CN111004854B - Rapid constant temperature detection method, primer set and kit for vibrio vulnificus and vibrio cholerae simultaneously - Google Patents
Rapid constant temperature detection method, primer set and kit for vibrio vulnificus and vibrio cholerae simultaneously Download PDFInfo
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Abstract
The invention discloses a rapid constant temperature detection method, a primer group and a kit for vibrio vulnificus and vibrio cholerae simultaneously. The method comprises the following steps: extracting genome DNA from a sample to be detected; taking the genome DNA as a template, taking a primer group capable of amplifying a common specific sequence of vibrio vulnificus and vibrio cholerae as a primer, and performing isothermal amplification reaction under an enzyme reaction system; and determining whether vibrio vulnificus and/or vibrio cholerae exist in the sample to be detected by judging whether the reaction result is positive. The detection method has the advantages of high sensitivity, high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.
Description
The application is filed 8/30/2016, and has the application number of 201610780489.8 and the invention name of: a method, primer and kit for simultaneously and quickly detecting vibrio vulnificus and vibrio cholerae at constant temperature are disclosed.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, a primer and a kit for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature.
Background
Vibrio vulnificus (Vibrio vulnificus) and Vibrio cholerae (Vibrio cholerae) are two gram-negative pathogens commonly found in marine foods and can cause primary sepsis, wound infection, acute gastroenteritis, cholera, and other conditions. Therefore, it is very important to prevent and detect these two food-borne pathogens.
The traditional vibrio vulnificus and vibrio cholerae detection method has the defects of relatively long detection period, relatively complex operation and lower detection efficiency, and is difficult to meet the requirements of the modern society on high flux, high sensitivity, high specificity, rapidness and convenience in the food-borne pathogenic bacteria detection process. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means such as PCR, but this method requires a special detection instrument, and is therefore not suitable for being widely applied to real-time in-situ detection performed in the basic detection departments, especially in the interior of an enterprise production line. In order to ensure food safety, a rapid, simple and accurate method for detecting Vibrio vulnificus and Vibrio cholerae in food is urgently required.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method developed in recent years, which designs 4 specific primers (comprising upstream and downstream outer primers F3 and B3 and upstream and downstream inner primers FIP and BIP, wherein FIP consists of F1C and F2, and BIP consists of B1C and B2) for 6 regions of a target sequence, and uses a DNA polymerase with strand displacement activity to perform a nucleic acid amplification reaction by incubating for about 60min under isothermal conditions, thereby generating macroscopic reaction byproducts-white magnesium pyrophosphate precipitate (see Notomi T, okayama H, masubechi H, yonekawa T, watanabe K, amino N, hase T.Loop-mediated isothermal amplification of DNA, nucleic Acids Research,2000 Jun 15;28 (12): E63). The technology has the advantages of no need of a PCR instrument or a fluorescent quantitative PCR instrument, completion at constant temperature, capability of judging the reaction result by naked eyes, high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.
At present, primers are designed for two pathogenic bacteria (such as vibrio vulnificus and vibrio cholerae) respectively, and a method for detecting the two pathogenic bacteria respectively by LAMP is not known, but a method and an example for simultaneously detecting the two pathogenic bacteria by using one set of LAMP primers are not known. Therefore, a method for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae at constant temperature is needed in the industry, and simultaneously, the requirements of basic detection departments on rapidness and convenience are met, so that real-time and on-site detection can be conveniently carried out in an enterprise production line.
Disclosure of Invention
The invention aims to solve the technical problem that a set of primers in the primer design of the prior LAMP technology can only detect one pathogenic bacteria, fully utilizes the sequence information of the microbial genome and the corresponding sequence analysis tools which are rich in the current public data resources, designs a primer group for specifically identifying vibrio vulnificus and vibrio cholerae simultaneously, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention provides a method for simultaneously and rapidly amplifying and detecting vibrio vulnificus and vibrio cholerae at constant temperature, a primer group and a kit based on a microbial genome data resource (data of 8 months and 5 days in 2013) in a GenBank database for designing LAMP primers of vibrio vulnificus and vibrio cholerae. The detection method for simultaneously detecting the vibrio vulnificus and the vibrio cholerae has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation and low cost.
