CN107227378B - RPA-IAC primer and method for detecting vibrio mimicus - Google Patents
RPA-IAC primer and method for detecting vibrio mimicus Download PDFInfo
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Abstract
The invention provides a primer pair and an internal standard amplification sequence, wherein the primer pair comprises SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2, wherein the internal standard amplification sequence comprises a nucleotide sequence shown in SEQ ID NO: 3. The invention also provides the application of the primer pair and the internal standard amplification sequence in the preparation of products for detecting or assisting in detecting the vibrio mimicus; and a method for detecting the vibrio mimicus based on the RPA-IAC technology. Experiments prove that the RPA-IAC primer has good specificity, high sensitivity, short detection time, no need of special instruments and wide application range, and the RPA-IAC detection method of vibrio mimicus established based on the primer and matched with an amplification internal standard is sensitive, accurate, simple, convenient and quick, and has guiding significance for quarantine of import and export related goods and products.
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 mimicus.
Background
The vibrio mimicus is a gram-negative vibrio, has single flagella and power, belongs to a normal marine flora like vibrio parahaemolyticus and vibrio cholerae, and is widely present in natural river water, seawater and marine products. The strains grew well in nutrient broth containing no sodium or only 1% sodium chloride and could be isolated from aquatic animals.
Mimotorhinovirus pathogenic reports are reported in the United states, North America, New Zealand, Bengal and African countries, diseases are also reported in Fujian, Beijing, Shanghai, Jiangsu and Zhejiang areas of China, and are mostly sporadic or fulminant, diseases are found all the year round, and summer is mostly used; the incubation period is 3-72 h, and the average time is 24 h; the main symptoms comprise diarrhea, vomit, abdominal pain, watery stool or mucous bloody stool; the age of onset is not limited, but is rare in children. Therefore, the method has important significance for detecting the vibrio mimicus in public health.
At present, the detection of vibrio mimicus still mainly depends on the traditional method, namely, bacteria are enriched by using a selective culture medium at first, and then the identification is carried out by combining a biochemical method and a serological method. 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, enzyme linked fluorescence immunoassay (VIDAS-CHL), Enzyme Immunoassay (EIA), Polymerase Chain Reaction (PCR), colloidal gold test strip method, API biochemical identification test strip method, loop-mediated isothermal amplification (LAMP) technology, real-time fluorescence PCR and other methods 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.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for sensitively, accurately, simply, conveniently and quickly detecting vibrio mimicus based on an RPA-IAC technology, and also provides an RPA-IAC primer, an internal standard amplification sequence and a corresponding detection kit which are used for the method, have good specificity, high sensitivity, short detection time, no need of special instruments and wide application range.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a primer pair comprising SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2, wherein the internal standard amplification fragment comprises a nucleotide sequence shown in SEQ ID NO: 3.
The second aspect of the invention provides the application of the primer pair and the internal standard amplification sequence in the preparation of products for detecting or assisting in detecting the vibrio mimicus;
in a preferred embodiment, the product for preparing the vibrio mimicus detection or auxiliary detection product comprises: preparing a product for detecting or assisting in detecting whether the biological sample is infected with the vibrio mimicus, and preparing a product for detecting or assisting in detecting whether the pathogenic bacteria are or are candidates for the vibrio mimicus.
In a third aspect, the invention provides a kit comprising the primer pair and a plasmid containing the internal standard amplification sequence.
In a preferred embodiment, the kit further comprises an RPA isothermal amplification reagent.
In a preferred example, the RPA isothermal amplification reagent comprises reagents contained in a TwistAmp Basic kit of an RPA amplification kit from TwistDX.
In a preferred embodiment, the kit further comprises a DNA extraction reagent.
In a preferred embodiment, the DNA extraction reagent comprises a reagent contained in an animal genome DNA extraction kit with the product number DP432 from Tiangen Biochemical technology, Inc.
In a preferred embodiment, the DNA extraction reagent further comprises a reagent contained in a bacterial genomic DNA extraction kit with the product number DP302 from Tiangen Biochemical technology, Inc.
In a preferred embodiment, a positive control and a negative control are also included.
The fourth aspect of the invention provides the application of the kit in detecting or assisting in detecting vibrio mimicus or preparing products for detecting or assisting in detecting vibrio mimicus.
In a preferred embodiment, the detecting or auxiliary detecting vibrio mimicus comprises: detecting or assisting in detecting whether the biological sample is infected with vibrio mimicus, and detecting or assisting in detecting whether the pathogenic bacteria is or is a candidate for vibrio mimicus.
