CN106191298A - A kind of method detecting vibrio parahaemolyticus Vibrio parahaemolyticus - Google Patents
A kind of method detecting vibrio parahaemolyticus Vibrio parahaemolyticus Download PDFInfo
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Abstract
The present invention provides a kind of method detecting vibrio parahaemolyticus Vibrio parahaemolyticus, and the sequence of the forward primer wherein used is SEQ ID NO:1, and the sequence of reverse primer is SEQ ID NO:2, and the sequence of probe is SEQ ID NO:3；The present invention uses the detection method of real time RPA method detection food-borne pathogens vibrio parahaemolyticus, to realize carrying out parahemolyticas field quick detection safe, special, quick, sensitive, simple, thus makes up the deficiency of existing traditional sensing techniques.And the method is applicable to Site Detection, it is possible in time, effectively suppress vibrio parahaemolytisus poisoning to cause the generation of epidemic situation, improve food safety security system.
Description
Technical field
The invention belongs to disease technical field of microbial detection, be specifically related to a kind of detection vibrio parahaemolyticus Vibrio
The method of parahaemolyticus.
Background technology
Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) belongs to antibacterial circle (Bacteria), Proteobacteria
(Proteobacteria), γ-deformation Gammaproteobacteria (Gammaproteobacteria), vibrio mesh (Vibrionales), vibrionaceae
(Vibrionaceae), vibrio (Vibrio), for a kind of Gram-negative halophilic bacteria, its main infection live in warm, contain
On river that salt is high or the aquatile of marine site mouth, have stronger pathogenic, be one of main food-borne pathogens.The mankind
If eating the marine product infected by it by mistake, it may appear that suffer from diarrhoea, suffer from abdominal pain, the gastroenteritis such as enterospasm, Nausea and vomiting and watery stool anti-
Should, the clinical response such as general spasticity and renal failure can be caused time serious.This bacterium at first by the Fujino etc. of Japan from food
Isolated in poisoning sufferer, has flagellum, without pod membrane, internal without spore, for polymorphic bacillus.Along with seafood trade in recent years
Globalization, the poisoning occurrence rate being induced by rises year by year.
Present stage research shows, vibrio parahaemolyticus produces thermostable direct hemolysin (thermostable direct
Hemolysin, TDH), TDH is correlated with hemolysin (TDH related hemolysin, TRH) and thermolability hemolysin
(thermolabile hemolysin, TLH) three kinds of important hemolysins.Generally vibrio parahaemolytious clinical separation strain produces heat-resisting
Property direct hemolysin hemolysin relevant with thermostable direct hemolysin, there are some researches show, the gastroenteritis that VP causes is molten with both
Sanguinin has close relationship, therefore, it is considered that TDH and TRH is the major virulent factor of vibrio parahaemolytious.TLH is a kind of non-
Typical phospholipids enzyme, can dissolve the erythrocyte of animal.
GB detection method is still the micro-biological process of antibacterial culturing, be divided into increasing bacterium, separate, identify and serology mirror
Fixed.The method operation complexity, just need to can obtain testing result through certain incubation time.The method of research has at present: 1. exempt from
Epidemic disease method, such as Enzyme-linked Immunosorbent Assay, immune colloidal gold technique etc.；2. molecular biosciences method: as PCR, real-time fluorescence PCR, Chao Shi PCR,
LAMP, nucleic probe method, biochip technology etc.；3. denaturing high-performance chromatography etc..For above several foods in GB
Borne pathogen uses the micro-biological process that comparison is traditional, and these methods are suitable for application in quickly detection.And it is another
On the one hand, the immunological method of rise and molecular biology for detection often have that instrument dependency, testing cost be high, technology is wanted
Ask harsh so that these seem method sensitive, special, efficient and can not be used widely and popularizing in market.The most urgently
A kind of new technique being applicable to Site Detection that can balance need to be found at these two aspects implementation methodology and economy, when first
Between detect pathogenic bacterium, in order in time take steps to remove and reduce with reduce consumer eat these microbial contamination products
Risk, food safety is significant.
Summary of the invention
The present invention provides a kind of method detecting vibrio parahaemolyticus Vibrio parahaemolyticus, i.e. one makes
By the detection method of real-time RPA method detection food-borne pathogens vibrio parahaemolyticus, to realize parahemolyticas is entered
Row field quick detection safe, special, quick, sensitive, simple, thus make up the deficiency of existing traditional sensing techniques.And
The method is applicable to Site Detection, it is possible in time, effectively suppress vibrio parahaemolytisus poisoning to cause the generation of epidemic situation, complete
Kind food safety security system.
