CN114507745A - RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof - Google Patents
RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof Download PDFInfo
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Abstract
The invention discloses an RPA-IAC primer for detecting vibrio alginolyticus, a kit, application and a method thereof, wherein an RPA primer pair is shown as SEQ ID NO.1 and SEQ ID NO. 2. The RPA primer pair has good amplification efficiency and amplification specificity to vibrio alginolyticus, and can improve the sensitivity of RPA detection; no special thermal cycle equipment is needed, the reaction time is short, compared with LAMP, the amplification product has no diffuse band, the RPA-IAC amplification product has a band with a specific size according to the design site of the primer, and the result is easy to judge. False negative results are effectively avoided by adding amplification internal standards.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method, a primer and a kit for detecting vibrio alginolyticus in shellfish aquatic products.
Background
The vibrio alginolyticus is an acapsular gram-negative vibrio halophilus, is widely distributed in shellfish and shellfish such as oyster, shrimp and crab in marine culture and shellfish aquatic animals such as crustacean, and is one of food-borne pathogenic bacteria with the highest popularity and harm degree.
At present, most coastal countries in the world have pathogenic reports of vibrio alginolyticus, the world also has more infection reports in Jiangzhe and Minyue coastal countries, and more cases of vibrio alginolyticus infection are one of the major threats to public dietary health, so the method has very important significance on the detection of vibrio alginolyticus on public health.
At present, the detection of vibrio alginolyticus still mainly depends on the traditional method, namely, the selective culture medium is firstly used for enrichment, and then biochemical and serological methods are combined for identification. The traditional method has accurate detection result, but has the defects of low detection efficiency, single detection target, low sensitivity, long time consumption, fussy operation and the like. In order to meet the requirement of rapid detection of pathogenic bacteria, the methods such as the VIDAS-enzyme linked fluorescence immunoassay (VIDAS-CHL), the Enzyme Immunoassay (EIA), the Polymerase Chain Reaction (PCR), the colloidal gold test strip method, the API biochemical identification test strip method, the loop-mediated isothermal amplification (LAMP) technology, the real-time fluorescence PCR and the like are developed. The VIDAS-CHL method, the EIA method, the colloidal gold test paper method and the API method have the defects of poor specificity, low sensitivity and the like, and are not widely applied; the LAMP method, as a new detection method, has high false positive rate although the detection efficiency is greatly improved and the detection cost is reduced. As a highly sensitive, rapid and simple detection method, the PCR technology is widely applied in a plurality of fields, especially, a revolutionary achievement is obtained in the aspect of pathogen detection, the PCR technology becomes a gold standard for rapid detection of nucleic acid, and on the basis, a DNA probe technology, a real-time fluorescence PCR technology, a PCR combined Denaturation High Performance Liquid Chromatography (DHPLC) technology and the like are developed, the design of a PCR primer is a key factor for restricting the success or failure of the detection method, the conventional PCR primer design not only needs to repeatedly compare the specificity of the primer, but also needs to optimize various parameters and reaction conditions of the primer, especially the annealing temperature, so as to prevent non-specific amplification, and the selected primer is easy to influence the effectiveness and specificity of the amplification under the restriction of the conditions. In addition, the fluorescent detection equipment is generally high in selling price, and the popularization and application of the detection method are limited to a certain extent.
Recombinase Polymerase Amplification (RPA) is a nucleic acid detection technique that can be used in place of PCR. RPA technology relies primarily on three enzymes: a recombinase capable of binding to single-stranded nucleic acids (oligonucleotide primers), a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase. RPA can be reacted at constant temperature, has high detection sensitivity, and can amplify trace amount of nucleic acid (especially DNA) template to detectable level to obtain about 10% from single template molecule12And (4) amplifying the product. Further, RPA is suitable for on-site detection in which nucleic acid cannot be extracted without complicated sample treatment. These features make RPAs increasingly popular.
As a relatively new amplification technique, RPA is much less mature than conventional PCR techniques. The key of the RPA analysis lies in the design of amplification primers and probes, and existing research shows that the influence of the RPA primers on the detection effect of the RPA is extremely important, and different RPA primers have extremely obvious difference aiming at the same species. The traditional PCR primer design method can not be applied to the design of RPA primer basically, because the RPA primer is longer than the general PCR primer, and usually needs to reach 30-38 bases. Too short primers can reduce the recombination rate and affect the amplification rate and detection sensitivity. In designing RPA primers, denaturation temperature is no longer a critical factor affecting amplification primers. The prior art also lacks a mature and reliable RPA primer design method, in particular an RPA primer design method with high amplification effect and good detection result.