In a preferred embodiment, the product for preparing the vibrio mimicus detection or auxiliary detection product comprises: preparing a product for detecting or assisting in detecting whether the biological sample is infected with the vibrio mimicus, and preparing a product for detecting or assisting in detecting whether the pathogenic bacteria are or are candidates for the vibrio mimicus.
The fifth aspect of the invention provides an RPA-IAC method for detecting or assisting in detecting vibrio mimicus, which comprises the step of using the primer pair and a plasmid containing the internal standard amplification sequence or the kit.
In a preferred embodiment, the detecting or auxiliary detecting vibrio mimicus comprises: detecting or assisting in detecting whether the biological sample is infected with vibrio mimicus, and detecting or assisting in detecting whether the pathogenic bacteria is or is a candidate for vibrio mimicus.
In a preferred embodiment, the method comprises the following steps:
1) RPA-IAC amplification
And (3) carrying out RPA-IAC amplification by using DNA contained in a biological sample or pathogenic bacteria as a template, the primer pair and the plasmid containing the internal standard amplification sequence or the kit.
2) And judging whether the biological sample is infected with the vibrio mimicus or not or whether the pathogenic bacteria are or are candidate to be the vibrio mimicus according to the RPA-IAC amplification result.
Optionally, step 1) is preceded by a step of extracting DNA from the biological sample or the pathogenic bacteria.
In a preferred embodiment, the extraction of DNA from the biological sample or the pathogenic bacteria is performed by using an animal genome DNA extraction kit of Tiangen Biochemical technology Co., Ltd, the product number of DP432, or a bacterial genome DNA extraction kit of Tiangen Biochemical technology Co., Ltd, the product number of DP 302.
In a preferred embodiment, the criteria for determining the result of RPA-IAC amplification are:
if the amplification product obtained by the RPA-IAC amplification only has 467bp target gene segment, or has 334bp, 467bp internal standard amplification segment and target gene segment, then the biological sample is judged to be infected by vibrio mimicus, or the pathogenic bacteria is or is a candidate of vibrio mimicus; if the amplification product obtained by RPA amplification only contains an internal standard amplification fragment with the size of 334bp and does not contain a target gene fragment with the size of 467bp, the biological sample is judged not to be infected with vibrio mimicus, or the pathogenic bacteria is not or is not candidate to be vibrio mimicus; if the amplification product obtained by RPA 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 467bp, the reaction is judged to be false negative, and the detection is required to be carried out again.
In a preferred embodiment, the reaction system for amplifying the RPA-IAC is as follows:
0.2mL twist Amp reaction tube containing freeze-dried enzyme powder
Rehydration buffer 29.5. mu.L
SEQ ID NO: 1 and SEQ ID NO: 2. mu.L of each primer shown in 2, the final concentration of each primer was 0.4. mu. mol/L
Comprises SEQ ID NO: 3 plasmid 1.83X 10 of internal standard amplification sequence3copies
Template DNA 50ng
Magnesium acetate solution 2.5 μ L with concentration of 280mmol/L
Supplementing deionized water to 50 mu L;
in a preferred embodiment, the reaction conditions for the amplification of the RPA-IAC are as follows: the temperature of the RPA-IAC amplification is 37 ℃, and the time is 40 min.
The sixth aspect of the present invention provides a system for screening Vibrio mimicus, comprising:
a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample or pathogenic bacteria;
the RPA-IAC amplification device is connected with the nucleic acid extraction device and is suitable for carrying out RPA-IAC amplification on the nucleic acid sample by the primer pair or the kit;
and the judging device is connected with the RPA-IAC amplification device so as to judge whether the biological sample is infected with the vibrio mimicus or whether the pathogenic bacteria are or are candidates for the vibrio mimicus based on the result of the RPA-IAC amplification.
In a preferred embodiment, the reaction system for amplifying the RPA-IAC is as follows:
0.2mL twist Amp reaction tube containing freeze-dried enzyme powder
Rehydration buffer 29.5. mu.L
SEQ ID NO: 1 and SEQ ID NO: 2. mu.L of each primer shown in 2, the final concentration of each primer was 0.4. mu. mol/L
Comprises SEQ ID NO: 3 plasmid 1.83X 10 of internal standard amplification sequence3copies
Template DNA 50ng
Magnesium acetate solution 2.5 μ L with concentration of 280mmol/L
Supplementing deionized water to 50 mu L;
optionally, the reaction conditions for the RPA-IAC amplification are as follows: the temperature of the RPA-IAC amplification is 37 ℃, and the time is 40 min.
