CN106434900B - Method, primer and kit for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature - Google Patents
Method, primer and kit for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature Download PDFInfo
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Abstract
The invention discloses a method, a primer group and a kit for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae 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 common specific sequences of the vibrio vulnificus and the vibrio cholerae as primers; and determining whether the sample to be detected has vibrio vulnificus and/or vibrio cholerae by judging whether the reaction result is positive. 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 simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature.
Background
Vibrio vulnificus (Vibrio vulgaris) and Vibrio cholerae (Vibrio cholerae) are two gram-negative pathogenic bacteria commonly found in marine foods and can cause diseases such as primary septicemia, wound infection, acute gastroenteritis, cholera and the like. Therefore, it is very important for the prevention and detection of these two food-borne pathogenic bacteria.
The traditional detection method for vibrio vulnificus and vibrio cholerae has the defects of long detection period, relatively complex operation and low detection efficiency, and is difficult to meet the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society. In recent years, researchers have developed detection means such as PCR with the development of nucleic acid molecule detection technology, but this method requires a special detection instrument, and is not suitable for real-time in-situ detection widely used in the basic detection department, particularly in the production line of enterprises. In order to ensure the safety of food, a rapid, simple and accurate method for detecting Vibrio vulnificus and Vibrio cholerae 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.
At present, there are methods for respectively designing primers aiming at two pathogenic bacteria (such as vibrio vulnificus and vibrio cholerae) and respectively carrying out LAMP detection on the two pathogenic bacteria, but a method and an example for simultaneously detecting the two pathogenic bacteria by using one set of LAMP primers are not found. Therefore, a method for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature is urgently needed in the industry, and meanwhile, the method meets the requirements of basic detection departments on rapidness and convenience, and can be conveniently developed for real-time on-site detection in an enterprise production line.
Disclosure of Invention
The invention aims to overcome the defect that only one pathogenic bacterium can be detected by one set of primers in the design of primers in 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 and simultaneously identifying vibrio vulnificus and vibrio cholerae, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs LAMP primers of Vibrio vulnificus and Vibrio cholerae 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 simultaneously and rapidly detecting the Vibrio vulnificus and the Vibrio cholerae by isothermal amplification. The detection method of the invention is adopted to simultaneously detect the vibrio vulnificus and the vibrio cholerae, and has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenient operation and low cost.
The invention provides a method for simultaneously and rapidly detecting strains of vibrio vulnificus and vibrio cholerae, which comprises 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 common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes as a primer;
(3) and determining whether the sample to be detected has vibrio vulnificus and/or vibrio cholerae by judging whether the reaction result is positive.
The invention relates to a method for simultaneously detecting strains of vibrio vulnificus and vibrio cholerae at constant temperature, which 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 taking a specific amplification primer group of the vibrio vulnificus and the vibrio cholerae as primers, and then determining whether the sample to be detected has the vibrio vulnificus and/or the vibrio cholerae 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 consensus specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes is 2698869-2699116 bp of the Vibrio vulnificus genome with GI number 320154846 and 216343-216590 bp of the Vibrio cholerae genome with GI number 147673035.
In the invention, the primer group capable of amplifying the common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes is part of nucleic acid sequences of 2698869-2699116 bp of the Vibrio vulnificus genome with GI number 320154846 and 216343-216590 bp of the Vibrio cholerae genome with GI number 147673035 or part of complementary chains thereof. Wherein, the Vibrio vulnificus and Vibrio cholerae genome consensus specific base sequence refers to a base sequence which is only shared and unique by Vibrio vulnificus and Vibrio cholerae genomes but is not contained by other microorganism genomes.
Wherein the primer group capable of amplifying the specific base sequences of the genomes of the Vibrio vulnificus and the Vibrio cholerae comprises but is not limited to a primer group A, or a primer group with 68.4 percent or more homology with a single sequence in the sequence of the primer group or the complementary strand sequence thereof.
Primer set a:
upstream outer primer F3_ a: 5'-CGAGACTTGTGACGAGCTG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TCCAAGCTGAGAAACGTCG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 4).
In the present invention, the primer set capable of amplifying the specific base sequences of the genomes of vibrio vulnificus and vibrio cholerae may further comprise a primer set having a homology of 68.4% or more with a single sequence in the sequence of each of the aforementioned primer sets or the sequence of the complementary strand thereof, and the primer set includes, but is not limited to, the following primer set B:
primer set B:
upstream outer primer F3_ B: 5'-CGAGACTTGTGACGAGCTG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GCGCTATCCAAGCTGAGAA-3' (SEQ ID NO: 6) (68.4% homology to primer B3_ A5 '-TCCAAGCTGAGAAACGTCG-3');
upstream inner primer FIP _ B: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 8).