An Internal Amplification Control (IAC) is an artificially constructed DNA sequence or a conserved gene sequence of a pathogenic bacterium added to a nucleic acid Amplification reaction system to indicate a false negative phenomenon. The main principle of the internal amplification standard is as follows: adding the amplification internal standard into a PCR reaction system to amplify together with the target gene, and if some inhibiting factors exist in the reaction system, inhibiting the amplification reactions of the amplification internal standard and the target gene so as to achieve the purpose of indicating false negative of the amplification reaction.
CN107227378A, CN107385057A, CN107227377A, CN106967816A and the like disclose different vibrio detection methods based on RPA-IAC, and the methods have better accuracy, but also further illustrate RPA, and particularly the RPA-IAC technology still needs to be further improved.
The development of an excellent-effect RPA-IAC technology of Vibrio alginolyticus is still a challenging task.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides an RPA-IAC primer, a kit, application and a method for detecting Vibrio alginolyticus.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
the RPA primer pair and the amplification internal standard combination for detecting the vibrio alginolyticus are combined, and the nucleotide sequence of the RPA primer pair is as follows:
collagenasecollagenase-F：5′-GCTTATCGAGTCGATGCGCCTGACAAGTGATC-3′；
collagenasecollagenase-R：5′-TCATAAGGGTCCCGTATGCGTACTTGCTAT-3′；
the 5 'end of the internal amplification standard is collagenancecholestagenase-F, the 3' end of the internal amplification standard is a reverse complementary sequence of collagenancecholestagenase-R, and the middle part of the internal amplification standard is at least a partial continuous sequence of a heterogeneous conserved gene.
In some examples of the combination of the RPA primer pair and the internal amplification standard, at least part of the continuous sequence of the heterogeneously conserved gene is 250-320 bp in length.
In some examples of combinations of RPA primer pairs and internal amplification standards, the nucleotide sequence of at least a portion of the contiguous sequence of the xenogeneic conserved gene is gttcgagaccttcaacaccccagccatgtacgtggccatccaggcggtgctgtccctgtacgcctctggccgcaccactggcatcgtgatggac tccggagacggggtcacccacacggtgcccatctacgaggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacct gaccgactacctcatgaagatcctgacggagcggggctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaagg.
In some examples of combinations of RPA primer pairs and internal amplification standards, the internal amplification standards are inserted within plasmids.
In a second aspect of the present invention, there is provided:
a kit for detecting Vibrio alginolyticus comprises an RPA primer pair and an amplification internal standard combination according to the first aspect of the invention.
In some examples of the kit, further comprising at least one of the following components:
1) RPA isothermal amplification reagent;
2) DNA extraction reagent;
3) a positive control and a negative control.
In a third aspect of the present invention, there is provided:
the RPA primer pair and the internal amplification label combination in the first aspect of the invention are applied to the preparation of vibrio alginolyticus detection reagents.
In some examples of applications, the vibrio alginolyticus detection reagent is used for detecting shellfish aquatic products.
In a fourth aspect of the present invention, there is provided:
a RPA-IAC method for detecting vibrio alginolyticus,
s1) obtaining DNA or RNA of a sample to be detected;
s2) carrying out RPA-IAC amplification by using the primer pair and the internal amplification standard combination of the first aspect of the invention;
s3) determining whether the Vibrio alginolyticus is present in the sample based on the RPA-IAC amplification result.
In some examples of the RPA-IAC method, the sample to be tested is a shellfish product.
In some examples of the RPA-IAC method, the system of the RPA-IAC amplification reaction is:
0.2mL twist Amp reaction tube containing freeze-dried enzyme powder
The reaction condition of the RPA-IAC amplification reaction is 37 ℃ for 40 min.
The invention has the beneficial effects that:
the RPA primer pair disclosed by the invention has good amplification efficiency and amplification specificity on vibrio alginolyticus, and can improve the sensitivity of RPA detection.
The kit of some embodiments of the invention has the advantages of high amplification efficiency, amplification specificity, high sensitivity and wide application range when being used for detecting the vibrio alginolyticus.