Present invention firstly provides a kind of primer sets detecting vibrio parahaemolyticus Vibrio parahaemolyticus, its
Sequence information is as follows:
Forward primer F-RPA:5 '-TTAGATTTGGCGAACGAGAACGCAGACATTACG-3 ' (SEQ ID NO:1)
Reverse primer R-RPA:5 '-AGATGTTGCCTGTATCAGACAAGCTGTCACCGA-3 ' (SEQ ID NO:2)
The present invention also provides for the probe for coordinating above-mentioned primer sets to use, and its sequence information is as follows:
Probe p:5 '-TTACGTTCTTCGCCGCTGACAATCGCTTCTCATACAACCACACGAT-3 ' (SEQ ID NO:
3)
The 30th bit base T of its middle probe P carries out CF 5(6)-Carboxyfluorescein FAM labelling, and the 33rd bit base T carries out BHQ (Black
Hole Quencher dyestuff) labelling, and the 31st bit base C abasic site analog THF replacement.
Above-mentioned primer sets and probe are for preparing the system of detection vibrio parahaemolyticus Vibrio parahaemolyticus
Product；
Another aspect of the invention provides one detection vibrio parahaemolyticus Vibrio parahaemolyticus method,
Comprise the following steps that
1) preparation RPA reaction system:
Each primer final concentration of: primers F-RPA and primer R-RPA each 420nmol/ μ L；Probe P 120nmol/ μ L；Slow
Rush liquid composition and concentration is: Tris-HCl (pH7.9) 50mmol/ μ L, KAc (potassium acetate) 100mmol/ μ L, DTT (two sulfur threoses
Alcohol) 2mmol/ μ L, 5%PEG 20mol/ μ L, dNTPs 200 μm ol/ μ L, ATP 3mmol/ μ L, pcr (creatine phosphokinase)
50mmol/ μ L, CK (creatine kinase) 100ng/ μ L, archaeal dna polymerase Bsu 30ng/ μ L, strand binding Protein G p32 300ng/ μ
L, recombinase UvsX 240ng/ μ L, auxiliary enzymes UvsY 60ng/ μ L, exonuclease exo 200ng/ μ L；Sample DNA templates is fitted
Amount, adds distilled water and makes unreacted mixed system (reaction mix) volume be 23.75 μ L；280mmol/ μ L MgAc (acetic acid
Magnesium) 1.25 μ L, making reaction system cumulative volume is 25 μ L；
2) RPA reaction system amplification:
Aseptic 1.5mL centrifuge tube is sequentially added into all reactive components in addition to magnesium acetate and template DNA, after vibration from
The heart；Add appropriate template DNA, centrifugal after vibration；Reaction mix micropipettor is transferred to aseptic fluorescent quantitation special
With in sample-adding pipe, add 280mmol/ μ L MgAc (magnesium acetate) 1.25 μ L on the supporting transparent cover of dedicated pipe, close the lid
Son, centrifugal；Rock 10 times, centrifugal；
3) fluorescence-causing substance detection: the fluorescent quantitation instrument selected in the present invention is Roche company of Germany
96System.Pre-setting response procedures on display screen, including reaction temperature, reaction volume, probe luminous signal is corresponding
Fluorescein.The every 30s of course of reaction gathers a signal, is denoted as a circulation of reaction.After completing sample-adding, rapidly sample is added
On the position that 96 orifice plates of instrument are suitable for, and clicking on start button controlling main interface, reaction starts, and instrument is by automatically by setting
Put collection fluorescence signal.
Detection primer group provided by the present invention and method have the advantage that
One, high sensitivity, can be to 0.4pg/ μ L to the detection minimum rate of accumulation of vibrio parahaemolyticus.
Two, strong specificity, according to the thermolability hemolysin of vibrio parahaemolyticus (thermolabile hemolysin,
TLH) encoding gene tlh is designed specific primer, and experiment proves detection high specificity.
Three, the detection time is shorter, within about 15 minutes, can obtain testing result, have the biggest compared with common detection methods
Advantage.
Four, instrument and equipment requires low, it is not necessary to thermocycler, PCR instrument, electrophresis apparatus or gel imaging system, only needs one
Individual portable fluorescence detector can complete detection.
