CN107227378A - Detect the RPA IAC primers and method of vibrio mimicus - Google Patents
Detect the RPA IAC primers and method of vibrio mimicus Download PDFInfo
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- CN107227378A CN107227378A CN201710682439.0A CN201710682439A CN107227378A CN 107227378 A CN107227378 A CN 107227378A CN 201710682439 A CN201710682439 A CN 201710682439A CN 107227378 A CN107227378 A CN 107227378A
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Abstract
The invention provides primer pair and amplification of internal standard sequence, the primer pair includes SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 1：Nucleotide sequence shown in 2, the amplification of internal standard sequence includes SEQ ID NO：Nucleotide sequence shown in 3.Present invention also offers the application of described primer pair and amplification of internal standard sequence in the product for preparing detection or auxiliary detection vibrio mimicus；And the method for the detection vibrio mimicus based on RPA IAC technologies.It the experiment proved that, the RPA IAC primer specificities of the present invention are good, sensitivity is high, detection time is short, do not need special instrument and applied widely, and based on the primer and coordinate amplification interior label set up vibrio mimicus RPA IAC detection methods it is sensitive, accurate, easy and quick, to import and export complementary goods and product quarantine have directive significance.
Description
Technical field
The present invention relates to field of biological detection, method, primer and the kit of vibrio mimicus are more particularly to detected.
Background technology
Vibrio mimicus system Grain-negative vibrios, it is dynamic with single flagellum, belong to as vibrio parahaemolytious, comma bacillus
Normal ocean flora, is widely present in nature river, seawater and marine product.Bacterial strain is without sodium or only containing 1% sodium chloride
Nutrient broth in well-grown, can be separated from aquatic animal.
There is vibrio mimicus to cause a disease in the U.S., North America, New Zealand, Bangladesh and African country to report, China Fujian, north
Also there is morbidity capital, Shanghai, Jiangsu and Zhejiang area, many in distributing or breaking out, and have morbidity throughout the year, are many using summer；It is latent
3~72h of volt phase, average 24h；Cardinal symptom has diarrhoea, vomiting, stomachache, and stool to be in water sample or mucous bloody stool；Age of onset
Do not limit, but children are rare.Therefore the detection to vibrio mimicus has being of great significance in public health.
At present, the detection to vibrio mimicus still relies primarily on conventional method, i.e., increase bacterium first with selective medium, enter
And combine biochemical and serological method and identified.Traditional technique in measuring result is accurate, but has that detection efficiency is low, detection mesh
Mark that single, sensitivity is low and time-consuming, the deficiency such as cumbersome.To meet the requirement of pathogenic bacteria quick detection, develop enzyme-linked
Immunofluorescence assay (VIDAS-CHL), enzyme immunoassay (EIA), PCR (PCR), colloidal gold strip method,
The methods such as API biochemical identification test strips method, loop-mediated isothermal amplification (LAMP) technology and real-time fluorescence PCR.Wherein, VIDAS-
There is poor specificity, the low defect of sensitivity in CHL methods, EIA methods, colloid gold test paper method and API methods, without obtaining extensively should
With；LAMP methods, as a kind of emerging detection method, although drastically increasing detection efficiency, reduce testing cost again,
It is that the false positive rate produced is higher.Round pcr is obtained as a kind of High sensitivity, fast and convenient detection method in numerous areas
Extensive use, especially achieves revolutionary achievement in terms of pathogen detection, as nucleic acid quick detection a goldstandard,
And developed DNA probe technology, real-time fluorescence PCR technology and PCR combination denaturing high-performance chromatographies again on basis herein
(DHPLC) technology etc., and PCR primer is designed to restrict the key factor of such detection method success or failure, conventional PCR
Design of primers, not only needs to compare the specificity of primer repeatedly, and needs the parameters and reaction condition of optimizational primer, especially
It is that annealing temperature is even more to be optimized repeatedly, to prevent non-specific amplification, the primer chosen under the restriction of these conditions
Easily influence the validity and specificity of amplification.In addition, the universal price of fluorescence detection device is higher, also limit to a certain extent
The popularization and application of this kind of detection method.
The content of the invention
It is sensitive, accurate, easy and quick based on RPA-IAC technologies the technical problem to be solved in the present invention is to provide one kind
Detection vibrio mimicus method, present invention also offers the specific good, sensitivity for this method is high, detection time is short,
Special instrument and RPA-IAC primers applied widely and amplification of internal standard sequence and corresponding detection kit are not needed.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
First aspect present invention provides primer pair and amplification of internal standard sequence, and the primer pair includes SEQ ID NO：1 institute
The nucleotide sequence and SEQ ID NO shown：Nucleotide sequence shown in 2, the amplification of internal standard fragment includes SEQ ID NO：3 institutes
The nucleotide sequence shown.