The kit of some embodiments of the invention does not need special thermal cycle equipment, has short reaction time, has no diffusion zone in the amplification product compared with LAMP, has a strip with a specific size in the RPA-IAC amplification product according to the design site of the primer, and is easy to judge the result.
The kit of some embodiments of the invention effectively avoids false negative results by adding amplification internal standards.
The RPA-IAC method of some embodiments of the invention has the advantages of high amplification efficiency, amplification specificity, high sensitivity and wide application range; the control requirement on the reaction condition is low, and the amplification time is short.
Drawings
FIG. 1 shows the result of the electrophoresis detection of the specificity test of the RPA-IAC primer pair in example 4 of the present invention. Wherein M is marker DL1000, 1: vibrio alginolyticus; 2: vibrio mimicus; 3: vibrio parahaemolyticus; 4: vibrio fluvialis; 5: vibrio vulnificus; 6: vibrio cholerae; 7: vibrio harveyi; 8: salmonella; 9: e.coli; 10: staphylococcus aureus bacteria; 11: listeria monocytogenes.
FIG. 2 shows the results of the electrophoretic detection of the sensitivity assay for RPA-IAC amplification in example 5 of the present invention. Wherein M is marker DL1000, 1-6: the template amount of Vibrio alginolyticus is 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng in sequence, and the amount of the internal amplification standard is 1.83 multiplied by 103copies。
FIG. 3 shows the results of the electrophoresis of the sensitivity test for RPA amplification in example 5 of the present invention. Wherein M is marker DL1000, 1-6: the amount of Vibrio alginolyticus template is 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng in sequence.
Detailed Description
Unless otherwise defined, all terms used herein have the same meaning as commonly understood in the art to which this invention belongs.
The present invention will now be described with reference to specific examples and figures, which are provided for illustrative purposes only and are not to be construed as limiting the invention. The specific techniques or conditions are not specified in the examples and are generally performed according to conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or according to the manufacturer's instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio fluvialis, Vibrio harveyi, Escherichia coli, Staphylococcus aureus, Salmonella, and Listeria monocytogenes used in the following examples are all provided by Microbiol research institute, Guangzhou.
Example 1
This example provides RPA primer pairs and amplification internal standard sequences and uses thereof.
The screening method of the RPA primer pair comprises the following steps: aiming at vibrio alginolyticus collagenase gene (GenBank ID: CP012739.1), a plurality of RPA primers are designed and screened in a large quantity, the specificity, the sensitivity, the functions between the primers and the suitability of the used primers and an RPA amplification kit are integrated, and the following RPA primer pairs which are good in specificity, good in repeatability and high in sensitivity and can be used for detecting the vibrio alginolyticus are screened finally:
collagenasecollagenase-F：5′-GCTTATCGAGTCGATGCGCCTGACAAGTGATC-3′(SEQ ID NO：1)；
collagenasecollagenase-R：5′-TCATAAGGGTCCCGTATGCGTACTTGCTAT-3′(SEQ ID NO：2)；
design and synthesis of amplification internal standard sequence: in order to avoid homologous interference of similar bacteria and individual nucleic acid pollution of detection personnel, by comprehensive analysis and consideration, on the basis of a partial nucleic acid sequence of a highly conserved porcine species specific gene beta-actin, two ends of the nucleic acid sequence are respectively connected with a primer sequence collagenancephalagenelagene-F and collagenancephalagenase-R to form an amplification internal standard sequence with the sequence size of 334bp as shown in the specification:
GCTTATCGAGTCGATGCGCCTGACAAGTGATCgttcgagaccttcaacaccccagccatgtacgtggccat ccaggcggtgctgtccctgtacgcctctggccgcaccactggcatcgtgatggactccggagacggggtcacccacacggtgcccatctacg aggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacctgaccgactacctcatgaagatcctgacggagcgggg ctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaaggATAGCAAGTACGCATACGGGACCCTTA TGA (Seq ID NO: 3). Seq ID NO: in 3, the upper case sequence is the original sequence of added collagenancephalagenelagene-F and the reverse complementary sequence of collagenancephalagenase-R, the lower case sequence 272bp is derived from a segment of sequence with Genbank accession number DQ452569.1 and title Sus scrofa beta Actin (ACTB) gene, 489-760 in partial cds, and homology verification by theory and practice is carried out.