Five, easy and simple to handle, result presents substantially, and whole detection process is not related to complex and expensive instrument and equipment, before sample
Process simple, it is not necessary to carrying out DNA purification, the DNA that boiling method slightly extracts just can reach testing requirement, slightly has molecular biology
The personnel on basis can complete whole operation；Amplified production accumulation Tong Bu with fluorescence signal, it is achieved monitor in real time, eliminate follow-up
Amplification detecting step, reduce the detection time.
Six, safer to experimenter and environment, detection process is made without gel electrophoresis therefore does not use EB etc. poisonous
Reagent.
Accompanying drawing explanation
The real-time RPA primer of Fig. 1: vibrio parahaemolyticus and probe design diagram, primer is irised out by square frame, visits
Pin is represented by italic overstriking, and the decorating site in probe is represented by underscore.
The canonical plotting of Fig. 2: amplification；
Fig. 3: specific detection figure.
Detailed description of the invention
RPA technology, full name is restructuring enzymatic polymerization enzymatic amplification technology (recombinase polymerase
Amplification, RPA), it is one and is participated in by multiple enzyme and albumen, under isothermal condition, realized nucleic acid exponential amplification
New technique.By expanding in simulation DNA body, under the isothermy of 37～42 DEG C, in 10min, produce purpose fragment, 40～
60min completes billions of DNA copy.Exploitation is applicable to the fluorescent quantitation RPA (real-of pathogenic bacterium detection on this basis
Time RPA) method is the key of the present invention.Real-time RPA passes through fluorescent dye or fluorescently-labeled specific spy
It is marked tracking, real time and on line monitoring course of reaction for RPA amplified production, product can be carried out in conjunction with corresponding software
Analyze.The present invention devises a fluorophor (Fluorophore) at probe 5 ' end, 3 ' ends devise a fluorescent quenching base
Group (Quencher), and devise an abasic site (abasic site) class in the centre position that two groups are connected
Like thing (tetrahydrofuran, THF).THF can by one from colibacillary exonuclease (exonuclease,
Exo) identify and shear.Fluorescence cannot be sent, when amplification to THF when Fluorophore and Quencher is on a chain
During site, exo identifies THF, and shears, and makes Fluorophore with Quencher separate, and fluorescence is accumulated, thus real
The accumulation synchronization with fluorescence signal of existing amplified production, reaches the effect monitored in real time.
In the actual application of RPA technology, primer and probe design play a key effect.Owing to RPA technology is for primer
There is with probe the design requirement being different from regular-PCR, in order to obtain optimized RPA primer, need to carry out primer screening, one
As can by primer sequence fine setting improve.A pair highly sensitive, high specificity, the primer of applicable RPA reaction system, undoubtedly
Make RPA method for quick more sensitive and accurate.
Having portable fluorescence detector at present can use, therefore the present invention provides a kind of real-time RPA method
The detection method of detection food-borne pathogens vibrio parahaemolyticus, carries out safe, special, quick, clever with realization to parahemolyticas
Field quick detection quick, simple, thus make up the deficiency of existing traditional sensing techniques.And the method is applicable to on-the-spot inspection
Survey, it is possible in time, effectively suppress vibrio parahaemolytisus poisoning to cause the generation of epidemic situation, improve food safety and ensure body
System.
Embodiment 1: primer and the screening of probe and the foundation of method
The present invention compiles according to the thermolability hemolysin (thermolabile hemolysin, TLH) of vibrio parahaemolyticus
Code gene tlh carries out the design of primer and probe:
Finding disclosed tlh gene order by NCBI, the primer of design, through Primer-BLAST comparison, has good
Specificity, for the purpose of the gene being positioned at tlh gene order 440-608 position, fragment carries out real-time RPA primer and spy
The design of pin.