The invention provides a method for simultaneously and rapidly detecting vibrio vulnificus and vibrio cholerae strains, which comprises 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 which can amplify a specific base sequence shared by the genomes of the vibrio vulnificus and the vibrio cholerae as a primer, and performing isothermal amplification reaction under an enzyme reaction system;
(3) And determining whether vibrio vulnificus and/or vibrio cholerae exist in the sample to be detected by judging whether the reaction result is positive.
The invention discloses a method for simultaneously detecting vibrio vulnificus and vibrio cholerae strains at constant temperature, which comprises the steps of extracting genome DNA from a sample to be detected, taking the genome DNA as a template, taking a vibrio vulnificus and vibrio cholerae specific amplification primer group as primers, carrying out constant temperature amplification reaction, and then determining whether the vibrio vulnificus and/or vibrio cholerae exist in the sample to be detected by judging whether a reaction result is positive. Wherein the enzyme reaction system includes, but is not limited to, a DNA polymerase reaction system.
In the invention, the common specific base sequence of the vibrio vulnificus and the vibrio cholerae genome is 2698869 ～ 2699116bp of the vibrio vulnificus genome with the GI number of 320154846 and 216343 ～ 216590bp of the vibrio cholerae genome with the GI number of 147673035.
In the invention, the primer group capable of amplifying the common specific base sequence of the vibrio vulnificus and the vibrio cholerae genome is a part of the nucleotide sequence of 2698869 ～ 2699116bp of the vibrio vulnificus genome with the GI number of 320154846 and 216343 ～ 216590bp of the vibrio cholerae genome with the GI number of 147673035 or a part of the complementary strand thereof. Wherein the common specific base sequence of the Vibrio vulnificus and Vibrio cholerae genome refers to a base sequence which is common and unique to only the Vibrio vulnificus and Vibrio cholerae genome and not contained in the genome of other microorganisms.
Wherein, the primer group capable of amplifying the specific base sequences of the genome of Vibrio vulnificus and Vibrio cholerae comprises, but is not limited to, a primer group A, or a primer group with 68.4% or more of homology with a single sequence in the primer group sequence or the complementary strand sequence thereof.
Primer group 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);
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 Vibrio vulnificus and Vibrio cholerae genomes may further comprise a primer set having a homology of 68.4% or more with a single sequence of the aforementioned primer set sequences or the complementary strand sequences thereof, the primer set including but not limited to the following primer set B:
primer group 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) (homology 68.4% with primer B3_A 5'-TCCAAGCTGAGAAACGTCG-3');
upstream inner primer FIP_B:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 7);
downstream inner primer bip_b:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 8).
In one embodiment of the method of the present invention, the isothermal amplification enzyme reaction system is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP and BIP primer, 0.15-0.3 mu mol/L F3 and B3 primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer may be 1 XThermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in an enzyme reaction system 2+ And (5) performing merging treatment.
In the method of the invention, the reaction program of the isothermal amplification reaction is (1) incubation for 10-90 min, preferably 10-60 min at 60-65 ℃; (2) the reaction is stopped for 2 to 20 minutes at the temperature of 80 ℃. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color development detection, and the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and can be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis chart shows characteristic ladder-shaped strips, the sample to be detected is positive and contains vibrio vulnificus and/or vibrio cholerae; if the electrophoresis chart does not show a characteristic ladder-shaped strip, the sample to be tested is negative and does not contain vibrio vulnificus and vibrio cholerae. The turbidity detection is to detect turbidity by naked eyes or a turbidity meter, and the sample to be detected is positive when the detection tube has obvious turbidity and contains vibrio vulnificus and/or vibrio cholerae; if no turbidity is found, the sample to be tested is negative, and does not contain vibrio vulnificus and vibrio cholerae. Or observing whether the reaction tube bottom has sediment or not by naked eyes after centrifugation, and if the reaction tube bottom has sediment, the sample to be tested is positive and contains vibrio vulnificus and/or vibrio cholerae; if the bottom of the reaction tube does not have sediment, the sample to be tested is negative and does not contain vibrio vulnificus and vibrio cholerae.