Second aspect of the present invention provides described primer pair and amplification of internal standard sequence and is preparing detection or aiding in detection to intend
Application in the product of state vibrios；
In a preference, the product for preparing detection or aiding in detection vibrio mimicus includes：Prepare detection or aid in
Whether detection biological sample infects the product of vibrio mimicus, and prepares detection or aid in detecting that pathogen is or candidate is mimicry
The product of vibrios.
Third aspect present invention provides a kind of kit, including described primer pair and includes described amplification of internal standard
The plasmid of sequence.
In a preference, described kit also includes RPA constant-temperature amplification reagents.
In a preference, the RPA constant-temperature amplifications reagent includes the RPA amplification kits from TwistDX companies
The reagent that TwistAmp Basic kits are included.
In a preference, in addition to DNA extracts reagents.
In a preference, it is DP432 that the DNA extracts reagents, which include the article No. from Tiangeng biochemical technology Co., Ltd,
The reagent that is included of Animal genome DNA extraction kit.
In a preference, it is DP302 that the DNA extracts reagents, which are also included from Tiangeng biochemical technology Co., Ltd article No.,
The reagent that is included of bacterial genomes DNA extraction kit.
In a preference, in addition to positive control and negative control.
Fourth aspect present invention provide described kit detect or aid in detection vibrio mimicus, or prepare detection or
Application in the product of auxiliary detection vibrio mimicus.
In a preference, the detection or auxiliary detection vibrio mimicus include：Detection or auxiliary detection biological sample are
No infection vibrio mimicus, and detection or auxiliary detection pathogen whether be or candidate is vibrio mimicus.
In a preference, the product for preparing detection or aiding in detection vibrio mimicus includes：Prepare detection or aid in
Whether detection biological sample infects the product of vibrio mimicus, and prepares detection or aid in detecting that pathogen is or candidate is mimicry
The product of vibrios.
Fifth aspect present invention provides a kind of RPA-IAC methods for detecting or aiding in detection vibrio mimicus, including the use of
The step of described primer pair and plasmid or the kit for including described amplification of internal standard sequence.
In a preference, the detection or auxiliary detection vibrio mimicus include：Detection or auxiliary detection biological sample are
No infection vibrio mimicus, and detection or auxiliary detection pathogen whether be or candidate is vibrio mimicus.
In a preference, described method comprises the following steps：
1) RPA-IAC is expanded
Amplification of internal standard of the DNA contained using biological sample or pathogen as template and described primer pair and described in including
The plasmid of sequence or the kit carry out RPA-IAC amplifications.
2) result expanded according to RPA-IAC judges whether biological sample infects vibrio mimicus, or pathogen whether be or
Candidate is vibrio mimicus.
Optional, in step 1) the step of extracting DNA in biological sample or pathogen is additionally included in before.
In a preference, the DNA that extracted in biological sample or pathogen is to use the limited public affairs of Tiangeng biochemical technology
The Animal genome DNA extraction kit or Tiangeng biochemical technology Co., Ltd article No. that the article No. of department is DP432 are the thin of DP302
Bacterium genome DNA extracting reagent kit is carried out.
In a preference, the standard that the result of the RPA-IAC amplifications judges is：
If the amplified production that the RPA-IAC amplifications are obtained only has size and is 467bp target gene fragment, or gathers around simultaneously
The amplification of internal standard fragment and target gene fragment for having size to be respectively 334bp, 467bp, then be determined as that biological sample infects mimicry
Vibrios, or pathogen is or candidate is vibrio mimicus；If the amplified production that the RPA amplifications are obtained only has size to be 334bp's
Amplification of internal standard fragment, not containing the target gene fragment that size is 467bp, is then determined as that biological sample is uninfected by vibrio mimicus,
Or pathogen is not or candidate is not vibrio mimicus；If the amplified production that the RPA amplifications are obtained is not 334bp's containing size
Amplification of internal standard fragment and not containing size be 467bp target gene fragment, be determined as reaction for false negative, it is necessary to re-start
Detection.
In a preference, the reaction system of the RPA-IAC amplifications is as follows：
0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
The μ L of rehydration buffer solution 29.5
SEQ ID NO：1 and SEQ ID NO：Each 2 μ L of primer shown in 2, the final concentration of primer is 0.4 μm of ol/L
Include SEQ ID NO：The plasmid 1.83 × 10 of amplification of internal standard sequence shown in 33copies
Template DNA 50ng
The μ L of magnesium acetate solution 2.5, concentration is 280mmol/L
Deionized water complements to 50 μ L；
In a preference, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of the RPA-IAC amplifications is 37
DEG C, the time is 40min.