The biological sample of the present invention is blood, cells, tissues, etc., or food in which blood, cells, tissues, etc. are mixed, etc.; the pathogenic bacteria is a pure strain or a mixture of at least two strains.
The internal standard amplification fragment is a section of artificially constructed DNA sequence or a section of conservative gene sequence of pathogenic bacteria which is added into a PCR reaction system to indicate 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 the false negative of the PCR reaction.
The beneficial effects of the invention include:
(1) the invention designs a specific RPA-IAC primer aiming at the vibrio mimicus, establishes a vibrio mimicus RPA-IAC detection method, and can carry out qualitative detection on the vibrio mimicus.
(2) The amplification internal standard sequence is not homologous with the vibrio mimicus genome and the human genome, so that the false negative detection and the individual nucleic acid interference detection of operators can be effectively avoided while the amplification efficiency of the target sequence is ensured.
(3) The invention only designs a pair of primers aiming at the vibrio mimicus to complete the amplification, thereby saving the complicated primer design process; the primer amplification of the invention only needs constant temperature reaction at 37 ℃, and does not need special thermal cycle equipment; the reaction time is only 40min, and the detection time is short; unlike the diffuse band of LAMP product, the RPA-IAC amplification product has a band with a specific size according to the designed site of the primer, and the result is easy to judge.
(4) The RPA-IAC amplification primer has good specificity, high sensitivity and wide application range.
(5) The method for detecting the vibrio mimicus RPA-IAC established by the invention is sensitive, accurate, simple, convenient and quick, and has guiding significance for inspection and quarantine of goods and products related to import and export.
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 mimicus; 2: vibrio vulnificus; 3: vibrio parahaemolyticus; 4: vibrio fluvialis; 5: vibrio alginolyticus; 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 amounts of the vibrio mimicus templates are 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng in sequence, and the amounts of the amplification internal standards are 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 amounts of the vibrio mimicus template are 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng in sequence.
Detailed Description
Unless otherwise defined, the terms used herein have the ordinary meanings 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 alginolyticus, Vibrio vulnificus, Vibrio mimicus, Vibrio fluvialis, Vibrio harveyi, Escherichia coli, Staphylococcus aureus, Salmonella, and Listeria monocytogenes used in the following examples are provided by the Shantou export-import inspection and quarantine office.
Example 1
This example provides RPA primer pairs and internal standard amplification sequences and uses thereof.
The screening method of the RPA primer pair comprises the following steps: aiming at the vibrio mimicus VMH gene (GenBank ID: CP016384.1), a plurality of RPA primers are designed and screened in large quantity, the specificity, the sensitivity, the functions between the primers and the suitability of each primer and an RPA amplification kit are synthesized, and the following RPA primer pairs which have good specificity, good repeatability and high sensitivity and can detect the vibrio mimicus are screened finally:
VMH-F：5′-GAGATATTGGCATCTTTACAAAATGGACGA-3′(SEQ ID NO：1)；
VMH-R：5′-GATTGGCAATCAGAAGAAAGCCAATTACCCAG-3′(SEQ ID NO：2)；
design and synthesis of internal standard amplification sequence: in order to avoid homologous interference of similar bacteria and individual nucleic acid pollution of detection personnel, through comprehensive analysis consideration and repeated experimental verification, 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 VMH-F and a primer sequence VMH-R to form an internal standard amplification sequence with the sequence size of 334bp as shown in the specification:
GAGATATTGGCATCTTTACAAAATGGACGAgttcgagaccttcaacaccccagccatgtacgtggccatccaggcggtgctgtccctgtacgcctctggccgcaccactggcatcgtgatggactccggagacggggtcacccacacggtgcccatctacgaggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacctgaccgactacctcatgaagatcctgacggagcggggctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaaggCTGGGTAATTGGCTTTCTTCTGATTGCCAATC(Seq ID NO：3)
the above Seq ID NO: 3, the upper case sequence is the original sequence of the added VMH-F and the reverse complement sequence of the VMH-R, the lower case sequence 272bp is derived from a section of sequence with Genbank accession number DQ452569.1 and the title of Sus scrofa beta Act (ACTB) gene, 489-.
The synthesis of the RPA-IAC primer pair and the internal standard amplification sequence is completed by Shanghai Biotechnology Limited company.
