarily able to ensure their versatility and/or specificity in practical applications. Table 1 of the present invention shows the problem that the versatility is not ensured in the prior art. That is, the detection sequence of the group of Vibrio cholerae O1 used in the prior art method is not actually common to the group of Vibrio cholerae O1, i.e., there is a possibility that a partial strain of the group of Vibrio cholerae O1 is missed. A similar problem exists in the confirmation of specificity, that is, there is a possibility that the group of non-Vibrio cholerae O1 is erroneously identified as the group of Vibrio cholerae O1. Therefore, the industry needs a vibrio cholerae O1 group detection method which can ensure specificity and universality, meets the requirements of basic detection departments on rapidness and convenience, and can conveniently carry out real-time on-site detection in an enterprise production line.
Disclosure of Invention
The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the prior LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying the vibrio cholerae O1 group, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a vibrio cholerae O1 group LAMP primer based on microbial genome data resources (data up to 2013, 8 and 5 days) in a GenBank database, and provides a method, a primer group and a kit for rapidly detecting a vibrio cholerae O1 group by constant-temperature amplification. The detection method for detecting the vibrio cholerae O1 group has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenience in operation and low cost.
The invention provides a method for rapidly detecting a vibrio cholerae O1 group strain, which comprises the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) 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 genome specific base sequence of the vibrio cholerae O1 group as a primer;
(3) and determining whether the vibrio cholerae O1 group exists in the sample to be detected by judging whether the reaction result is positive or not.
The method for detecting the vibrio cholerae O1 group strain at constant temperature comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by taking the genome DNA as a template and a vibrio cholerae O1 group specific amplification primer group as a primer, and then determining whether the vibrio cholerae O1 group exists in the sample to be detected by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the invention, the genome specific base sequence of the vibrio cholerae O1 group is 170082-171001 bp bit sequence of the vibrio cholerae O1 group with the GI number of 15640032.
In the invention, the primer group capable of amplifying the genome specific base sequence of the vibrio cholerae O1 group is a part of the nucleotide sequence of 170082-171001 bp of the genome (GI No. 15640032) or a part of the complementary strand of the nucleotide sequence. Wherein the genome-specific nucleotide sequence of Vibrio cholerae O1 is a nucleotide sequence unique to the genome of Vibrio cholerae O1 and not contained in the genome of other microorganisms.
Wherein the primer set capable of amplifying the genome specific base sequence of Vibrio cholerae O1 group includes, but is not limited to, any one selected from the following primer sets A-B, or any one selected from the primer sets having 59% or more homology 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'-AACAAACTGGCGTTGGCT-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TCGTTATCCACACGCTGC-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-AGACCGGGTCAGCGTCGTAATGGCTTGAGTTTTGGTGCG-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-CTGCAACTGTCGCACATGGTCTTCAAGATTCGGCCAGACGAG-3' (SEQ ID NO: 4);
primer set B:
upstream outer primer F3_ B: 5'-CCTCTGGTCTCTTTGACTAAGG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-AGTTTCACGCCTTGCTCAC-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-TCGGGAAAATGTAGGTCGCCACCGAAATCAAAACGGCTGGC-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-CGGCGGATGGTAAAAACAAAGCAGAAATGGCCCGGTACCAGAG-3' (SEQ ID NO: 8).