Sixth aspect present invention provides a kind of system for screening vibrio mimicus, including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus is used to extract the nucleic acid sample in the biological sample or pathogen
This；
RPA-IAC amplification devices, the RPA-IAC amplification devices are connected with the nucleic acid-extracting apparatus, it is adaptable to described
Primer pair or the kit to the sample of nucleic acid carry out RPA-IAC amplifications；
Judgment means, the judgment means are connected with the RPA-IAC amplification devices, so as to what is expanded based on RPA-IAC
As a result, judge whether the biological sample infects vibrio mimicus, or pathogen whether be or candidate is vibrio mimicus.
In a preference, the reaction system of the RPA-IAC amplifications is as follows：
0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
The μ L of rehydration buffer solution 29.5
SEQ ID NO：1 and SEQ ID NO：Each 2 μ L of primer shown in 2, the final concentration of primer is 0.4 μm of ol/L
Include SEQ ID NO：The plasmid 1.83 × 10 of amplification of internal standard sequence shown in 33copies
Template DNA 50ng
The μ L of magnesium acetate solution 2.5, concentration is 280mmol/L
Deionized water complements to 50 μ L；
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of the RPA-IAC amplifications is 37 DEG C, time
For 40min.
The biological sample of the present invention is blood, cell, tissue etc., or mixed the food of blood, cell, tissue etc. etc.
Deng；And pathogen is then the mixing of a kind of pure culture or at least more than two kinds of strain.
" amplification of internal standard fragment " of the present invention is added in PCR reaction systems to indicate the one of false negative phenomenon
The conserved genetic sequences of the artificial constructed DNA sequence dna of section either one section of pathogenic bacteria.Amplification interior label effect cardinal principle be：It will expand
Increase internal standard to be added in PCR reaction systems, be allowed to carry out coamplification with target gene, if in reaction system existed
Restraining factors, then the amplified reaction of amplification interior label and target gene will be all suppressed, so as to reach instruction PCR reaction false negatives
Purpose.
Beneficial effects of the present invention include：
(1) present invention designs specificity RPA-IAC primers for vibrio mimicus, establishes vibrio mimicus RPA-IAC detections
Method, can carry out qualitative detection to vibrio mimicus.
(2) amplification interior label sequence and vibrio mimicus genome and human genome of the invention are non-homogeneous, can ensure
Effectively evade the generation of detection false negative and the individual nucleic acid Interference Detection of operating personnel while target sequence amplification efficiency.
(3) present invention only devises pair of primers for vibrio mimicus can complete amplification, and the primer for eliminating complexity is set
Meter process；The primer amplification of the present invention only needs to isothermal reaction at 37 DEG C, it is not necessary to special heat circulating equipment；During reaction
Between only need 40min, detection time is short；Unlike the dispersion plating of LAMP products, RPA-IAC amplified productions have according to design of primers site
There is the band of particular size, its result is easy to judge.
(4) RPA-IAC amplimers specificity of the invention is good, sensitivity is high and applied widely.
(5) the vibrio mimicus RPA-IAC detection methods that the present invention is set up, sensitive, accurate, easy and quick, to importing and exporting
Complementary goods and examination and test of products quarantine have directive significance.
Brief description of the drawings
Fig. 1 is the specific test electrophoresis detection result of RPA-IAC primer pairs in the embodiment of the present invention 4.Wherein M is
Marker DL1000,1：Vibrio mimicus；2：Vibrio vulnificus；3：Vibrio parahaemolytious；4：Vibrio fluvialis；5：Vibrio alginolyticus；6：Cholera
Vibrios；7：Vibrio harveyi；8：Salmonella；9：Escherichia coli；10：Staphylococcus aureus；11：Listeria monocytogenes.
Fig. 2 is the sensitivity test electrophoresis detection result of RPA-IAC amplifications in the embodiment of the present invention 5.Wherein M is marker
DL1000,1-6：Vibrio mimicus template amount is followed successively by 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng, amplification interior label
Consumption be 1.83 × 103copies。
Fig. 3 is the sensitivity test electrophoresis detection result of RPA amplifications in the embodiment of the present invention 5.Wherein M is marker
DL1000,1-6：Vibrio mimicus template amount is followed successively by 100ng, 10ng, 1ng, 0.1ng, 0.01ng and 0.001ng.
Embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiments and the drawings, the present invention will be described, it is necessary to which explanation, these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to normal
Advise experiment condition, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular
cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
The vibrio parahaemolytious that is used in following examples, comma bacillus, vibrio alginolyticus, Vibrio vulnificus, vibrio mimicus, river
Vibrios, Vibrio harveyi, Escherichia coli, staphylococcus aureus, salmonella and Listeria monocytogenes are entered and left the border by Shantou
Inspection and quarantine bureau provides.
Embodiment 1
Present embodiments provide RPA primer pairs and amplification of internal standard sequence and its application.