In the method of the present invention, in a specific embodiment, 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. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic step-shaped strip, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative and does not contain Vibrio vulnificus and Vibrio cholerae. The turbidity detection is carried out by observing with naked eyes or detecting turbidity by a turbidity meter, and if the detecting tube is obviously turbid, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae; if no turbidity is found, the sample to be detected is negative and does not contain vibrio vulnificus and vibrio cholerae. Or the reaction tube bottom can be visually observed whether the sediment exists after the centrifugation, if the sediment exists at the reaction tube bottom, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae; if no precipitate is left at the bottom of the reaction tube, the sample to be detected is negative and does not contain vibrio vulnificus and vibrio cholerae.
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 and does not contain vibrio vulnificus and vibrio cholerae; if the color after the reaction is green, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae. 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 and does not contain vibrio vulnificus and vibrio cholerae; if the color after the reaction is sky blue, the sample to be detected is positive and contains Vibrio vulnificus and/or Vibrio cholerae. The color development detection can be carried out in real time or end point detection reaction results through a detection instrument besides the reaction results observed by naked eyes, and by reasonably setting a threshold value of negative reaction, when the reaction result of a sample to be detected is lower than or equal to the threshold value, the sample to be detected is negative and does not contain vibrio vulnificus and vibrio cholerae; and when the reaction result of the sample to be detected is greater than the threshold value, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae. 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 simultaneously detecting the strains of the vibrio vulnificus and the vibrio cholerae at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of Vibrio vulnificus and Vibrio cholerae genomes, and includes but is not limited to, the sequence of the primer is part of nucleic acid sequences of 2698869-2699116 bp of the Vibrio vulnificus genome with GI number 320154846 and 216343-216590 bp of the Vibrio cholerae genome with GI number 147673035 or part of complementary chains of the nucleic acid sequences.
Wherein the primer group capable of amplifying the common specific base sequences of the genomes of the Vibrio vulnificus and the Vibrio cholerae is selected from any one of the following primer groups, or is selected from any one of the primer groups with single sequence homology of 68.4% or more in the sequences of the primer groups or the complementary strand sequences thereof. Wherein, the primer group includes but is not limited to the following primer group A. The primer set having a homology of 68.4% or more with a single sequence in the aforementioned primer set sequence or its complementary strand sequence includes, but is not limited to, the following primer set B.
Primer set a:
upstream outer primer F3_ a: 5'-CGAGACTTGTGACGAGCTG-3', respectively;
downstream outer primer B3_ a: 5'-TCCAAGCTGAGAAACGTCG-3', respectively;
upstream inner primer FIP _ A: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3', respectively;
the downstream inner primer BIP _ A: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-CGAGACTTGTGACGAGCTG-3', respectively;
downstream outer primer B3_ B: 5'-GCGCTATCCAAGCTGAGAA-3', respectively;
upstream inner primer FIP _ B: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3', respectively;
the downstream inner primer BIP _ B: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' are provided.
The invention also provides a kit used in the method for simultaneously detecting the strains of the vibrio vulnificus and the vibrio cholerae at constant temperature, which comprises the primer group capable of amplifying the specific base sequences of the genomes of the vibrio vulnificus and the vibrio cholerae. In the kit, the primer group capable of amplifying the common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes includes but is not limited to using part of the nucleic acid sequence of 2698869-2699116 bp of the Vibrio vulnificus genome with GI number 320154846 and 216343-216590 bp of the GI number 147673035 genome or part of the complementary strand thereof as the primer sequence; the primer includes but is not limited to the primer set a. But not limited to, a primer group having 68.4% or more homology to a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer set B.
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 vulnificus and/or vibrio cholerae, or a vector comprising the whole genomic DNA or a partial genomic DNA of vibrio vulnificus and/or vibrio cholerae, and the genomic DNAs of the two pathogenic bacteria may be mixed together or may be separately packaged.
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.
In the kit of the present inventionContains a color developer including but 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 vector, which comprises any one primer selected from the primer group A, B. The vector contains DNA sequences with the common specificity of the vibrio vulnificus and the vibrio cholerae, so 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 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, and the like.
The invention also provides application of the primers selected from any one of the primer groups A, B in simultaneous isothermal detection of Vibrio vulnificus and Vibrio cholerae.
The invention also provides application of the kit in simultaneous constant-temperature detection of vibrio vulnificus and vibrio cholerae.
The invention also provides application of the vector in simultaneous constant-temperature detection of vibrio vulnificus and vibrio cholerae.
The invention provides a simple, rapid and sensitive method for simultaneously detecting vibrio vulnificus and vibrio cholerae, 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 vulnificus and the vibrio cholerae has the advantages of capability of simultaneously detecting two pathogenic bacteria by one reaction, 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 for Vibrio vulnificus and Vibrio cholerae of example 7 of the present invention.