The synthesis of the RPA-IAC primer pair and the amplification internal standard sequence is completed by Shanghai Biotechnology Co.
In application, the primer pair and the internal amplification standard sequence can be applied to the preparation of products for detecting or assisting in detecting the vibrio alginolyticus, for example, the internal amplification standard sequence is prepared into a plasmid containing the internal amplification standard sequence, and then the internal amplification standard sequence is combined with the primer pair or the like to form the products for detecting or assisting in detecting the vibrio alginolyticus, so that the products can be directly used for detecting or assisting in detecting whether the biological sample is infected with the vibrio alginolyticus.
Example 2
This embodiment provides a kit and its application, the kit includes:
(1) SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2 (both from example 1), and a polypeptide comprising the sequence as shown in SEQ ID NO: 3, amplifying internal standard sequence plasmid;
(2) DNA extraction reagent;
(3) a tube containing lyophilized enzyme powder;
(4) rehydrating the buffer;
(5) and (3) magnesium acetate solution.
The lyophilized enzyme-containing powder tube, Rehydration Buffer (Rehydration Buffer), and magnesium acetate solution (280mmol/L) were reagents from the RPA amplification KIT twist Basic KITs (twist DX, Cat. TABAS03 KIT).
The DNA extraction reagent is a reagent in an animal genome DNA extraction kit from Tiangen Biochemical technology Co., Ltd, the commodity number of DP432, and a reagent contained in a bacterial genome DNA extraction kit from Tiangen Biochemical technology Co., Ltd, the commodity number of DP 302.
The polypeptide containing the amino acid sequence shown in SEQ ID NO: 3, construction of a plasmid of an amplification internal standard sequence shown in the specification: the amplification internal standard sequence synthesized in example 1 was ligated to the universal vector PUC57 to obtain a nucleic acid sequence containing the sequence shown in SEQ ID NO: 3, and amplifying internal standard sequences.
The construction work of the amplification internal standard sequence plasmid is realized by Shanghai Biotechnology company Limited by adopting the conventional T4 bacteriophage DNA ligase to connect to the universal vector PUC57 and preparing the positive plasmid freeze-dried powder. According to the copy number of N copies/. mu.L ═ PCR fragment mass (g/. mu.L)/(650 g/mol. times.base number) × (6.02X 10)23) Calculating, diluting the positive plasmid dry powder to 1.83X 103copies/μL
Using SEQ ID NO: 1. SEQ ID NO: 2 and the above peptide having the sequence shown in SEQ ID NO: 3, carrying out RPA-IAC amplification on the plasmid of the amplification internal standard sequence shown in the step 3 aiming at the vibrio alginolyticus, wherein the sizes of the obtained RPA-IAC target gene amplification product and the amplification internal standard product are respectively 234bp and 334 bp.
Experiments prove that the DNA extraction reagent, the tube containing freeze-dried enzyme powder, the rehydration buffer solution and the magnesium acetate solution, and the DNA extraction reagent containing the DNA extraction reagent and the rehydration buffer solution are mixed with the DNA extraction reagent of SEQ ID NO: 1. SEQ ID NO: 2 and a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 3, the effect is more superior by the combined use of plasmids of the amplification internal standard sequence, which is particularly shown in strong specificity, good repeatability, high sensitivity and good quality control effect.
In application, the kit can be applied to detection or auxiliary detection of vibrio alginolyticus, for example, detection or auxiliary detection of whether a biological sample is infected with vibrio alginolyticus or detection or auxiliary detection of whether a biological sample to be detected is or is candidate for vibrio alginolyticus; it can also be used for preparing products for detecting or assisting in detecting Vibrio alginolyticus, such as products for detecting or assisting in detecting whether a biological sample is infected with Vibrio alginolyticus, or products for detecting or assisting in detecting whether a pathogenic bacterium is or is selected as Vibrio alginolyticus.
Example 3
This example provides a method for detecting or aiding in the detection of Vibrio alginolyticus, such as detecting or aiding in the detection of whether a biological sample is infected with Vibrio alginolyticus, or detecting or aiding in the detection of whether a pathogen is or is a candidate for Vibrio alginolyticus, using the primer pair of example 1 or the kit of example 2.
The method comprises the following steps:
the method comprises the following steps: RPA-IAC amplification
Adding the DNA of a biological sample or pathogenic bacteria as a template into the DNA containing the nucleotide sequence shown in SEQ ID NO: 3, adopting the plasmid with an amplification internal standard sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain an RPA-IAC amplification product, and setting a blank control (the template is ultrapure water).