The color development test is to add a color developing agent including, but not limited to, calcein (50. Mu.M) or SYBR GreenI (30-50X), or hydroxynaphthol blue (i.e., HNB, 120-150. Mu.M) into the reaction tube. When calcein or SYBR Green I is adopted as a color developing agent, if the color is orange after the reaction, the sample to be tested is negative, and the sample does not contain vibrio vulnificus and vibrio cholerae; if the color is green after the reaction, the sample to be tested is positive and contains vibrio vulnificus and/or vibrio cholerae. When hydroxyl naphthol blue is adopted as a color developing agent, if the color after the reaction is violet, the sample to be tested is negative, and the sample does not contain vibrio vulnificus and vibrio cholerae; if the color after the reaction is sky blue, the sample to be tested is positive and contains vibrio vulnificus and/or vibrio cholerae. Besides the reaction result observed by naked eyes, the color development detection can also be carried out by detecting the reaction result in real time or through a detecting instrument, and 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 and does not contain vibrio vulnificus and vibrio cholerae; when the reaction result of the sample to be tested is larger than the threshold value, the sample to be tested is positive and contains vibrio vulnificus and/or vibrio cholerae. Such detection instruments include, but are not limited to, fluorescence spectrophotometers, fluorescent quantitative PCR instruments, isothermal amplification microfluidic chip nucleic acid analyzers, genie II isothermal amplification fluorescent detection systems, and the like.
In the color development detection, if calcein or hydroxynaphthol blue is adopted as the color developing agent, the calcein or hydroxynaphthol blue can be added before the isothermal amplification reaction, or after the isothermal amplification reaction is finished, preferably before the isothermal amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is used as the developer, it is added after the isothermal amplification reaction is complete. If calcein is used as the color-developing agent, 50. Mu.M calcein and 0.6-1mM Mn are added to the enzyme reaction system 2+ ]For example, 0.6-1mM MnCl 2 。
The invention also provides primers for use in a method for simultaneously detecting Vibrio vulnificus and Vibrio cholerae strains at constant temperature. The primer includes a primer set capable of amplifying specific base sequences of Vibrio vulnificus and Vibrio cholerae genomes, including, but not limited to, a portion of a nucleic acid sequence of 2698869 ～ 2699116bp of Vibrio vulnificus genome with GI number 320154846 and 216343 ～ 216590bp of Vibrio cholerae genome with GI number 147673035 or a portion of a complementary strand thereof.
Wherein the primer set capable of amplifying a base sequence common to the genomes of Vibrio vulnificus and Vibrio cholerae is selected from any one of the following primer sets or from any one of the primer sets having a homology of 68.4% or more with a single sequence in the sequences of the primer sets or the complementary strand sequences thereof. Wherein the primer set includes, but is not limited to, the following primer set A. The primer set having a homology of 68.4% or more to 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 group a:
upstream outer primer f3_a:5'-CGAGACTTGTGACGAGCTG-3';
downstream outer primer b3_a:5'-TCCAAGCTGAGAAACGTCG-3';
upstream inner primer FIP_A:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3';
downstream inner primer bip_a:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3';
primer group B:
upstream outer primer f3_b:5'-CGAGACTTGTGACGAGCTG-3';
downstream outer primer b3_b:5'-GCGCTATCCAAGCTGAGAA-3';
upstream inner primer FIP_B:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3';
downstream inner primer bip_b:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3'.