The screening technique of RPA primer pairs is：For vibrio mimicus VMH genes (GenBank ID:CP016384.1), design
A plurality of RPA primers have simultaneously carried out a large amount of screenings, the effect between comprehensive its specificity, sensitivity, primer and primer, Yi Jisuo
The suitability of each primer for using and RPA amplification kits, finally filter out specific good, reproducible and sensitivity it is high as
The RPA primer pairs of vibrio mimicus are can detect down：
VMH-F：5′-GAGATATTGGCATCTTTACAAAATGGACGA-3′(SEQ ID NO：1)；
VMH-R：5′-GATTGGCAATCAGAAGAAAGCCAATTACCCAG-3′(SEQ ID NO：2)；
The design and synthesis of amplification of internal standard sequence：To avoid the homologous interference of close bacterium and the individual nucleic acid of testing staff
Pollution, is considered and experimental verification repeatedly by comprehensive analysis, with highly conserved pig detection β-actin part
Based on nucleotide sequence, two ends connect primer sequence VMH-F and VMH-R respectively, and it is 334bp's to form sequence size as follows
Amplification of internal standard sequence：
GAGATATTGGCATCTTTACAAAATGGACGAgttcgagaccttcaacaccccagccatgtacgtggccatccaggcgg
tgctgtccctgtacgcctctggccgcaccactggcatcgtgatggactccggagacggggtcacccacacggtgccc
atctacgaggggtacgccctgccccacgccatcctgcgtctggacctggctggccgggacctgaccgactacctcat
gaagatcctgacggagcggggctacagcttcaccaccacggccgagcgggagatcgtgcgggacatcaaggCTGGGT
AATTGGCTTTCTTCTGATTGCCAATC(Seq ID NO：3)
Above-mentioned Seq ID NO：In 3, original series and VMH-R reverse complemental sequence that big write sequence is the VMH-F of addition
Row, small write sequence 272bp is DQ452569.1 and entitled Sus scrofa beta actin from Genbank accession number
(ACTB) 489-760 one section of sequence in gene, partial cds, and the homology checking Jing Guo theory and practice.
The synthetic work of above-mentioned RPA-IAC primer pairs and amplification of internal standard sequence, the limited public affairs of work biotechnology are given birth to by Shanghai
Department completes.
Upper in application, above-mentioned primer pair and amplification of internal standard sequence can be applied to prepare detection or auxiliary detects vibrio mimicus
Product, for example, be prepared into the plasmid containing this amplification of internal standard sequence by amplification of internal standard sequence, then with primer pair or other combinations
Get up to turn into the product of detection or auxiliary detection vibrio mimicus, be used directly to detect or aid in whether detection biological sample infects plan
State vibrios.
Embodiment 2
A kind of kit and its application are present embodiments provided, the kit includes：
(1)SEQ ID NO：1、SEQ ID NO：2 (coming from embodiment 1), and contain such as SEQ ID NO：Internal standard shown in 3
The plasmid of extension increasing sequence；
(2) DNA extracts reagents；
(3) tube cell containing lyophozyme；
(4) rehydration buffer solution；
(5) magnesium acetate solution.
Above-mentioned tube cell containing lyophozyme, rehydration buffer solution (Rehydration Buffer) and magnesium acetate solution
(280mmol/L) is that (TwistDX companies, article No. is from RPA amplification kit TwistAmp Basic kits
TABAS03KIT the reagent in).
The Animal genome DNA for the Tiangeng biochemical technology Co., Ltd that it is DP432 from article No. that the DNA extracts reagents, which are,
Reagent in extracts kit and the bacterial genomes DNA from the Tiangeng biochemical technology Co., Ltd that article No. is DP302 are extracted
The reagent that kit is included.
It is described to contain such as SEQ ID NO：The structure of the plasmid of amplification of internal standard sequence shown in 3：The internal standard that embodiment 1 is synthesized
Extension increasing sequence is connected on universal support PUC57, is obtained containing such as SEQ ID NO：The positive matter of amplification of internal standard sequence shown in 3
Grain.
The structure of above-mentioned amplification of internal standard sequence plasmid：Universal support is connected to using conventional T4 phage DNAs ligase
PUC57, and it is prepared into positive plasmid freeze-dried powder.This structure is complete by Shanghai Sheng Gong Bioisystech Co., Ltd in the present invention
Into.According to quality (g/ μ L)/(650g/mol × base number) × (6.02 × 10 of copy number N copies/ μ L=PCR fragments23)
Calculate, positive plasmid dry powder is diluted to 1.83 × 103copies/μL。
Utilize SEQ ID NO：1、SEQ ID NO：2 and above-mentioned contain such as SEQ ID NO：Amplification of internal standard sequence shown in 3
Plasmid carries out RPA-IAC amplifications for vibrio mimicus, obtained RPA-IAC target genes amplified production and amplification interior label product
Size is respectively 467bp, 334bp.