The preparation method of the RPA-IAC amplification system comprises the following steps: to a 0.2mL twist Amp reaction tube containing lyophilized enzyme powder, 29.5. mu.L of Rehydration Buffer (Rehydration Buffer), 2. mu.L each of the upstream and downstream primers (collagenancephalagenase-F and collagenancephalagenase-R of example 1) (final concentration of each primer is 0.4. mu. mol/L), containing the nucleotide sequence as set forth in SEQ ID NO: 3 (1.83) as a template103copies), template DNA1ul (50ng), and finally 2.5. mu.L of magnesium acetate solution (280mmol/L) was added, made up to 50. mu.L with deionized water.
RPA-IAC amplification reaction conditions: and (3) fully and uniformly mixing the RPA-IAC amplification system, and placing the mixture on a metal bath at 37 ℃ for reaction for 40min to obtain an RPA-IAC amplification product.
Step two: electrophoretic detection of RPA-IAC amplification products
After the RPA-IAC reaction is finished, adding 50 mu L of phenol/chloroform (1:1) solution into the amplification product, fully mixing uniformly, centrifuging at 12000rpm for 2min, taking 5 mu L of supernatant to perform electrophoresis on 1.5% agarose gel, observing the result on a gel imaging system, and sequencing the RPA-IAC amplification product (sequencing verification is performed in the establishment process, and the description is omitted).
The standard for judging the result of RPA-IAC amplification is as follows:
if the amplification product obtained by RPA-IAC amplification only has a target gene segment with the size of 226bp, or simultaneously has an internal standard amplification segment and a target gene segment with the sizes of 334bp and 226bp respectively, then the amplification product is judged to contain vibrio alginolyticus, which prompts the biological sample to infect the vibrio alginolyticus, or the pathogenic bacteria is or is a candidate of the vibrio alginolyticus; if the amplification product obtained by RPA-IAC amplification only has an internal standard amplification fragment with the size of 334bp and does not contain a target gene fragment with the size of 226bp, the biological sample is judged to contain no vibrio alginolyticus, and the biological sample is prompted to be not infected with the vibrio alginolyticus, or pathogenic bacteria are not or are not candidate to be vibrio alginolyticus; and if the amplification product obtained by RPA-IAC amplification does not contain the internal standard amplification fragment with the size of 334bp and does not contain the target gene fragment with the size of 226bp, judging the reaction to be false negative, and prompting that the detection needs to be carried out again.
If the plant to be detected or the pathogenic bacteria to be detected has not extracted DNA, the method step (1) of RPA-IAC amplification may further comprise a step of extracting DNA from the biological sample or the pathogenic bacteria to be detected by using an animal genome DNA extraction kit (purchased from Tiangen Biochemical technology Co., Ltd., product number DP432) or a bacterial genome DNA extraction kit (purchased from Tiangen Biochemical technology Co., Ltd., product number DP302) according to the kit instructions.
Example 4
This example verifies the validity and specificity of the primer pair and the internal amplification standard sequence of example 1, and the used test materials are as follows: vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio fluvialis, Vibrio harveyi, Escherichia coli, Staphylococcus aureus, Salmonella and Listeria monocytogenes, which are standard strains purchased and strictly verified, are all stored in the inspection and quarantine technical center of Shantou entry and exit inspection and quarantine bureau.
The method comprises the following steps: extraction of genomic DNA
The genome DNA of Vibrio alginolyticus, Vibrio mimicus, Vibrio fluvialis, Vibrio harveyi, Escherichia coli, Staphylococcus aureus, Salmonella and Listeria monocytogenes are respectively extracted by using a bacterial genome DNA extraction kit with the product number DP302 of Tiangen Biochemical technology (Beijing) Co.
Step two: RPA-IAC amplification
Various genomic DNA templates were used:
Using groups 1 to 11 as templates, RPA-IAC amplification was performed in the same manner as in step one of example 3.
Step three: electrophoretic detection of RPA-IAC amplification products
The method for electrophoretic detection of the RPA-IAC amplification product is the same as the second step of example 3.