In the present invention, the primer set capable of amplifying the specific base sequence of the genome of vibrio cholerae group O1 may further include a primer set having a homology of 59% or more with a single sequence in the sequences of the primer sets or the complementary strand sequences thereof, and the primer set includes, but is not limited to, any one of the following primer sets C to D:
primer set C:
upstream outer primer F3_ C: 5'-CAGCATGAATACAGCAAGA-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-GCCAGTTTGTTTTTCATGG-3' (SEQ ID NO: 10) (61% homology to the complementary strand 5'-AGCCAACGCCAGTTTGTT-3' of primer F3_ A);
upstream inner primer FIP _ C: 5'-AGTTGGTCCATGGTTAACGGTGAATTTACAGGCTTACGC-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-GGTTGGCGATTGTTACAAAAGCATAACTCCTTATGCGGGAAT-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-AATGGAATTTGAACCTCGTC-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-TCATCCACCTTAGTCAAAGA-3' (SEQ ID NO: 14) (59% homology to the complementary strand 5'-CCTTAGTCAAAGAGACCAGAGG-3' of primer F3_ B);
upstream inner primer FIP _ D: 5'-ATCATTGCCAGAGTGGAACTTGTGTGGATAACGAAACCAT-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D: 5'-GTGGTATGGACGTTCAATCGTTCAGAGGTTCAAAAATCCCTT-3' (SEQ ID NO: 16).
In the method of the present invention, the primer set capable of amplifying the genome-specific base sequence of Vibrio cholerae O1 group may include, but is not limited to, a single loop primer. Preferably, the loop primer is one, including loop primer LB. The primer group capable of amplifying the genome specific base sequence of the vibrio cholerae O1 group is a primer group B'; or any one of primer sets having a homology of 59% or more with a single sequence in the sequence of the primer set B' or the sequence of the complementary strand thereof:
a primer set B':
upstream outer primer F3_ B: 5'-CCTCTGGTCTCTTTGACTAAGG-3', respectively;
downstream outer primer B3_ B: 5'-AGTTTCACGCCTTGCTCAC-3', respectively;
upstream inner primer FIP _ B: 5'-TCGGGAAAATGTAGGTCGCCACCGAAATCAAAACGGCTGGC-3', respectively; the downstream inner primer BIP _ B: 5'-CGGCGGATGGTAAAAACAAAGCAGAAATGGCCCGGTACCAGAG-3', respectively;
downstream loop primer LB _ B: 5'-AGCACGGTAACTCTTTTGCCTCTG-3' (SEQ ID NO: 17).
In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBstDNA 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 LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer，2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to the improvement of the reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L of LTris-HCl (pH 8.8), 10mmol/L of KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic stepped strip, the sample to be detected is positive to a vibrio cholerae O1 group and contains a vibrio cholerae O1 group; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative to the vibrio cholerae O1 group. The turbidity detection is 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 the vibrio cholerae O1 group and contains the vibrio cholerae O1 group; if no turbidity is found, the sample to be tested is negative to the vibrio cholerae O1 group. Or the reaction tube bottom can be visually observed whether the sediment exists or not after the centrifugation, if the sediment exists at the reaction tube bottom, the sample to be detected is positive to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group; if no precipitate is left at the bottom of the reaction tube, the sample to be detected is negative to the vibrio cholerae O1 group.
The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or 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 cholerae O1 group; if the color is green after the reaction, the sample to be detected is positive to the vibrio cholerae O1 group and contains the vibrio cholerae O1 group. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to vibrio cholerae O1 group; if the color after the reaction is sky blue, the sample to be detected is positive to the vibrio cholerae O1 group. The 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 the vibrio cholerae O1 group; and when the reaction result of the sample to be detected is greater than the threshold value, the sample to be detected is positive to the vibrio cholerae O1 group. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.
In the color development detection, if calcein or hydroxynaphthol blue is 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 cholerae O1 group strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of the genome of the vibrio cholerae O1 group, and the sequence of the primer is part of the nucleic acid sequence of 170082-171001 bp position of the genome of the vibrio cholerae O1 group with the GI number of 15640032 or part of a complementary strand of the nucleic acid sequence.