The invention also provides a kit for the method for simultaneously detecting vibrio vulnificus and vibrio cholerae strains at constant temperature, which comprises the primer group capable of amplifying the specific base sequences of the vibrio vulnificus and vibrio cholerae genomes. In the kit of the invention, the primer set capable of amplifying the specific base sequence shared by the Vibrio vulnificus and Vibrio cholerae genomes comprises, but is not limited to, a part of the 2698869 ～ 2699116bp nucleic acid sequence of the Vibrio vulnificus genome with the GI number of 320154846 and the 216343 ～ 216590bp nucleic acid sequence of the genome with the GI number of 147673035 or a part of the complementary strand thereof as the primer sequence; the primer includes, but is not limited to, the primer set a. Also included are, but not limited to, primer sets having 68.4% or more homology to a single sequence of the aforementioned primer sequences or the complementary strand sequences thereof; including but not limited to primer set B.
The kit also comprises Bst DNA polymerase buffer solution, bst DNA polymerase, dNTP solution and Mg 2+ (MgSO 4 Or MgCl 2 ) And one or more of betaines. In one embodiment, the enzymatic reaction system of the kit of the present invention comprises 1 XBst DNA polymerizationEnzyme reaction buffer solution, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP and BIP primer, 0.15-0.3 mu mol/L F3 and B3 primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer may be 1 XThermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in an enzyme reaction system 2+ And (5) performing merging treatment.
The kit also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, whole genomic DNA, partial genomic DNA of Vibrio vulnificus and/or Vibrio cholerae, or a vector comprising whole genomic DNA or partial genomic DNA of Vibrio vulnificus and/or Vibrio cholerae, and the genomic DNA of two pathogenic bacteria may be mixed together or packaged separately.
The kit of the invention further comprises a negative control template, wherein the negative control template comprises, but is not limited to, double distilled water.
The kit of the invention further comprises a color-developing agent, wherein the color-developing agent comprises, but is not limited to, calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn ] 2+ ]For example, mnCl 2 。
The kit of the invention also comprises double distilled water.
The kit of the invention also comprises a nucleic acid extraction reagent.
The invention also provides a vector comprising any one of the primers selected from the primer set A, B. The vector contains a DNA sequence with common specificity of vibrio vulnificus and vibrio cholerae, so that the vector can be applied to the research fields of microbiology, 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 chromosomal 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, etc. A vector lambda phage-A comprising any one of the primers of primer set A, a vector lambda phage-B comprising any one of the primers of primer set B, etc.
The invention also provides application of the primer 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 isothermal detection of vibrio vulnificus and vibrio cholerae.
The invention also provides application of the carrier in simultaneous isothermal 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 method for detecting the vibrio vulnificus and the vibrio cholerae has the advantages of capability of detecting two pathogenic bacteria simultaneously by one-time reaction, strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the existing common detection method, the constant-temperature amplification method can be carried out under the constant-temperature condition, only a simple constant-temperature device is needed, expensive instruments in a PCR experiment are not needed, and steps such as electrophoresis detection are not needed on amplified products, so that the method is very suitable for being widely applied to popularization and use in various communities including basic food safety detection departments, and can be fully applied even under the environment with relatively insufficient molecular biology expertise and skill foundation. The above preferred conditions may be arbitrarily combined based on the common knowledge in the art, and all the conditions fall within the scope of the present invention.
Drawings
FIG. 1 shows the specificity of the isothermal detection method of Vibrio vulnificus and Vibrio cholerae according to 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, to which the present invention is not limited. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited.
EXAMPLES 1-6 Vibrio vulnificus and Vibrio cholerae isothermal reaction systems and detection methods
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 China center for type culture collection of industrial microorganisms, with the number of CICC10383 (=ATCC 27562), and the vibrio cholerae strain is derived from China center for type culture collection of common microorganisms, with the number of CGMCC 1.8676. Extracting genome DNA and DNA OD from 1mL bacterial culture with bacterial nucleic acid extraction kit of Beijing Tiangen bioengineering company 260 /OD 280 The concentration is between 1.8 and 2.0, and the concentration is 210.8 ng/. Mu.L and 30.4 ng/. Mu.L respectively.