And (4) analyzing results:
as shown in FIG. 1, the results of the analysis are shown in groups 1 to 11, which correspond to lanes 1 to 11, respectively, lane 1 shows that there are amplified bands at 334bp and 226bp, and the judgment standard of example 3 shows that Vibrio alginolyticus is positive, i.e., Vibrio alginolyticus is successfully detected, and the other lanes 2 to 11 show that there are amplified bands only at 334bp, so that the judgment standard of example 3 eliminates the possibility of false negative, and further improves the reliability of the results. In conclusion, the positive judgment result and the negative judgment result are accurate, and all groups of amplification internal standards are successfully amplified, so that the primer pair and the amplification internal standard sequence in the embodiment 1 have effectiveness, high specificity and good indication property; furthermore, the method for detecting or assisting in detecting the vibrio alginolyticus, which is established based on the primer pair, the amplification internal standard sequence and the kit, can accurately detect the vibrio alginolyticus.
Example 5
This example performed sensitivity verification of the primer set and internal amplification standard sequence of example 1, and compared with the case where NO internal amplification standard was added (i.e., the case where the plasmid containing the internal amplification standard sequence shown in SEQ ID NO: 3 in example 2 was not added), the following test materials were used: vibrio alginolyticus, which is a standard strain purchased and strictly verified, is stored in the inspection and quarantine technical center of Shantou entry and exit inspection and quarantine bureau.
The method comprises the following steps: extraction of genomic DNA
Respectively extracting the genomic DNA of the vibrio alginolyticus by adopting a bacterial genomic DNA extraction kit with the product number DP302 of Tiangen Biochemical technology (Beijing) Limited company according to a kit specification, and performing 10-fold gradient dilution on the obtained genomic DNA to prepare the genomic DNA of the vibrio alginolyticus with different concentrations.
Step two: RPA amplification and RPA-IAC amplification
The genomic DNA of Vibrio alginolyticus at different concentrations was used as a template:
group 1: taking 100 ng/. mu.L of vibrio alginolyticus genome DNA (1. mu.L) as a template;
group 2: taking 10 ng/. mu.L of vibrio alginolyticus genome DNA (1. mu.L) as a template;
group 3: taking 1 ng/muL vibrio alginolyticus genome DNA (1 muL) as a template;
group 4: taking 0.1 ng/. mu.L of vibrio alginolyticus genome DNA (1. mu.L) as a template;
group 5: taking 0.01 ng/. mu.L of vibrio alginolyticus genome DNA (1. mu.L) as a template;
group 6: 0.001 ng/. mu.L of Vibrio alginolyticus genomic DNA (1. mu.L) was used as a template.
Respectively carrying out RPA amplification and RPA-IAC amplification by taking the groups 1 to 6 as templates, wherein the RPA-IAC amplification method is the same as the first step of the example 3, and the difference between the RPA amplification and the RPA-IAC amplification is that the amplification system does not contain the nucleotide sequence shown in SEQ ID NO: 3, and amplifying the internal standard sequence.
Step three: electrophoretic detection of RPA amplification product and RPA-IAC amplification product
The electrophoretic detection method of the amplification products is the same as the second step of example 3.
And (4) analyzing results:
the results of RPA-IAC amplification are shown in FIG. 2, where groups 1-4 all have a band at 226bp and groups 2-6 have a band at 334bp, which indicates that the results of groups 1-6 are all valid and groups 5-6 have no possibility of false negative, and also indicates that the primer pair and the internal amplification standard sequence of example 1 of the present invention have high sensitivity, and can detect that the sample only contains 0.1ng of trace DNA; the results of RPA amplification are shown in FIG. 3, with sensitivity results similar to that of RPA-IAC amplification. In conclusion, it is clear that the addition of the internal amplification standard does not reduce the sensitivity of the detection.
Comparative example 1
In fact, before seeking the primer pairs of the present invention, the plasmid (from example 2) containing the internal amplification standard sequence of the present invention was tried to be combined with a very large number of primer pairs, and only some of them were extracted and described in this comparative example, and the rest of them are not described in detail, specifically:
50 shellfish aquatic products were detected by the method for detecting or assisting in detecting Vibrio alginolyticus established in example 3 using the Primer pair of example 1 and Primer Premier 5.0 software design and selection of the Primer pair with the front row, and the separation and systematic biochemical identification of Vibrio alginolyticus were performed on 50 seafood with reference to national food hygiene microbiological inspection standard (standard number: GB4789.7-2013), the sampling and template concentration gradient settings of the sensitivity test were performed as in example 5, and the detection results are shown in Table 1.