It is demonstrated experimentally that above-mentioned DNA extracts reagents, tube cell containing lyophozyme, rehydration buffer solution, magnesium acetate solution, Yi Jiqi
With SEQ ID NO：1、SEQ ID NO：2 and contain such as SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3 is used in combination,
Effect is more superior, is in particular in that high specificity, reproducible, sensitivity are high and Quality Control effect is good.
Upper in application, mentioned reagent box can be applied to detect or aid on detection vibrio mimicus, for example, detect or aid in inspection
Survey whether biological sample infects vibrio mimicus, or detection or auxiliary detection biological sample to be measured whether be or candidate is mimicry arc
Bacterium；It can also be used to the product for preparing detection or auxiliary detection vibrio mimicus, for example, prepare detection or auxiliary detection biological sample
The product of vibrio mimicus whether is infected, or prepares detection or aids in the product that detection pathogen is or candidate is vibrio mimicus.
Embodiment 3
A kind of method for detecting or aiding in detection vibrio mimicus is present embodiments provided, for example, detects or aid in detection biology
Whether sample infects vibrio mimicus, or whether detection or auxiliary detection pathogen are or candidate is vibrio mimicus that this method is used
The primer pair or the kit of embodiment 2 of embodiment 1.
The above method comprises the following steps：
Step one：RPA-IAC is expanded
Using biological sample or the DNA of pathogen as template, contain such as SEQ ID NO in addition embodiment 2：Internal standard shown in 3
The plasmid of extension increasing sequence, using SEQ ID NO：1 and SEQ ID NO：The primer pair of 2 compositions carries out RPA-IAC amplifications, obtains
RPA-IAC amplified productions, while setting blank control (template is ultra-pure water).
The compound method of RPA-IAC amplification systems is as follows：Into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Add rehydration buffer solution (Rehydration Buffer) 29.5 μ L, upstream and downstream primer (VMH-F and VMH-R of embodiment 1)
Each 2 μ L (final concentration of primer is 0.4 μm of ol/L), contain such as SEQ ID NO：The μ L of plasmid 1 of amplification of internal standard sequence shown in 3
(1.83×103Copies), template DNA 1ul (50ng), finally adds the μ L (280mmol/L) of magnesium acetate solution 2.5, spends
Ionized water complements to 50 μ L.
RPA-IAC amplification reaction conditions：Above-mentioned RPA-IAC amplification systems are fully mixed, on the metal bath for being placed in 37 DEG C
40min is reacted, RPA-IAC amplified productions are obtained.
Step 2：The electrophoresis detection of RPA-IAC amplified productions
After RPA-IAC reactions terminate, 50 μ L phenol/chloroforms (1 are added into above-mentioned amplified production:1) solution, is fully mixed
12000rpm centrifuges 2min afterwards, takes 5 μ L of supernatant liquid in 1.5% agarose gel electrophoresis, result is observed on gel imaging system,
And RPA-IAC amplified productions are sequenced and (sequence verification have been carried out during method for building up, repeat no more herein).
The standard that the result of RPA-IAC amplification judges is：
If the amplified production that the RPA-IAC amplifications are obtained only has size and is 467bp target gene fragment, or gathers around simultaneously
The amplification of internal standard fragment and target gene fragment for having size to be respectively 334bp, 467bp, then be judged to, containing vibrio mimicus, pointing out
The biological sample infects vibrio mimicus, or pathogen is or candidate is vibrio mimicus；If the expansion that the RPA-IAC amplifications are obtained
Volume increase thing only has the amplification of internal standard fragment that size is 334bp, not containing the target gene fragment that size is 467bp, is then determined as
Do not contain vibrio mimicus, point out the biological sample to be uninfected by vibrio mimicus, or pathogen is not or candidate is not vibrio mimicus；
If the RPA-IAC expands the amplification of internal standard fragment that obtained amplified production is not 334bp containing size and is not containing size
467bp target gene fragment, is judged to reacting false negative, points out needs to re-start detection.
If plant to be measured or pathogen to be measured do not extract DNA also, before above method step (1) RPA-IAC amplifications also
Can be including the use of Animal genome DNA extraction kit (being purchased from Tiangeng biochemical technology Co., Ltd, article No. is DP432) or thin
Bacterium genome DNA extracting reagent kit (being purchased from Tiangeng biochemical technology Co., Ltd, article No. is DP302) is according to kit specification pair
The step of DNA in biological sample or pathogen to be measured is extracted.