(2) The genome DNA of the vibrio vulnificus and the vibrio cholerae to be detected are used as templates, self-matched kits (see table 1 and table 2) are respectively adopted, a reaction system is prepared according to the conditions described in table 2, and the specific amplification primer group of the vibrio vulnificus and the vibrio cholerae is used as a primer to carry out isothermal amplification reaction. The primers in examples 1 to 6 are primer sets A, A, A, B, B, B, respectively.
(3) The amplification results were confirmed by electrophoresis detection, turbidity detection or chromogenic detection under the conditions described 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 vibrio vulnificus and vibrio cholerae and obtain the detection result. Thus, the present invention can be applied to detect whether or not a sample contains Vibrio vulnificus and/or Vibrio cholerae.
EXAMPLE 7 Vibrio vulnificus and Vibrio cholerae specific detection
Vibrio vulnificus and Vibrio cholerae 28 strains (1 to 25, 27 to 28 and 30 in Table 3 and FIG. 1) were collected, cultured with Vibrio vulnificus and Vibrio cholerae strains (26 and 29 in Table 3), 1mL of bacterial liquid was taken, bacterial DNA was extracted using a kit IA, and LAMP amplification (primer set A) and observation with addition of a color-developer 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 to 25 are Staphylococcus aureus, staphylococcus aureus subspecies aureobacteria, staphylococcus epidermidis, rhodococcus equi, bacillus mycoides, listeria monocytogenes, listeria, salmonella enterica subspecies enterica, salmonella typhimurium, salmonella paratyphi B, shigella dysenterica, shigella pallidum, shigella flexneri, escherichia coli (containing clostridium botulinum type A gene), pathogenic Escherichia coli, diarrhea causing Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, yersinia enterocolitica, yersinia pseudotuberculosis, 27 to 28 are respectively Vibrio parahaemolyticus and Freund's, 30 is Shigella sonnei, NTC: negative control, P is a mixed solution of strain DNA of 26 and 29 (V.vulnificus and V.cholerae, respectively, no. 26 and No. 29), i.e., a mixed sample of V.vulnificus and V.cholerae. In FIG. 1, only the products after the amplification reaction of the mixed sample tube containing Vibrio vulnificus and Vibrio cholerae appear bright green, and are positive results, as shown in the tube P. The products after amplification reaction of other non-traumatic vibrio and cholera vibrio strains and negative control are orange, and are negative results, such as the tubes 1 to 25, 27 to 28, 30 and NTC negative control tubes.
As can be seen from the results of FIG. 1 and Table 3, the detection kit and the detection method of the present invention have good strain specificity of Vibrio vulnificus and Vibrio cholerae, i.e., only Vibrio vulnificus and Vibrio cholerae strains amplify positively, and other non-Vibrio vulnificus and Vibrio cholerae strains are negative.
Preparing a detection kit, wherein a primer adopted in the kit is a primer group B, and the same detection result is obtained according to the specific detection method, namely, the products after amplification reaction of vibrio vulnificus and vibrio cholerae strains and negative control are negative results, and the products after amplification reaction of vibrio vulnificus and vibrio cholerae strains are positive results.