Design 4 groups of primers to and 1 probe carry out optimal primer screening, primer sequence for fine setting sequence, be combined with probe
Sequence be respectively positioned on the centre of 4 pairs of primer amplified region, primer sets and probe sequence are as follows:
Forward primer F1-RPA:5 '-CAACATTAGATTTGGCGAACGAGAACGCAGAC-3 ' (32bp)
Reverse primer R1-RPA:5 '-TTAAAGATGTTGCCTGTATCAGACAAGCTGT-3 ' (31bp)
Product length: 169bp
2nd group:
Forward primer F2-RPA:5 '-CAACATTAGATTTGGCGAACGAGAACGCAGACA-3 ' (33bp)
Reverse primer R2-RPA:5 '-TTGCCTGTATCAGACAAGCTGTCACCGAGTG-3 ' (31bp)
Product length: 160bp
3rd group:
Forward primer F3-RPA:5 '-TTAGATTTGGCGAACGAGAACGCAGACATTACG-3 ' (33bp)
Reverse primer R3-RPA:5 '-AGATGTTGCCTGTATCAGACAAGCTGTCACCGA-3 ' (33bp)
Product length: 160bp
4th group:
Forward primer F4-PRA:5 '-TTAGATTTGGCGAACGAGAACGCAGACATT-3 ' (30bp)
Reverse primer R4-RPA:5 '-GTTGCCTGTATCAGACAAGCTGTCACCGAGTG-3 ' (32bp)
Product length: 156bp
Probe p:5 '-TTACGTTCTTCGCCGCTGACAATCGCTTCTCATACAACCACACGAT-3 ' (46bp)
Probe modification: the 30th bit base [T] of probe P carries out CF 5(6)-Carboxyfluorescein FAM labelling, and the 33rd bit base [T] is carried out
BHQ (Black Hole Quencher dyestuff) labelling, and the 31st bit base [C] abasic site analog [THF] replaces
Generation.
Probe P after modification:
5’-TTACGTTCTTCGCCGCTGACAATCGCTTC[FAM-dT][THF]A[BHQ-dT]ACAACCACACGAT-
3′。
Step 2) primer, probe screening: use set up " foundation of 2 recombinase polymerase isothermal amplification methods ", according to
RPA reaction system preparation reactant liquor, carry out RPA reaction system amplification, carry out fluorescence-causing substance detection simultaneously.Respectively with the 4 of design
The system that group primer pair and probe form expands, and amplified production is estimated by amplification curve.
Preliminary result shows, same reaction conditions, and the 3rd group of amplification efficiency of primer, product purity are better than other 3 groups and draw
Thing, therefore using the 3rd group of primer as preferably, renames primer:
Forward primer F-RPA:5 '-TTAGATTTGGCGAACGAGAACGCAGACATTACG-3 ' (33bp)
Reverse primer R-RPA:5 '-AGATGTTGCCTGTATCAGACAAGCTGTCACCGA-3 ' (33bp)
The foundation of 2 recombinase polymerase isothermal amplification methods
Including the steps
1) preparation RPA reaction system:
Each primer final concentration of: primers F-RPA and primer R-RPA each 420nmol/ μ L；Probe P 120nmol/ μ L；Slow
Rush liquid composition and concentration is: Tris-HCl (pH7.9) 50mmol/ μ L, KAc (potassium acetate) 100mmol/ μ L, DTT (two sulfur threoses
Alcohol) 2mmol/ μ L, 5%PEG 20mol/ μ L, dNTPs 200 μm ol/ μ L, ATP 3mmol/ μ L, pcr (creatine phosphokinase)
50mmol/ μ L, CK (creatine kinase) 100ng/ μ L, archaeal dna polymerase Bsu 30ng/ μ L, strand binding Protein G p32 300ng/ μ
L, recombinase UvsX 240ng/ μ L, auxiliary enzymes UvsY 60ng/ μ L, exonuclease exo 200ng/ μ L；Sample DNA templates is fitted
Amount, adds distilled water and makes unreacted mixed system (reaction mix) volume be 23.75 μ L；280mmol/ μ L MgAc (acetic acid
Magnesium) 1.25 μ L, making reaction system cumulative volume is 25 μ L.Magnesium acetate can make the beginning that is swift in response, it is therefore desirable to is eventually adding.
2) RPA reaction system amplification:
Aseptic 1.5mL centrifuge tube is sequentially added into all reactive components in addition to magnesium acetate and template DNA, after vibration from
The heart；Add appropriate template DNA, centrifugal after vibration；Reaction mix micropipettor is transferred to aseptic fluorescent quantitation special
With in sample-adding pipe, add 280mmol/ μ L MgAc (magnesium acetate) 1.25 μ L on the supporting transparent cover of dedicated pipe, close the lid
Son, centrifugal；Rock 10 times, centrifugal.
3) fluorescence-causing substance detection: the fluorescent quantitation instrument selected in the present invention is Roche company of Germany
96System.Pre-setting response procedures on display screen, including reaction temperature, reaction volume, probe luminous signal is corresponding
Fluorescein.The every 30s of course of reaction gathers a signal, is denoted as a circulation of reaction.After completing sample-adding, rapidly sample is added
On the position that 96 orifice plates of instrument are suitable for, and clicking on start button controlling main interface, reaction starts, and instrument is by automatically by setting
Put collection fluorescence signal.