TABLE 1
Note: the detection rate was obtained by repeating the detection three times, and the sensitivity was measured in accordance with the detection amount of example 5.
The results show that: in view of the fact that the detection result of the primer pair of the invention is completely consistent with the detection results of the reference standard and the disclosed detection method under the condition that the plasmid containing the amplification internal standard sequence exists, the primer pair of the invention has strong combination specificity, high sensitivity and good repeatability, and the combination of a plurality of primers randomly selected by adopting primer design software has various defects of weak specificity, weak sensitivity, poor repeatability, interference with the amplification internal standard and the like more or less.
Comparative example 2
This comparative example provides a comparison of the results of the detection of Vibrio alginolyticus using different reagents in combination with the primer set of the present invention (from example 1) and the plasmid containing an internal amplification standard sequence (from example 1).
Various reagents were combined with the primer pairs of the invention and plasmids containing the amplification internal standard sequences:
combination 1: the invention relates to a primer pair, a plasmid containing an amplification internal standard sequence, a commercially available animal genome DNA extraction kit 1 and a commercially available RPA amplification kit 1
And (3) combination 2: the invention relates to a primer pair, a plasmid containing an amplification internal standard sequence, a certain commercially available animal genome DNA extraction kit 2 and a certain commercially available RPA amplification kit 2
And (3) combination: the invention relates to a primer pair, a plasmid containing an amplification internal standard sequence, a certain commercially available animal genome DNA extraction kit 3 and a certain commercially available RPA amplification kit 3
The combination of the invention: the primer pair and the plasmid containing the amplification internal standard sequence of the invention + the reagent contained in the animal genome DNA extraction KIT with the product number DP432 from Tiangen Biochemical technology Limited company + the reagent contained in the RPA amplification KIT with the product number TABAS03KIT from TwistDX company are provided.
When the combination of the invention is used for detecting Vibrio alginolyticus, the method is performed by the method in example 3. The sampling and template concentration gradient settings for the sensitivity assay were performed as in example 5.
When the combination 1, the combination 2 and the combination 3 are used for detecting the vibrio alginolyticus, the commercial kits are operated according to the kit instructions, and other conditions are the same as the combination. And tested by a standard published comparative method (see comparative example 1).
The tested sample is the same as 50 shellfish aquatic products in comparative example 1.
The results are shown in Table 2.
TABLE 2
Note: the detection rate was obtained by repeating the detection three times, and the sensitivity was measured in accordance with the detection amount of example 5.
The results show that: the detection results of the combination of the primer pair, the plasmid containing the amplification internal standard sequence and each specific reagent are completely consistent with the detection results of European Union standards and the disclosed detection methods, which shows that the kit formed by the combination of the primer pair, the amplification internal standard sequence and each specific reagent has the advantages of strong specificity, high sensitivity and good repeatability, and under the condition that the amplification internal standard sequence exists, the primer pair and the combination of other randomly selected reagents have various defects of weak specificity, weak sensitivity and poor repeatability to a greater or lesser extent.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
SEQUENCE LISTING
<110> Guangzhou microbial research institute Co., Ltd
Guangdong Huawei Testing Co., Ltd.
<120> RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 32
<212> DNA
<213> Artificial sequence
<400> 1
gcttatcgag tcgatgcgcc tgacaagtga tc 32
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
tcataagggt cccgtatgcg tacttgctat 30
<210> 3
<211> 334
<212> DNA
<213> Artificial sequence
<400> 3
gcttatcgag tcgatgcgcc tgacaagtga tcgttcgaga ccttcaacac cccagccatg 60
tacgtggcca tccaggcggt gctgtccctg tacgcctctg gccgcaccac tggcatcgtg 120
atggactccg gagacggggt cacccacacg gtgcccatct acgaggggta cgccctgccc 180
cacgccatcc tgcgtctgga cctggctggc cgggacctga ccgactacct catgaagatc 240
ctgacggagc ggggctacag cttcaccacc acggccgagc gggagatcgt gcgggacatc 300
aaggatagca agtacgcata cgggaccctt atga 334
Claims (10)
1. The combination of the RPA primer pair and the amplification internal standard for detecting the vibrio alginolyticus is characterized in that: the nucleotide sequence of the RPA primer pair is as follows:
collagenasecollagenase-F：5′- GCTTATCGAGTCGATGCGCCTGACAAGTGATC -3′；
collagenasecollagenase-R：5′- TCATAAGGGTCCCGTATGCGTACTTGCTAT -3′；
the 5 'end of the internal amplification standard is collagenancecholestagenase-F, the 3' end of the internal amplification standard is a reverse complementary sequence of collagenancecholestagenase-R, and the middle part of the internal amplification standard is at least a partial continuous sequence of a heterogeneous conserved gene.