In addition, the specificity of the primer groups A-B is respectively subjected to theoretical analysis, the method comprises the steps of performing Bowtie comparison on sequences between the primers F3 and B3 and genomes of vibrio vulnificus and/or vibrio cholerae in a GenBank database to determine a detection area, and then performing Blast comparison on non-vibrio vulnificus and vibrio cholerae in public database resources on the sequences of the detection area to detect the matching degree of the primer areas and the genome sequences. If the matching degree is higher, the specificity is poorer; if the primers cannot be simultaneously compared with the strain of the non-detection strain, the specificity is good. As a result, it was found that, when each primer set allowed up to three mismatches, 6 primers were simultaneously aligned to Vibrio vulnificus and Vibrio cholerae, which was Vibrio furnishii (GI= 375129161), but there were more mismatches in the primer regions (two mismatches in each of B3, F2 and F1c, and one mismatch in each of F3, B2 and B1 c), indicating that the specificity of each primer set was good.
Example 8 sensitivity detection
The DNA of bacteria CICC10383 (Vibrio vulnificus) and CGMCC1.8676 (Vibrio cholerae) was extracted as in example 2, and the kit IIB was used, and DNA gradients of 0.5fg, 5fg, 50fg and 500fg were added to the reaction system, and LAMP amplification (primer set A) and visualization with the addition of a color-developer were performed, respectively, under other reaction conditions as described in example 2 of Table 2. As shown in FIG. 2, 1-4 are 0.5fg, 5fg, 50fg and 500fg, respectively, NTC: negative control. In FIG. 2, the reaction products of the treatments of 0.5fg, 5fg, 50fg and 500fg, which are treated with the Vibrio vulnificus DNA as a template, appear bright green, are positive results, and the reaction products of the negative control appear orange, are negative results; the reaction products treated with 5fg, 50fg and 500fg, treated with the vibrio cholerae DNA as a template, appeared bright green, as a positive result, and the reaction products of 0.5fg and the negative control appeared orange, as a negative result. The detection result shows that the lowest DNA content of 0.5 fg-5 fg (equivalent to 1 bacteria) in each reaction tube can be detected, and the sensitivity is higher.
According to the detection method, other steps and conditions are the same, and the DNA with the length of 5fg in each reaction tube can be detected by using the primer group B, so that the detection sensitivity is higher.
Example 8 versatility testing
The primer sets A-B are subjected to theoretical analysis respectively, the sequence between the primers F3 and B3 is subjected to Bowtie comparison with the genome of vibrio vulnificus and/or vibrio cholerae in a GenBank database to determine a detection region, then the vibrio vulnificus (3 genomes) and the vibrio cholerae (8 genomes) in public database resources are subjected to Blast comparison with the detection region sequence, the matching degree of the primer region and the genome sequence is detected, and if the primer region is completely matched, the universality is good. As a result, it was found that the primer regions of each primer set were completely matched with the 11 genomes of Vibrio vulnificus and Vibrio cholerae described above, indicating that the versatility of each primer set was good.
TABLE 1 kit type and main composition for simultaneously detecting Vibrio vulnificus and Vibrio cholerae at constant temperature
TABLE 2 EXAMPLES 1-6 reaction conditions and detection results in the method of simultaneously detecting Vibrio vulnificus and Vibrio cholerae at constant temperature of the present invention
TABLE 3 strains used in the experiments and the results of the tests
Note that: a) CGMCC: china general microbiological culture Collection center, CICC: china center for type culture Collection (CMCC): china medical bacterial culture Collection center. b) ++. Positive result, -: negative results. c) The DNA template used was a mixture of the DNA of strains 26 and 29 (1: 1).