Wherein the primer group capable of amplifying the genome-specific nucleotide sequence of Vibrio cholerae O1 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 primer group 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 59% or more with a single sequence in the aforementioned primer set sequence or its complementary strand sequence 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'-AACAAACTGGCGTTGGCT-3', respectively;
downstream outer primer B3_ a: 5'-TCGTTATCCACACGCTGC-3', respectively;
upstream inner primer FIP _ A: 5'-AGACCGGGTCAGCGTCGTAATGGCTTGAGTTTTGGTGCG-3', respectively;
the downstream inner primer BIP _ A: 5'-CTGCAACTGTCGCACATGGTCTTCAAGATTCGGCCAGACGAG-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-CCTCTGGTCTCTTTGACTAAGG-3', respectively;
downstream outer primer B3_ B: 5'-AGTTTCACGCCTTGCTCAC-3', respectively;
upstream inner primer FIP _ B: 5'-TCGGGAAAATGTAGGTCGCCACCGAAATCAAAACGGCTGGC-3', respectively; the downstream inner primer BIP _ B: 5'-CGGCGGATGGTAAAAACAAAGCAGAAATGGCCCGGTACCAGAG-3' are provided.
Primer set C:
upstream outer primer F3_ C: 5'-CAGCATGAATACAGCAAGA-3', respectively;
downstream outer primer B3 — C: 5'-GCCAGTTTGTTTTTCATGG-3', respectively;
upstream inner primer FIP _ C: 5'-AGTTGGTCCATGGTTAACGGTGAATTTACAGGCTTACGC-3', respectively;
the downstream inner primer BIP _ C: 5'-GGTTGGCGATTGTTACAAAAGCATAACTCCTTATGCGGGAAT-3', respectively;
primer set D:
upstream outer primer F3_ D: 5'-AATGGAATTTGAACCTCGTC-3', respectively;
downstream outer primer B3_ D: 5'-TCATCCACCTTAGTCAAAGA-3', respectively;
upstream inner primer FIP _ D: 5'-ATCATTGCCAGAGTGGAACTTGTGTGGATAACGAAACCAT-3', respectively;
the downstream inner primer BIP _ D: 5'-GTGGTATGGACGTTCAATCGTTCAGAGGTTCAAAAATCCCTT-3' are provided.
In the primers used in the method for detecting the vibrio cholerae O1 group at the constant temperature, the primer group capable of amplifying the specific base sequence of the genome of the vibrio cholerae O1 group can also be but not limited to a loop primer; preferably, the loop primer is one and is LB. The primer group capable of amplifying the genome specific base sequence of the vibrio cholerae O1 group is a primer group B'; or any one selected from the group consisting of primer groups having a homology of 59% or more with a single sequence in the sequence of the primer group B' or the sequence of the complementary strand thereof:
a primer set B':
upstream outer primer F3_ B: 5'-CCTCTGGTCTCTTTGACTAAGG-3', respectively;
downstream outer primer B3_ B: 5'-AGTTTCACGCCTTGCTCAC-3', respectively;
upstream inner primer FIP _ B: 5'-TCGGGAAAATGTAGGTCGCCACCGAAATCAAAACGGCTGGC-3', respectively; the downstream inner primer BIP _ B: 5'-CGGCGGATGGTAAAAACAAAGCAGAAATGGCCCGGTACCAGAG-3', respectively;
downstream loop primer LB _ B: 5'-AGCACGGTAACTCTTTTGCCTCTG-3' are provided.
In a specific embodiment, the primers are respectively FIP, BIP, F3, B3 and LB primers or primers with 59% or more homology with single primer in the primer sequence or the complementary strand sequence.
The invention also provides a kit used in the method for detecting the vibrio cholerae O1 group strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the genome of the vibrio cholerae O1 group. In the kit, the primer group capable of amplifying the genome specific base sequence of the vibrio cholerae O1 group comprises but is not limited to a primer sequence which is a part of a nucleic acid sequence of 170082-171001 bp of a genome (GI No.: 15640032) or a part of a complementary strand thereof; 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 59% or more with a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer sets C-D, etc.