3 reaction temperature optimizations
The vibrio parahaemolyticus DNA extracted with reference to DNA extraction kit description.Arrange 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C,
41 DEG C, 42 DEG C, 6 thermogrades, with 200ng extract vibrio parahaemolyticus DNA as template, add the present invention design primer
And probe, carry out nucleic acid amplification according to aforementioned loading methods.Fluorescent value according to amplification curve and signal value of taking off judges the suitableeest
Reaction temperature.Result shows, under the same terms, during 40 DEG C of reaction temperatures, the amplified signal departure time is shorter, and fluorescence signal
By force, therefore optimum temperature is reacted with 40 DEG C for subsequent experimental.
4 detection sensitivities are tested and prepare standard curve
It is that the vibrio parahaemolyticus genomic DNA sterilized water of 100ng/ μ L carries out gradient dilution to concentration so that each
In reaction system (25 μ L), the content of vibrio parahaemolyticus genomic DNA is respectively 100ng, 10ng, 1ng, 100pg, 10pg,
1pg and 0.Each concentration carries out three groups of parallel laboratory tests.Utilize the primers F-RPA and R-RPA of design, and probe P, to above-mentioned dilute
The vibrio parahaemolyticus genomic DNA being interpreted into variable concentrations carries out fluorescence RPA amplification respectively.
Result shows, along with the reduction of target amplification mrna concentration, the curve departure time little by little extends and fluorescence letter
Number value occur decline, still have amplification curve to generate when template content is 10pg in reaction system, 1pg and 0 nothing are taken off；When instead
Answering template Genome DNA content in system to have amplification curve clearly when reaching at or above 100pg, the departure time and fluorescence are strong
Degree fully meets Site Detection requirement.Visible, the sensitivity of the detection vibrio parahaemolyticus that this method is set up has reached 0.4pg/
uL。
The curve departure time according to fluorescence detector record, with the concentration of the vibrio parahaemolyticus of gradient dilution in system
Logarithm is abscissa, with average departure time of three parallel laboratory tests of homologous thread as vertical coordinate, draws standard curve.Such as Fig. 2
Shown in, the standard curve obtained is y=-1.8138x+12.067, coefficient R2It is 0.9202.
The limitation existed due to RPA reaction itself, gets started when reacting after Mg ion addition system, again because expanding
It is swift in response, so, the real-time RPA of the present invention can not accomplish the most accurate amplified production is carried out quantitative aspect.
But this does not affect its application quickly detected at the scene.
5 detection specificitys
Select vibrio alginolyticus, Vibrio vulnificus, vibrio cholera, Vibro harveyi, Vibrio anguillarum, Listeria monocytogenes, golden yellow
Staphylococcus, Salmonella judge experimental strain as specificity, and the DNA of above bacterial strain is by Ningbo City's inspection and quarantining for import/export
Office provides.According to aforementioned loading methods, the DNA profiling amount of various experimental bacteria is 10pg, and the DNA of above bacterial strain is carried out real-
Time RPA detects, and using vibrio parahaemolyticus as positive controls, sterilized water as negative control group.
Result is as it is shown on figure 3, only DNA profiling is that vibrio parahaemolyticus group response curve shows amplification, rather than pair is molten
All there is not amplification in courageous and upright vibrio group and negative control group.Result shows, the method that the present invention sets up can realize secondary haemolysis
The specific detection of property vibrio, not with other relevant pathogenic bacteria generation cross reactions.
The detection of actual sample is applied by embodiment 2
1 material
9 parts of aquatic products come from Distribution of Aquatic Products market, including 2 portions of extra large Semen Benincasae, 2 parts of white extra large Semen Benincasae, 2 parts of colored clams, 3 parts of razor clams
Son (10g net content/part).
2 reagent
3% sodium chloride basic peptone water (3%APW, vibrio parahaemolyticus selective enrichment is cultivated), sterilized water is (seedless
Acid enzyme)
3 sample treatment
Sample handling procedure is at " GB 4789.7-2013 national food safety standard food microbiological examination parahemolyticas
Vibrio check " on the basis of be modified slightly.
Put immediately after 3.1 non-frozen sample collectings in 7 DEG C～10 DEG C of cryopreservation casees and preserve, check the most early.
3.2 shellfishes take entire contents, including shellfish meat and body fluid.Band shell shellfish is then first scrubbed shell in tap water and gets rid of
Dry surface moisture, then opens shell with sterile working, takes appropriate section by above-mentioned requirements.