FIG. 2 shows the sensitivity of the method for detecting Vibrio vulnificus and Vibrio cholerae 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 isothermal reaction System and detection method of Vibrio vulnificus and Vibrio cholerae
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 derived from the China center for industrial microorganism strain preservation management with the serial number CICC10383 (ATCC 27562), and the vibrio cholerae strain is derived from the China center for common microorganism strain preservation with the serial number CGMCC 1.8676. Extracting 1mL of bacterial culture with bacterial nucleic acid of Beijing Tiangen bioengineering companyExtracting genome DNA and DNA OD from the kit260/OD280The concentration is 1.8-2.0, and the concentration is 210.8 ng/muL and 30.4 ng/muL respectively.
(2) Taking genomic DNAs of vibrio vulnificus and vibrio cholerae to be detected as templates, respectively adopting self-prepared kits (shown in tables 1 and 2), preparing a reaction system according to the conditions in table 2, and carrying out isothermal amplification reaction by taking specific amplification primer groups of vibrio vulnificus and vibrio cholerae as primers. The primers in examples 1 to 6 were primer sets A, A, A, B, B, and B, respectively.
(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 2.
As can be seen from Table 2, the detection method and the primer set and the reaction system adopted by the detection method can well amplify the common specific fragments of the Vibrio vulnificus and the Vibrio cholerae and obtain the detection results. Therefore, the present invention can be applied to the detection of the presence or absence of Vibrio vulnificus and/or Vibrio cholerae in a sample.
Example 7 Vibrio vulnificus and Vibrio cholerae specific assays
28 strains of Vibrio atraumatic and Vibrio cholerae (1 to 25, 27 to 28 and 30 in Table 3 and FIG. 1) were collected, these strains were cultured with the Vibrio vulnificus and Vibrio cholerae strains (26 and 29 in Table 3), 1mL of the bacterial solution was taken, bacterial DNA was extracted using the kit IA, and LAMP amplification (primer set A) and visualization by addition of a color developing agent were performed, respectively, with reference to the reaction system and conditions of example 1.
The detection results are shown in Table 3 and FIG. 1, in FIG. 1, 1-25 are Staphylococcus aureus, Staphylococcus aureus Chryseozoinum species, Staphylococcus epidermidis, Rhodococcus equi, Bacillus mycoides, Listeria monocytogenes, Listeria inokii, Listeria ehelii, Salmonella enterica, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, enterotoxigenic Escherichia coli, Escherichia coli enterotoxigenic, Escherichia coli hemorrhagic, Yersinia enterocolitica, Yersinia pseudotuberculosis, 27-28 are Vibrio parahaemolyticus and Vibrio shigella and 30 is Shigella sonnei, NTC: negative control, P is the mixture of the DNA of strains No. 26 and No. 29 (No. 26 and No. 29 are Vibrio vulnificus and Vibrio cholerae, respectively), i.e., the mixed sample of Vibrio vulnificus and Vibrio cholerae. In FIG. 1, the amplification reaction product obtained from the mixed sample tube containing only Vibrio vulnificus and Vibrio cholerae showed a bright green color, which is a positive result, as shown in the P-tube. The products of other strains of Vibrio non-traumatic and Vibrio cholerae and the negative control amplification reaction are orange, which are negative results, such as tubes No. 1-25, No. 27-28, and No. 30, and NTC negative control tubes.
As can be seen from the results shown in FIG. 1 and Table 3, the detection kit and the detection method of the present invention have good specificity for the strains of Vibrio vulnificus and Vibrio cholerae, i.e., only the strains of Vibrio vulnificus and Vibrio cholerae are amplified positively, and other strains of Vibrio non-vulnificus and Vibrio cholerae are negative.
Preparing a detection kit, wherein a primer adopted in the kit is a primer group B, and obtaining the same detection result according to the specific detection method, namely, the product obtained after the amplification reaction of the strains of the vibrio vulnificus and the vibrio cholerae and the negative control is a negative result, and the product obtained after the amplification reaction of the strains of the vibrio vulnificus and the vibrio cholerae is a positive result.