In the kit of the present invention, the primer set capable of amplifying the genome specific base sequence of vibrio cholerae O1 group may include, but is not limited to, a loop primer; the loop primer serves as an optional component. Preferably, the loop primer is one and is LB. The primer set including the loop primer LB includes, but is not limited to, a primer set B' and the like. In a specific embodiment, the kit of the present invention may comprise 0.4 to 1.0. mu. mol/L of LB loop primer. In a specific embodiment, the sequences of the primer sets are respectively the primers shown by FIP, BIP, F3, B3 and LB, or the primers with 59% or more homology to the aforementioned sequences or their complementary strand sequences.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg2+(MgSO4Or MgCl2) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/LdNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L BstDNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
The kit of the invention also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, the whole genomic DNA, a partial genomic DNA of vibrio cholerae group O1, or a vector comprising the whole genomic DNA or a partial genomic DNA of vibrio cholerae group O1.
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 of the invention also comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein and SYBR GreenI 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 vector, which comprises any one primer selected from the primer group A-B, C-D, B'. The vector contains a DNA sequence with the specificity of the vibrio cholerae O1 group, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application field of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one primer of primer set A, vector pBR322-B containing any one primer of primer set B, vector pBR322-B 'containing any one primer of primer set B' … …, and the like. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, … … a vector lambda phage-B 'containing any one of the primers of the primer set B', and the like.
The invention also provides the application of any one primer selected from the primer groups A-B, C-D, B' in constant temperature detection of the vibrio cholerae O1 group.
The invention also provides application of the kit in constant temperature detection of the vibrio cholerae O1 group.
The invention also provides application of the vector in constant temperature detection of the vibrio cholerae O1 group.
The invention provides a simple, rapid and sensitive method for detecting vibrio cholerae O1 group, a primer/primer group and a detection reagent/kit for the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the detection method of the vibrio cholerae O1 group has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the constant temperature amplification method adopted by the invention can be carried out under the constant temperature condition, only a simple constant temperature device is needed, expensive instruments in PCR experiments are not needed, and the steps of carrying out electrophoresis detection on the amplified products and the like are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments for popularization and use, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base of molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.
Drawings
FIG. 1 shows the specificity of the isothermal detection method for Vibrio cholerae O1 population in example 7 of the present invention.
FIG. 2 shows the sensitivity of the detection method of Vibrio cholerae O1 population in example 8 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art 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.
Example 1-6 Vibrio cholerae O1 group 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 cholerae O1 group strain for detection is derived from China general microbiological culture collection center with the number of CGMCC 1.8676. 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co260/OD280At a concentration of 1.8, 30.4 ng/. mu.L.
(2) The genome DNA of the vibrio cholerae O1 group 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 constant-temperature amplification reaction is carried out by taking the specific amplification primer group of the vibrio cholerae O1 group as a primer. The primers in examples 1 to 6 were primer sets A, B, B', C, D, and 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 fragment of the vibrio cholerae O1 group and obtain the detection result. In addition, when the detection is performed by using a detector, the detection effect is good when the reaction time is shortened to 10min (as in example 6). Therefore, the invention can be applied to detecting whether the sample contains the vibrio cholerae O1 group.
Example 7 detection of Vibrio cholerae O1 group specificity
28 strains of the Vibrio cholerae O1 group (1-28 in Table 4 and FIG. 1) were collected, and cultured separately from the Vibrio cholerae O1 group (29 in Table 4 and FIG. 1), 1mL of the bacterial solution was taken, and bacterial DNA was extracted using the 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 to 28 are respectively Staphylococcus aureus, Staphylococcus aureus Chryseozoinum species, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria inoke, Listeria eheli, Salmonella enterica subspecies, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, Escherichia coli diarrheal, enterotoxigenic Escherichia coli, Escherichia enterohemorrhagic Escherichia coli, Cronobacter sakazakii, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio freundii, NTC: negative control, 29: vibrio cholerae O1 group. In FIG. 1, the product obtained after the amplification reaction of only the Vibrio cholerae O1 group strain showed a bright green color as a positive result, as shown in tube 29. The products of the other strains of Vibrio cholerae O1 and the negative control after the amplification reaction are orange, which is a negative result, as shown in tubes No. 1-28 and NTC negative control tube.