In application, the primer pair and the internal standard amplification sequence can be applied to the preparation of products for detecting or assisting in detecting the vibrio mimicus, for example, the internal standard amplification sequence is prepared into a plasmid containing the internal standard amplification sequence, and then the internal standard amplification sequence is combined with the primer pair or other products for detecting or assisting in detecting the vibrio mimicus to directly detect or assist in detecting whether a biological sample is infected with the vibrio mimicus.
Example 2
This embodiment provides a kit and its application, the kit includes:
(1) SEQ ID NO: 1. SEQ ID NO: 2 (from example 1), and a polypeptide comprising the sequence set forth in SEQ ID NO: 3, a plasmid of an internal standard amplification sequence;
(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 plasmids of internal standard amplification sequences: the internal amplification sequence synthesized in example 1 was ligated to the universal vector PUC57 to yield a nucleic acid sequence containing the sequence shown in SEQ ID NO: 3, and (3) a positive plasmid of an internal standard amplification sequence.
Construction of the above internal standard amplification sequence plasmid: the plasmid was ligated to the universal vector PUC57 using conventional T4 phage DNA ligase and prepared as a positive plasmid lyophilized powder. This construction is carried out by Shanghai Biotechnology Co., Ltd. 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 internal standard amplification sequence aiming at the vibrio mimicus to obtain the amplified products of the RPA-IAC target genes and the amplified internal standard products with the sizes of 467bp and 334bp respectively.
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 the plasmids of the internal standard amplification 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 mimicus, for example, detection or auxiliary detection of whether a biological sample is infected with vibrio mimicus or detection or auxiliary detection of whether a biological sample to be detected is or is candidate for vibrio mimicus; the method can also be used for preparing products for detecting or assisting in detecting the vibrio mimicus, for example, products for detecting or assisting in detecting whether a biological sample is infected with the vibrio mimicus or products for detecting or assisting in detecting pathogenic bacteria as or for being selected as the vibrio mimicus.
Example 3
This example provides a method for detecting or aiding in the detection of Vibrio mimicus, such as detecting or aiding in the detection of whether a biological sample is infected with Vibrio mimicus, or detecting or aiding in the detection of whether a pathogenic bacterium is or is a candidate for Vibrio mimicus, 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 of an internal standard amplification 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 was added 29.5. mu.L of Rehydration Buffer (Rehydration Buffer), 2. mu.L each of the upstream and downstream primers (VMH-F and VMH-R of example 1) (final concentration of each primer was 0.4. mu. mol/L), containing the sequence as set forth in SEQ ID NO: 3 plasmid 1. mu.L (1.83X 10) of internal amplification sequence shown in3copies), template DNA1ul (50ng), and finally 2.5. mu.L of magnesium acetate solution (280mmol/L) was added and 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 467bp target gene segment, or has 334bp, 467bp internal standard amplification segment and target gene segment, then determining that the biological sample contains Vibrio mimicus, prompting the biological sample to infect Vibrio mimicus, or the pathogenic bacteria is or is candidate as Vibrio mimicus; if the amplification product obtained by the 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 467bp, the biological sample is judged to contain no vibrio mimicus, and the biological sample is prompted to be not infected with the vibrio mimicus or pathogenic bacteria are not or are not candidate to be the vibrio mimicus; 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 467bp, 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 standard amplification sequence of example 1, and the used test materials are as follows: the vibrio parahaemolyticus, vibrio cholerae, vibrio alginolyticus, vibrio vulnificus, vibrio mimicus, vibrio fluvialis, vibrio harveyi, escherichia coli, staphylococcus aureus, salmonella and listeria monocytogenes, wherein the strains are standard strains which are purchased and strictly verified and 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 vulnificus, Vibrio mimicus, Vibrio fluvialis, Vibrio harveyi, Escherichia coli, Staphylococcus aureus, Salmonella and Listeria monocytogenes is 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 1-11, which correspond to lanes 1-11, respectively, lane 1 shows that there are amplified bands at 334bp and 467bp, and the Vibrio mimicus is successfully detected if it is positive according to the determination criteria of example 3, and the other lanes 2-11 show that there are amplified bands only at 334bp, which eliminates the possibility of false negative according to the determination criteria of example 3, 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 internal standard amplification sequence in the embodiment 1 have effectiveness, high specificity and good indication property; furthermore, the method for detecting or assisting in detecting the vibrio mimicus, which is established based on the primer pair, the internal standard amplification sequence and the kit, can accurately detect the vibrio mimicus.