Embodiment 4
The present embodiment has carried out validity and specificity verification to the primer pair and amplification of internal standard sequence of embodiment 1, used
Material to be tested it is as follows：Vibrio parahaemolytious, comma bacillus, vibrio alginolyticus, Vibrio vulnificus, vibrio mimicus, vibrio fluvialis, Kazakhstan Vickers
Vibrios, Escherichia coli, staphylococcus aureus, salmonella and Listeria monocytogenes, these bacterial strains are for purchase and through strictly testing
The reference culture of card, is maintained in Shantou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center.
Step one：The extraction of genomic DNA
The article No. of TIANGEN Biotech (Beijing) Co., Ltd. is used for DP302 bacterial genomes DNA extraction kit
Extract vibrio alginolyticus, Vibrio vulnificus, vibrio mimicus, vibrio fluvialis, Vibrio harveyi, large intestine bar respectively according to kit specification
Bacterium, staphylococcus aureus, the genomic DNA of salmonella and Listeria monocytogenes.
Step 2：RPA-IAC is expanded
Using various genomic DNA templates：
Group 1 is using the genomic DNA pools of vibrio mimicus as template (positive control).
Group 2 is using the genomic DNA of Vibrio vulnificus as template.
Group 3 is using the genomic DNA of vibrio parahaemolytious as template.
Group 4 is using the genomic DNA of vibrio fluvialis as template.
Group 5 is using the genomic DNA of vibrio alginolyticus as template.
Group 6 is using the genomic DNA of comma bacillus as template.
Group 7 is using the genomic DNA of Vibrio harveyi as template.
Group 8 is using the genomic DNA of salmonella as template.
Group 9 is using the genomic DNA of Escherichia coli as template.
Group 10 is using the genomic DNA of staphylococcus aureus as template
Group 11 is using the genomic DNA of Listeria monocytogenes as template.
It is respectively template with a group 1- groups 11, the step of carrying out RPA-IAC amplifications, the method be the same as Example 3 of RPA-IAC amplifications
One.
Step 3：The electrophoresis detection of RPA-IAC amplified productions
The step of method be the same as Example 3 of the electrophoresis detection of RPA-IAC amplified productions two.
Interpretation of result：
As a result as shown in figure 1, group 1- groups 11 correspond to swimming lane 1-11 respectively, swimming lane 1, which is shown at 334bp and 467bp, to be had
Amplified band, according to the criterion of embodiment 3, is positive, i.e., smoothly detects vibrio mimicus, remaining swimming lane 2- swimming lanes 11 are shown
Only there is amplified band at 334bp, according to the criterion of embodiment 3, this eliminates false negative possibility, further carry
The high reliability of result.To sum up, it is above-mentioned it is positive judge and negative accurate and all group of result of determination amplification interior label into
Work(is expanded, and thus proves the primer pair and amplification of internal standard sequence of the embodiment of the present invention 1 and has validity, high specific and good instruction
Property；Further, the present invention is set up based on primer pair and amplification of internal standard sequence and kit detection or auxiliary detection mimicry arc
The method of bacterium can be detected accurately to vibrio mimicus.
Embodiment 5
The present embodiment has carried out sensitivity checking to the primer pair and amplification of internal standard sequence of embodiment 1, and with without expansion
Increase internal standard (i.e. without in embodiment 2 containing such as SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3) it is compared, it is used
Material to be tested it is as follows：Vibrio mimicus, this bacterial strain is purchase and the reference culture through strictly verifying, is stored in Shantou entry and exit inspection
Test Quarantine Bureau's inspection and quarantine technique center.
Step one：The extraction of genomic DNA
The article No. of TIANGEN Biotech (Beijing) Co., Ltd. is used for DP302 bacterial genomes DNA extraction kit
Extract the genomic DNA of vibrio mimicus respectively according to kit specification, and 10 times of gradients of obtained genomic DNA progress are dilute
Release, prepare the genomic DNA of the vibrio mimicus of various concentrations.
Step 2：RPA is expanded and RPA-IAC amplifications
Template is used as using the genomic DNA of the vibrio mimicus of various concentrations：
Group 1：With 100ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
Group 2：With 10ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
Group 3：With 1ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
Group 4：With 0.1ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
Group 5：With 0.01ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
Group 6：With 0.001ng/ μ L vibrio mimicus genomic DNA (1 μ L) for template.
It is respectively template with a group 1- groups 6, RPA amplifications and RPA-IAC amplifications is carried out respectively, the method for RPA-IAC amplifications is same
What the step of embodiment 3 one, RPA amplifications and RPA-IAC were expanded is distinguished as in amplification system containing such as without in embodiment 2
SEQ ID NO：The plasmid of amplification of internal standard sequence shown in 3.
Step 3：The electrophoresis detection of RPA amplified productions and RPA-IAC amplified productions
The step of method be the same as Example 3 of the electrophoresis detection of the two amplified production two.