As can be seen from the results shown in FIG. 1 and Table 4, the detection kit and the detection method of the present invention have good Vibrio cholerae O1 group strain specificity, i.e., only the Vibrio cholerae O1 group strain is amplified positively, and the other non-Vibrio cholerae O1 group strains are negative.
Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B, primer groups C-D and a primer group B', and the same detection results are obtained according to the specific detection method, namely, the products after the amplification reaction of the non-vibrio cholerae O1 group strain and the negative control are negative results, and the products after the amplification reaction of the vibrio cholerae O1 group strain are positive results.
In addition, theoretical analysis was performed on the specificities of the primer sets a to B, the primer sets C to D, and the primer set B' according to the method described in table 1, and as a result, it was found that, in the case where three mismatches were allowed at most for each primer, two primers were simultaneously compared to vibrio cholerae non-O1, indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNA of the bacterium CGMCC1.8676 was extracted by the method of example 1, and LAMP amplification (primer set A) and visualization by adding a color developing agent were carried out by using kit IB and by gradient addition of 2ng, 400pg, 80pg, 16pg, 320fg and 64fg DNA to the reaction system according to the method of example 1 of Table 3 under other reaction conditions. As shown in fig. 2, 1 to 6 are 2ng, 400pg, 80pg, 16pg, 320fg and 64fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 2ng, 400pg, 80pg, 16pg and 320fg treatments appeared bright green and as positive results, and the reaction products of 64fg treatment and negative control appeared orange and as negative results. As a result of the examination, DNA was detected in each reaction tube at a minimum of 320fg (about 720 bacteria).
According to the detection method, the DNA as low as 16 pg-320 fg in each reaction tube can be detected by using the primer group B, the primer groups C-D and the primer group B' respectively according to other steps and conditions.
Example 9 commonality testing
According to the method described in table 1, the primer sets a to B, C to D, and B' were theoretically analyzed for their versatility, and as a result, it was found that the primer regions of the primer sets completely match 8 chromosomes (GI numbers are 15640032, 147671401, 227080237, 227116370, 229606122, 360034408, 379739908, and 384423362, respectively) of the vibrio cholerae O1 group strain 1, and that they could be theoretically used for the detection of the 8 vibrio cholerae O1 group strains, indicating that the primer sets have good versatility.
TABLE 1 analysis of the universality and specificity of primers in the existing detection method of the Vibrio cholerae O1 population
Note: a) each Vibrio cholerae strain has two chromosomes, and the position of the detection region in the genome of GI No. 15640032#1/15600771#2 is determined by Bowtie alignment of the sequence between primers F3 and B3 with the 16 chromosomal genomic sequences of 8 strains of the Vibrio cholerae O1 group, #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 simultaneously aligned to the non-cholera arc strain, the specificity is good.
TABLE 2 types and main components of reagent kit for isothermal detection of Vibrio cholerae O1 group
TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal testing of Vibrio cholerae O1 population according to the present invention
TABLE 4 strains used in the test and the results
Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china medical bacteria strain preservation and management center. b) +: positive result, -: and (5) negative result.