2. The RPA primer pair and internal amplification standard combination according to claim 1, wherein: at least part of continuous sequences of the heterogenous conserved genes are 250-320 bp in length.
3. The RPA primer pair and internal amplification standard combination of claim 2, wherein: the nucleotide sequence of at least part of the continuous sequence of the heterogeneously conserved gene is gttcgagaccttcaacaccccagccatgtacgtggccatccaggcggtgctgtccctgtacgcctctggccgcaccactggcatcgtgatggactccggagacggggtcacccacacggtgcccatctacgaggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacctgaccgactacctcatgaagatcctgacggagcggggctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaagg.
4. The combination of the RPA primer pair and the internal amplification standard according to any one of claims 1-3, wherein: the internal amplification standard is inserted into a plasmid.
5. A kit for detecting vibrio alginolyticus is characterized in that: comprising the combination of the RPA primer pair of any one of claims 1-4 and an internal amplification standard.
6. The kit of claim 5, wherein: further comprises at least one of the following components:
RPA isothermal amplification reagent;
DNA extraction reagent;
a positive control and a negative control.
7. The use of the combination of the primer set of any one of claims 1 to 4 and an internal amplification standard in the preparation of a Vibrio alginolyticus detection reagent.
8. A RPA-IAC method for detecting vibrio alginolyticus is characterized in that:
s1) obtaining DNA or RNA of a sample to be detected;
s2) carrying out RPA-IAC amplification by using the combination of the primer pair and the internal amplification standard according to any one of claims 1-4;
s3) determining whether the Vibrio alginolyticus is present in the sample based on the RPA-IAC amplification result.
9. The RPA-IAC method according to claim 8, characterized in that: the sample to be detected is a shellfish aquatic product.
10. The RPA-IAC method according to claim 8 or 9, characterized in that: the system of the RPA-IAC amplification reaction is as follows:
0.2mL twist Amp reaction tube containing freeze-dried enzyme powder
Rehydration buffer 29.5. mu.L
The final concentration of the RPA primer pair is 0.4 mu mol/L
Plasmid 1.83X 10 containing internal amplification control3 copies
Template DNA 50ng
Magnesium acetate solution 2.5 μ L with concentration of 280mmol/L
Supplementing deionized water to 50 mu L; and/or
The reaction condition of the RPA-IAC amplification reaction is 37 ℃ for 40 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227377A (en) * | 2017-08-10 | 2017-10-03 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of Vibrio vulnificus |
| CN107254556A (en) * | 2017-08-10 | 2017-10-17 | 中国检验检疫科学研究院 | Method, RPA IAC primers and kit based on RPA IAC technology examination bean mosaic virus 4s |
| CN107385057A (en) * | 2017-08-10 | 2017-11-24 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of comma bacillus |
| CN111139307A (en) * | 2019-12-18 | 2020-05-12 | 江苏海洋大学 | Specific primer pair for rapidly detecting vibrio alginolyticus by RPA-LFS, kit and application thereof |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227377A (en) * | 2017-08-10 | 2017-10-03 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of Vibrio vulnificus |
| CN107254556A (en) * | 2017-08-10 | 2017-10-17 | 中国检验检疫科学研究院 | Method, RPA IAC primers and kit based on RPA IAC technology examination bean mosaic virus 4s |
| CN107385057A (en) * | 2017-08-10 | 2017-11-24 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of comma bacillus |
| CN111139307A (en) * | 2019-12-18 | 2020-05-12 | 江苏海洋大学 | Specific primer pair for rapidly detecting vibrio alginolyticus by RPA-LFS, kit and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| DONG ET AL.: "Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method", 《BMC VETERINARY RESEARCH》 * |
| GENG ET AL.: "Development and evaluation of a rapid and sensitive RPA assay for specific detection of Vibrio parahaemolyticus in seafood", 《BMC MICROBIOLOGY》 * |
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