Example 5
This example demonstrates the sensitivity of the primer pair and internal standard amplification sequence of example 1 and compares it with the absence of the internal amplification standard (i.e., the absence of the plasmid containing the internal standard amplification sequence shown in SEQ ID NO: 3 in example 2) using the following test materials: the vibrio mimicus is a standard strain which is purchased and strictly verified, and is stored in the Shantou entry and exit inspection and quarantine technical center.
The method comprises the following steps: extraction of genomic DNA
The method comprises the steps of respectively extracting the genomic DNA of vibrio mimicus by adopting a bacterial genomic DNA extraction kit with the product number DP302 of Tiangen Biochemical technology (Beijing) Co., Ltd according to the kit specification, and performing 10-fold gradient dilution on the obtained genomic DNA to prepare the genomic DNA of vibrio mimicus with different concentrations.
Step two: RPA amplification and RPA-IAC amplification
The genomic DNA of Vibrio mimicus at different concentrations was used as a template:
group 1: 100 ng/. mu.L of Vibrio mimicus genomic DNA (1. mu.L) was used as a template.
Group 2: 10 ng/. mu.L of Vibrio mimicus genomic DNA (1. mu.L) was used as a template.
Group 3: 1 ng/. mu.L of Vibrio mimicus genomic DNA (1. mu.L) was used as a template.
Group 4: 0.1 ng/. mu.L of Vibrio mimicus genomic DNA (1. mu.L) was used as a template.
Group 5: 0.01 ng/. mu.L of Vibrio mimicus genomic DNA (1. mu.L) was used as a template.
Group 6: 0.001 ng/. mu.L of Vibrio mimicus 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 internal standard amplification 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 result of RPA-IAC amplification is shown in FIG. 2, where groups 1-4 all have a band at 467bp and groups 2-6 have a band at 334bp, which indicates that the results of groups 1-6 are all valid and that groups 5-6 have no possibility of false negative, and also indicates that the primer pair of example 1 of the present invention has high sensitivity, and can detect that the sample contains only 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 containing the internal standard amplification sequence of the present invention (from example 2) 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 again, specifically:
the Primer pair of example 1 and the Primer pair which is designed and selected by the software of Primer Premier 5.0 and is arranged at the front are utilized, 50 parts of export marine products are respectively detected by the method for detecting or assisting to detect the vibrio mimicus established in example 3, meanwhile, the separation and the systematic biochemical identification of the vibrio mimicus are carried out on the 50 parts of marine products in reference to the national standard (GB 4789.7-2013) of food hygiene microbiological inspection, the sampling of the sensitivity test and the setting of the template concentration gradient are carried out according to example 5, and the detection results are shown in Table 2.
TABLE 2
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 internal standard amplification 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 internal standard amplification and the like more or less.
Comparative example 2
This comparative example provides a comparison of the results of detection of Vibrio mimicus using different reagents in combination with the primer set of the present invention (from example 1) and a plasmid containing an internal standard amplification sequence (from example 1).
Various reagents were combined with the primer pairs of the invention and plasmids containing internal standard amplification sequences:
combination 1: the invention relates to a primer pair, a plasmid containing an internal standard amplification sequence, a certain commercially available animal genome DNA extraction kit 1 and a certain commercially available RPA amplification kit 1
And (3) combination 2: the invention relates to a primer pair, a plasmid containing an internal standard amplification 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 internal standard amplification 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 an internal standard amplification sequence + 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 disclosed.
When the combination of the present invention is used for detecting Vibrio mimicus, the method is performed by the method of 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 mimicus, the operation is carried out according to the kit instruction of the commercial kit, and the other conditions are the same as the combination of the invention. And tested by a standard published comparative method (see comparative example 1).
The test sample was identical to 50 parts of export seafood as in comparative example 1.
The results are shown in Table 3.