<110> Shanghai bioinformatics research center
<120> method, primer set and kit for rapid isothermal detection of Vibrio vulnificus and Vibrio cholerae simultaneously
<160> 8
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 42
<212> DNA
<213> artificial sequence
<400> 3
tgcaactgct gcattcgctg tataaggcct ttagtcgcca tg 42
<210> 4
<211> 39
<212> DNA
<213> artificial sequence
<400> 4
ccagcagcga ttgcagcaac acaatgctag ccgtcgttc 39
<210> 5
<211> 19
<212> DNA
<213> artificial sequence
<400> 5
<210> 6
<211> 19
<212> DNA
<213> artificial sequence
<400> 6
<210> 7
<211> 42
<212> DNA
<213> artificial sequence
<400> 7
tgcaactgct gcattcgctg tataaggcct ttagtcgcca tg 42
<210> 8
<211> 39
<212> DNA
<213> artificial sequence
<400> 8
ccagcagcga ttgcagcaac acaatgctag ccgtcgttc 39
Claims (9)
1. A rapid isothermal detection method for vibrio vulnificus and vibrio cholerae simultaneously for non-diagnostic purposes, which 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 which can amplify the specific base sequence shared by the genomes of the vibrio vulnificus and the vibrio cholerae as a primer, and performing isothermal amplification reaction under an enzyme reaction system;
(3) Determining whether vibrio vulnificus and/or vibrio cholerae exist in the sample to be detected by judging whether the reaction result is positive;
wherein, the specific base sequence shared by the vibrio vulnificus and the vibrio cholerae genome is 2698869 ～ 2699116bp bit of the vibrio vulnificus genome with the GI number of 320154846 and 216343 ～ 216590bp bit of the vibrio cholerae genome with the GI number of 147673035;
wherein, the primer group capable of amplifying the specific base sequence shared by the vibrio vulnificus and the vibrio cholerae genome is a primer group B;
primer group 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);
upstream inner primer FIP_B:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 7);
downstream inner primer bip_b:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 8).
2. The method of claim 1, wherein in step (2), the enzyme reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP_B and BIP_B primer, 0.15-0.3 mu mol/L F3_B and B3_B primer, 0.16-0.64U/mu L Bst DNA polymerase, 0-1.5mol/L betaine.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: (1) incubating for 10-90 min at 60-65 ℃; (2) the reaction is stopped for 2 to 20 minutes at the temperature of 80 ℃.
4. The primer in the rapid isothermal detection method for vibrio vulnificus and vibrio cholerae is characterized by comprising a primer group capable of amplifying a common specific base sequence of vibrio vulnificus and vibrio cholerae genomes, wherein the sequence is a part of a nucleic acid sequence of 2698869 ～ 2699116bp bit of vibrio vulnificus with a GI number of 320154846 and 216343 ～ 216590bp bit of the vibrio cholerae genome with a GI number of 147673035 or a part of a complementary strand thereof;
wherein, the primer group capable of amplifying the specific base sequence shared by the vibrio vulnificus and the vibrio cholerae genome is a primer group B;
primer group 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);
upstream inner primer FIP_B:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3' (SEQ ID NO: 7);
downstream inner primer bip_b:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3' (SEQ ID NO: 8).
5. A rapid isothermal detection kit for vibrio vulnificus and vibrio cholerae simultaneously, which is characterized by comprising a primer group capable of amplifying a specific base sequence shared by vibrio vulnificus and vibrio cholerae genomes; the primer group capable of amplifying the specific base sequence shared by the vibrio vulnificus and vibrio cholerae genomes is a primer group B;
primer group B:
upstream outer primer f3_b:5'-CGAGACTTGTGACGAGCTG-3';
downstream outer primer b3_b:5'-GCGCTATCCAAGCTGAGAA-3';
upstream inner primer FIP_B:5'-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3';
downstream inner primer bip_b:5'-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3'.
6. The kit of claim 5, further comprising Bst DNA polymerase reaction buffer, bst DNA polymerase, dNTP solution, mg 2+ One or more of betaine.
7. The kit of claim 5, wherein the enzyme reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP_B and BIP_B primers, 0.15-0.3 mu mol/L F3_B and B3_B primers, 0.16-0.64U/. Mu.L Bst DNA polymerase, and 0-1.5mol/L betaine.
8. Use of a primer for isothermal detection of Vibrio vulnificus and Vibrio cholerae while not being diagnostic, wherein the primer is the primer according to claim 4.
9. The use of the kit according to any one of claims 5 to 7 for the isothermal detection of vibrio vulnificus and vibrio cholerae while not being diagnostic.
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