<110> Shanghai Wangwang food group Co., Ltd., Shanghai Marine industry and technology research institute
Rapid constant-temperature detection method, primer group and kit for <120> vibrio cholerae O1 group
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Claims (10)
1. A rapid constant temperature detection method for vibrio cholerae O1 group is characterized by comprising the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) taking the genome DNA as a template, taking a primer group capable of amplifying the genome specific base sequence of the vibrio cholerae O1 group as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;
(3) determining whether the vibrio cholerae O1 group exists in the sample to be detected by judging whether the reaction result is positive or not;
wherein the specific base sequence of the genome of the vibrio cholerae O1 group is 170082-171001 bp bit sequence of the genome of the vibrio cholerae O1 group with the GI number of 15640032;
wherein the primer group capable of amplifying the genome-specific base sequence of Vibrio cholerae O1 group is selected from any one of the following primer groups C-D;
primer set C:
upstream outer primer F3_ C: 5'-CAGCATGAATACAGCAAGA-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-GCCAGTTTGTTTTTCATGG-3' (SEQ ID NO: 10);
upstream inner primer FIP _ C: 5'-AGTTGGTCCATGGTTAACGGTGAATTTACAGGCTTACGC-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-GGTTGGCGATTGTTACAAAAGCATAACTCCTTATGCGGGAAT-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-AATGGAATTTGAACCTCGTC-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-TCATCCACCTTAGTCAAAGA-3' (SEQ ID NO: 14);
upstream inner primer FIP _ D: 5'-ATCATTGCCAGAGTGGAACTTGTGTGGATAACGAAACCAT-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D: 5'-GTGGTATGGACGTTCAATCGTTCAGAGGTTCAAAAATCCCTT-3' (SEQ ID NO: 16).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0. mu. mol/L FIP and BIP primers, 0.15-0.3. mu. mol/L F3 and B3 primers, 0.16-0.64U/. mu.L Bst DNA polymerase, 0-1.5mol/L betaine, including or not including 0.4-1.0. mu. mol/L LB primers.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by incubating at ① 60-65 ℃ for 10-90 min and terminating at ② 80 ℃ for 2-20 min.
4. The primer in the rapid constant temperature detection method for the vibrio cholerae O1 group is characterized by comprising a primer group capable of amplifying a specific base sequence of a vibrio cholerae O1 group genome, wherein the sequence is a part of a nucleic acid sequence of 170082-171001 bp of a vibrio cholerae O1 group genome with the GI number of 15640032 or a part of a complementary chain of the nucleic acid sequence;
wherein the primer group capable of amplifying the genome-specific base sequence of Vibrio cholerae O1 group is selected from any one of the following primer groups C-D;
primer set C:
upstream outer primer F3_ C: 5'-CAGCATGAATACAGCAAGA-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-GCCAGTTTGTTTTTCATGG-3' (SEQ ID NO: 10);
upstream inner primer FIP _ C: 5'-AGTTGGTCCATGGTTAACGGTGAATTTACAGGCTTACGC-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-GGTTGGCGATTGTTACAAAAGCATAACTCCTTATGCGGGAAT-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-AATGGAATTTGAACCTCGTC-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-TCATCCACCTTAGTCAAAGA-3' (SEQ ID NO: 14);
upstream inner primer FIP _ D: 5'-ATCATTGCCAGAGTGGAACTTGTGTGGATAACGAAACCAT-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D: 5'-GTGGTATGGACGTTCAATCGTTCAGAGGTTCAAAAATCCCTT-3' (SEQ ID NO: 16).
5. A rapid isothermal detection kit for vibrio cholerae O1, comprising the primer of claim 4.
6. The kit of claim 5, further comprising a Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg2+And one or more of betaine.
7. A rapid constant temperature detection kit for vibrio cholerae O1 group is characterized in that an enzyme reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0. mu. mol/L FIP and BIP primers, 0.15-0.3. mu. mol/L F3 and B3 primers, with or without 0.4-1.0. mu. mol/L LB primer, 0.16-0.64U/. mu.L Bst DNA polymerase, and 0-1.5mol/L betaine.
8. A vector comprising the primer of claim 4.
9. Use of a primer for isothermal detection of vibrio cholerae group O1, wherein the primer is according to claim 4.
10. Use of the kit according to any one of claims 5 to 7 or the vector according to claim 8 for isothermal detection of the vibrio cholerae group O1.
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