Interpretation of result：
The result of RPA-IAC amplifications is as shown in Fig. 2 group 1-4 has band at 467bp and group 2-6 has bar at 334bp
Band, this explanation group 1-6 result effectively and organizes 5- groups 6 without false negative possibility, while also illustrating drawing for the embodiment of the present invention 1
Thing is to higher sensitivity, can detect that in sample the only minim DNA containing 0.1ng；Result such as Fig. 3 institutes of RPA amplifications
Show, sensitivity results are expanded with RPA-IAC.Addition amplification interior label does not reduce the sensitivity of detection in summary.
Comparative example 1
In fact, seeking to before the primer pair of the present invention, that has attempted the present invention includes amplification of internal standard sequence
Plasmid (come from embodiment 2) is combined with very many primer pair, only won in this comparative example it is wherein some be illustrated, its
Remaining it will not go into details, is specially：
Using the primer pair of embodiment 1 and with the Software for Design of Primer Premier 5.0 and to choose wherein row point forward
Primer pair, with embodiment 3 set up detection or auxiliary detect vibrio mimicus method to 50 parts outlet marine products carry out respectively
Detection, with reference to microbiological test of food hygiene national standard (standard No.：GB4789.7-2013 to this 50 parts of seas in)
Product has carried out point discrete system biochemical identification of vibrio mimicus, and the sampling of sensitivity test and template concentrations gradient, which are set, presses
Carried out according to embodiment 5, testing result is as shown in table 2.
Table 2
As a result show：In view of the presence of the detection knot of primer pair of the present invention in the case of the plasmid for including amplification of internal standard sequence
Fruit and the testing result of normative reference and published detection method are completely the same, and this illustrates primer pair combination specificity of the present invention
By force, sensitivity is high and repeatability very well, and the combination of the several primer randomly selected using primer-design software then or it is many or
Strong, sensitivity is not high, repeatability is bad and disturbs the various defects such as amplification interior label for few presence specificity.
Comparative example 2
This comparative example provides the primer pair (come from embodiment 1) of different reagents and the present invention, includes amplification of internal standard
The plasmid (coming from embodiment 1) of sequence combines testing result contrast during detection vibrio mimicus.
Various reagents and primer pair of the invention and the plasmid combinations for including amplification of internal standard sequence:
Combination 1：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 1 of kit 1+
Combination 2：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 2 of kit 2+
Combination 3：Primer pair of the present invention and plasmid+certain the commercially available Animal genome DNA extraction for including amplification of internal standard sequence
The commercially available RPA amplification kits 3 of kit 3+
Present invention combination：Primer pair of the present invention and include the plasmid of amplification of internal standard sequence+have from Tiangeng biochemical technology
The reagent that limit company article No. is included by the DP432 Animal genome DNA extraction kit+article No. from TwistDX companies
The reagent included by TABAS03KIT RPA amplification kit TwistAmp Basic kits.
During using the invention described above combine detection vibrio mimicus, its method uses the method in embodiment 3 to carry out.Sensitivity
The sampling of experiment and the setting of template concentrations gradient are carried out according to embodiment 5.
It is every to use commercial reagent box when detecting vibrio mimicus using combinations thereof 1, combination 2 and combination 3, according to it
Kit specification carries out operation progress, and remaining condition is with present invention combination.Simultaneously be marked with accurate published control methods (see
Comparative example 1) detected.
By sample product with 50 portions of outlet marine products in comparative example 1.
Testing result is as shown in table 3.
Table 3
As a result show：The inspection of the combination of primer pair of the present invention, the plasmid for including amplification of internal standard sequence and each particular agent
Survey result and the testing result of EU criteria and published detection method is completely the same, this illustrates primer pair of the present invention, internal standard
The kit that the combination of extension increasing sequence and each particular agent is constituted has high and reproducible excellent of high specificity, sensitivity
Gesture, and in the case where there is amplification of internal standard sequence using primer pair of the present invention and the combination of other some reagents randomly selected
Then more or less presence specificity not strong, not high and repeated bad various defects of sensitivity.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu；Shantou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine skill
Art center
<120>Detect the RPA-IAC primers and method of 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. primer pair and amplification of internal standard sequence, it is characterised in that the primer pair includes SEQ ID NO：Nucleotides sequence shown in 1
Row and SEQ ID NO：Nucleotide sequence shown in 2, the amplification of internal standard fragment includes SEQ ID NO：Nucleotides sequence shown in 3
Row.
2. primer pair according to claim 1 and amplification of internal standard sequence are preparing detection or the production of auxiliary detection vibrio mimicus
Application in product；
Optional, the product for preparing detection or aiding in detection vibrio mimicus includes：Prepare detection or the biological sample of auxiliary detection
Whether product infect the product of vibrio mimicus, and prepare detection or aid in the production that detection pathogen is or candidate is vibrio mimicus
Product.