TABLE 3
The results show that: the detection result of the combination of the primer pair, the plasmid containing the internal standard amplification sequence and each specific reagent is completely consistent with the detection results of European Union standards and the disclosed detection method, which shows that the kit formed by the combination of the primer pair, the internal standard amplification sequence and each specific reagent has the advantages of strong specificity, high sensitivity and good repeatability, and the combination of the primer pair and other randomly selected reagents has various defects of weak specificity, weak sensitivity and poor repeatability more or less under the condition that the internal standard amplification sequence exists.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> inspection and quarantine technology center of Guangdong entry-exit inspection and quarantine bureau; inspection and quarantine technology center of Shantou entry and exit inspection and quarantine bureau
<120> RPA-IAC primer and method for detecting vibrio mimicus
<130> 17CN
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：1
<400> 1
gagatattgg catctttaca aaatggacga 30
<210> 2
<211> 32
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：2
<400> 2
gattggcaat cagaagaaag ccaattaccc ag 32
<210> 3
<211> 334
<212> DNA
<213> Artificial
<220>
<223> SEQ ID NO ：3
<400> 3
gagatattgg catctttaca aaatggacga gttcgagacc ttcaacaccc cagccatgta 60
cgtggccatc caggcggtgc tgtccctgta cgcctctggc cgcaccactg gcatcgtgat 120
ggactccgga gacggggtca cccacacggt gcccatctac gaggggtacg ccctgcccca 180
cgccatcctg cgtctggacc tggctggccg ggacctgacc gactacctca tgaagatcct 240
gacggagcgg ggctacagct tcaccaccac ggccgagcgg gagatcgtgc gggacatcaa 300
ggctgggtaa ttggctttct tctgattgcc aatc 334
Claims (10)
1. A primer pair and an internal standard amplification sequence, wherein the primer pair is SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2, and the internal standard amplification sequence is SEQ ID NO: 3.
2. The primer pair and the internal standard amplification sequence according to claim 1 are applied to preparation of products for detecting or assisting in detecting vibrio mimicus.
3. A kit comprising the primer pair of claim 1 and a plasmid comprising the internal amplification sequence of claim 1.
4. The kit of claim 3, further comprising RPA isothermal amplification reagents.
5. The kit according to claim 4, wherein the RPA isothermal amplification reagent is a reagent contained in the TwistAmp Basic kit of the RPA amplification kit from TwistDX.
6. The kit of claim 4, further comprising a DNA extraction reagent.
7. The kit according to claim 6, wherein the DNA extraction reagent further comprises a reagent contained in a genomic DNA extraction kit from a bacterium having a cargo number DP302 from Tiangen Biochemical technology Ltd.
8. The kit of claim 4, further comprising a positive control and a negative control.
9. Use of the kit of any one of claims 3 to 8 for the detection or assisted detection of vibrio mimicus, or for the preparation of a product for the detection or assisted detection of vibrio mimicus, for non-diagnostic purposes.
10. A method for detecting or assisting in detecting RPA-IAC of Vibrio mimicus for non-diagnostic purposes, which is characterized by comprising the following steps:
1) RPA-IAC amplification:
using a DNA contained in a biological sample or a pathogenic bacterium as a template, performing RPA-IAC amplification using the primer set according to claim 1 and a plasmid comprising an internal amplification sequence according to claim 1, or using the kit according to any one of claims 3 to 4;
2) judging whether the biological sample is infected with vibrio mimicus or not or whether the pathogenic bacteria are or are candidates for vibrio mimicus according to the result of RPA-IAC amplification;
before the step 1), the method also comprises the step of extracting DNA in a biological sample or pathogenic bacteria;
the standard for judging the result of RPA-IAC amplification is as follows:
if the amplification product obtained by the RPA-IAC amplification only has 467bp target gene segment, or has 334bp, 467bp internal standard amplification segment and target gene segment, then the biological sample is judged to be infected by vibrio mimicus, or the pathogenic bacteria is or is a candidate of vibrio mimicus; if the amplification product obtained by the 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 467bp, the biological sample is judged not to be infected by vibrio mimicus, or the pathogenic bacteria is not or is not candidate to be vibrio mimicus; if the amplification product obtained by RPA-IAC amplification does not contain an internal standard amplification fragment with the size of 334bp and does not contain a target gene fragment with the size of 467bp, the reaction is judged to be false negative, and detection is required to be carried out again;
the reaction system for RPA-IAC amplification is as follows:
a 0.2mL twist Amp reaction tube containing lyophilized enzyme powder; rehydration buffer: 29.5 μ L; SEQ ID NO: 1 and SEQ ID NO: 2, the final concentration of each primer is 0.4 mu mol/L; comprises SEQ ID NO: 3 internal standard amplification sequence: 1.83X 103copies; template DNA: 50 ng; magnesium acetate solution: 2.5 mu L, the concentration is 280 mmol/L; deionized water: make up to 50 μ L;
the reaction conditions for the amplification of the RPA-IAC are as follows: the temperature of the RPA-IAC amplification is 37 ℃, and the time is 40 min.
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