In addition, theoretical analysis is respectively carried out on the specificity of the primer groups A to B, the method comprises the steps of carrying out Bowtie comparison on the sequence between the primers F3 and B3 and the genome of Vibrio vulnificus and/or Vibrio cholerae in a GenBank database to determine a detection region, carrying out Blast comparison on non-vibrio vulnificus and Vibrio cholerae in public database resources of the detection region sequence, and detecting the matching degree of the detection region and the genome sequence. If the matching degree is higher, the specificity is poorer; if the primers can not be compared with the strains of the non-detection strains at the same time, the specificity is good. As a result, it was found that, in the case where each primer allowed up to three mismatches, each primer set had 6 primers simultaneously aligned to Vibrio vulnificus and Vibrio cholerae, which were Vibrio furnisii (GI. RTM. 375129161), but had more mismatches in the primer regions (two mismatches in each of B3, F2, F1c, and one mismatch in each of F3, B2, and B1 c), indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNAs of bacteria CICC10383 (Vibrio vulnificus) and CGMCC1.8676 (Vibrio cholerae) were extracted according to the method of example 2, and added to the reaction system using kit IIB according to DNA gradients of 0.5fg, 5fg, 50fg and 500fg, respectively, and LAMP amplification (primer set A) and visualization by adding color reagent were performed according to the method of example 2 of Table 2 under other reaction conditions. As shown in fig. 2, 1 to 4 are 0.5fg, 5fg, 50fg and 500fg, respectively, NTC: and (5) negative control. In FIG. 2, in the case of the treatment using Vibrio vulnificus DNA as a template, the reaction products of the 0.5fg, 5fg, 50fg and 500fg treatments were bright green and a positive result, and the reaction product of the negative control was orange and a negative result; the reaction products of the 5fg, 50fg and 500fg treatments were bright green as a positive result, and the reaction products of 0.5fg and negative control were orange as a negative result, with the Vibrio cholerae DNA as the template. The detection result shows that the DNA of 0.5 fg-5 fg (equivalent to 1 bacterium) can be detected in each reaction tube, and the sensitivity is higher.
According to the above detection method, other steps and conditions are as above, with the primer set B, DNA as low as 5fg in each reaction tube can be detected, and the detection sensitivity is high.
Example 8 commonality testing
Theoretical analysis is carried out on the universality of the primer groups A-B respectively, the method comprises the steps of carrying out Bowtie comparison on the sequences between the primers F3 and B3 and the genomes of Vibrio vulnificus and/or Vibrio cholerae in a GenBank database, determining a detection region, carrying out Blast comparison on the Vibrio vulnificus (3 genomes) and the Vibrio cholerae (8 genomes) in public database resources of the detection region sequence, detecting the matching degree of the primer region and the genome sequence, and having good universality if the primer regions are completely matched. As a result, the primer regions of the primer groups are completely matched with the 11 genomes of the Vibrio vulnificus and the Vibrio cholerae, which indicates that the primer groups have better universality.
TABLE 1 types and main components of reagent kit for simultaneous isothermal detection of Vibrio vulnificus and Vibrio cholerae
TABLE 2 examples 1-6 reaction conditions and test results in the method for simultaneously isothermal testing of Vibrio vulnificus and Vibrio cholerae of the present invention
TABLE 3 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. c) The DNA template used was a mixture of 26 and 29 strain DNA (1: 1).
Claims (5)
1. A method for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature for non-diagnosis purposes is characterized by comprising the following steps of:
(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 common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;
(3) determining whether the sample to be detected has vibrio vulnificus and/or vibrio cholerae by judging whether the reaction result is positive;
wherein the common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genomes is 2698869-2699116 bp of the Vibrio vulnificus genome with GI number 320154846 and 216343-216590 bp bit sequence of the Vibrio cholerae genome with GI number 147673035;
wherein the primer group capable of amplifying the common specific base sequences of the genomes of the vibrio vulnificus and the vibrio cholerae is a primer group A;
primer set a:
upstream outer primer F3_ a: 5'-CGAGACTTGTGACGAGCTG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TCCAAGCTGAGAAACGTCG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 4).
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 and 0-1.5mol/L betaine.
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 for simultaneously detecting Vibrio vulnificus and Vibrio cholerae at constant temperature according to claim 1, wherein the primer comprises a primer group capable of amplifying a common specific base sequence of the genomes of Vibrio vulnificus and Vibrio cholerae, the sequence of the primer group is the nucleic acid sequence of 2698869-2699116 bp of Vibrio vulnificus with GI number of 320154846 and 216343-216590 bp of Vibrio cholerae with GI number of 147673035;
wherein the primer group capable of amplifying the common specific base sequences of the genomes of the vibrio vulnificus and the vibrio cholerae is a primer group A;
primer set a:
upstream outer primer F3_ a: 5'-CGAGACTTGTGACGAGCTG-3', respectively;
downstream outer primer B3_ a: 5'-TCCAAGCTGAGAAACGTCG-3', respectively;
upstream inner primer FIP _ A: 5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3', respectively;
the downstream inner primer BIP _ A: 5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' are provided.
5. Use of a primer for the isothermal detection of Vibrio vulnificus and Vibrio cholerae simultaneously for non-diagnostic purposes, wherein the primer is according to claim 4.
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| CN105219845A (en) * | 2015-07-28 | 2016-01-06 | 青岛农业大学 | The dual LAMP method of Vibrio parahaemolyticus and Vibrio vulnificus can be detected simultaneously |
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