3. a kind of kit, it is characterised in that including the primer pair described in claim 1 and include described in claim 1
The plasmid of amplification of internal standard sequence.
4. kit according to claim 3, it is characterised in that also including RPA constant-temperature amplification reagents；
Optional, the RPA constant-temperature amplifications reagent includes the RPA amplification kits TwistAmp from TwistDX companies
The reagent that Basic kits are included；
Optional, in addition to DNA extracts reagents；
Optional, the DNA extracts reagents include the animal gene that the article No. from Tiangeng biochemical technology Co., Ltd is DP432
The reagent that group DNA extraction kit is included；
Optional, the DNA extracts reagents are also included from the bacterial gene that Tiangeng biochemical technology Co., Ltd article No. is DP302
The reagent that group DNA extraction kit is included；
Optional, in addition to positive control and negative control.
5. the kit described in claim 3 or 4 is detecting or aided in detection vibrio mimicus, or preparation detection or auxiliary detection to intend
Application in the product of state vibrios；
Optional, the detection or auxiliary detection vibrio mimicus include：Whether detection or auxiliary detection biological sample infect mimicry
Vibrios, and detection or auxiliary detection pathogen whether be or candidate is vibrio mimicus；
Optional, the product for preparing detection or aiding in detection vibrio mimicus includes：Prepare detection or the biological sample of auxiliary detection
Whether product infect the product of vibrio mimicus, and prepare detection or aid in the production that detection pathogen is or candidate is vibrio mimicus
Product.
6. a kind of RPA-IAC methods for detecting or aiding in detection vibrio mimicus, it is characterised in that including the use of claim 1 institute
The primer pair stated and include any one of the plasmid or claim 3-4 of amplification of internal standard sequence described in claim 1
The step of kit；
Optional, the detection or auxiliary detection vibrio mimicus include：Whether detection or auxiliary detection biological sample infect mimicry
Vibrios, and detection or auxiliary detection pathogen whether be or candidate is vibrio mimicus.
7. method according to claim 6, it is characterised in that：Comprise the following steps：
1) RPA-IAC is expanded
The DNA contained using biological sample or pathogen is the primer pair described in template and claim 1 and includes claim 1
Kit any one of the plasmid or claim 3-4 of described amplification of internal standard sequence carries out RPA-IAC amplifications；
2) result expanded according to RPA-IAC judges whether biological sample infects vibrio mimicus, or pathogen whether be or candidate
For vibrio mimicus；
Optional, in step 1) the step of extracting DNA in biological sample or pathogen is additionally included in before；
It is optional, it is described extracted in biological sample or pathogen DNA be use the article No. of Tiangeng biochemical technology Co., Ltd for
DP432 Animal genome DNA extraction kit or Tiangeng biochemical technology Co., Ltd article No. is DP302 bacterial genomes
DNA extraction kit is carried out；
Optional, the standard that the result of the RPA-IAC amplifications judges is：
If the amplified production that the RPA-IAC amplifications are obtained only has size and is 467bp target gene fragment, or possesses big simultaneously
Small is respectively 334bp, 467bp amplification of internal standard fragment and target gene fragment, then is determined as that biological sample infects vibrio mimicus,
Or pathogen is or candidate is vibrio mimicus；If the amplified production that the RPA amplifications are obtained only has the internal standard that size is 334bp to expand
Increase fragment, not containing the target gene fragment that size is 467bp, be then determined as that biological sample is uninfected by vibrio mimicus, or cause of disease
Bacterium is not or candidate is not vibrio mimicus；If the amplified production that the RPA amplifications are obtained does not expand containing size for 334bp internal standard
Increase fragment and not containing size be 467bp target gene fragment, be determined as reaction for false negative, it is necessary to re-start detection.
8. method according to claim 7, it is characterised in that：The reaction system of the RPA-IAC amplifications is as follows：
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of RPA-IAC amplification is 37 DEG C, and the time is
40min。
9. a kind of system for screening vibrio mimicus, it is characterised in that including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus is used to extract the sample of nucleic acid in the biological sample or pathogen；
RPA-IAC amplification devices, the RPA-IAC amplification devices are connected with the nucleic acid-extracting apparatus, it is adaptable to use right
It is required that kit carries out RPA-IAC expansions to the sample of nucleic acid any one of primer pair or claim 3-4 described in 1
Increase；
Judgment means, the judgment means are connected with the RPA-IAC amplification devices, so as to the result expanded based on RPA-IAC,
Judge whether the biological sample infects vibrio mimicus, or pathogen whether be or candidate is vibrio mimicus.
10. system according to claim 9, it is characterised in that：
The reaction system of the RPA-IAC amplifications is as follows：
Optional, the reaction condition of the RPA-IAC amplifications is as follows：The temperature of RPA-IAC amplification is 37 DEG C, and the time is
40min。
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