3.3 carry out sample homogenizing with Soviet Union's your swivel knife chip meat grinder of pool: take sample 10g with sterile working and put into Minced Steak cup
In, add a small amount of 3%APW, with low speed homogenizing 1min, the aseptic conical flask of 250mL put into by sample after grinding, then uses a small amount of 3%APW
Residual sample on flushing blade and in Minced Steak cup 1-2 time, washing liquid pours conical flask into, and residue 3%APW is finally all put into cone
Shape bottle, fully vibrates, and prepares the even liquid of sample of 1:10.
4 increase bacterium
4.1 draw 1:10 sample even liquid 1mL with pipettor, inject containing 9mL 3% sodium chloride basic peptone water
In 10mL centrifuge tube, shaking centrifuge tube mixing, prepare the even liquid of sample of 1:100.
The most separately take 10mL sterile centrifugation tube, by 3.1 operation sequences, be sequentially prepared 10 times of even liquid of series of diluted samples.
4.4.3 according to the estimation to sample pollution condition, 3 suitable serial dilution degree, each dilution factor inoculation 3 are selected
The 10mL centrifuge tube of Zhi Hanyou 9mL 3%APW, often pipe inoculation 1mL.Put in 36 DEG C ± 1 DEG C calorstat, cultivate 18h.
4.5 thick DNA extraction
The 1:10 sample even liquid 1mL taken in " prepared by 4.3 samples " boils 10min, is saved in-20 DEG C.
4.6 results are identified
Sample is carried out three kinds of method inspections: the DNA increasing the extraction of the sample even liquid before bacterium carries out real-time PCR respectively
The qualification of the real-time RPA that method and the present invention set up, the Vibrio parahaemolylicus pollusion then root in sample after increasing bacterium
According to " inspection of GB 4789.7-2013 national food safety standard food microbiological examination vibrio parahaemolyticus " by every g sample
Middle vibrio parahaemolyticus most probable number (MPN) is estimated.
Regulation in " pathogenic bacterium limitation in GB 29921-2013 national food safety standard food ", vibrio parahaemolyticus exists
Limitation in instant raw controlling the water circulation product is 1 detection vibrio parahaemolyticus of 5 sampling plans limitations, can accept of pathogenic bacterium index
The Limited Doses of level is 100MPN/g, the highest Safe limits 1000MPN/g.4.6.1Real time PCR detects parahemolyticas
Vibrio: with reference to pertinent literature, according to the thermolability hemolysin of vibrio parahaemolyticus (thermolabile hemolysin,
TLH) encoding gene tlh carries out real-time PCR primer design.
Forward primer F-PCR:5 '-ACTCAACACAAGAAGAGATCGACAA-3 ' (25bp)
Reverse primer R-PCR:5 '-GATGAGCGGTTGATGTCCAA-3 ' (20bp)
Probe P-PCR:5 '-CGCTCGCGTTCACGAAACCGT-3 ' (21bp)
Probe modification is 5 ' Texas Red, 3 ' BHQ2
Purpose fragment length: 208bp
Real-time PCR reaction system:
Table 1
As template, real-time PCR amplification is carried out with " the even liquid of 1:10 sample " the thick DNA before increasing bacterium.Real-time
PCR (two-step method) constant temperature circulating system is Roche company of Germany96System.Join according to document
Examining, the response procedures of optimization is: 95 DEG C, 10min；45cycles (95 DEG C, 5s；59 DEG C, 45s).Fluorescent value expands in each circulation
The terminal increased reads, and fluorescent labeling is Texas Red.
4.6.2 three kinds of methods are to the testing result of vibrio parahaemolyticus in sample:
Table 2
Note :+. positive；. negative
The above results show real-time RPA primer sets that the method designs and the result that detection method is obtained with
The result of real-time PCR is consistent, pollution level in National Standard Method is reached to the sample of 35MPN/g vibrio parahaemolyticus, front
The result of two kinds of methods, all shown as the positive, has reached Standard, it was demonstrated that primed probe of the present invention and method can
By property.
In sum, this method has more raw than existing conventional art PCR detection etc. has the dependent molecule of stronger instrument
Thing detection method, the higher specificity of method of food-borne pathogens vibrio parahaemolyticus, sensitivity, practicality and convenient
Property, in actual on-site field use, can favorably prevent and control the generation of vibrio parahaemolyticus security incident, carry out pre-in advance
Anti-.Use this method Site Detection to food-borne pathogens vibrio parahaemolyticus, pathogenic bacterium harm food can be stoped in time
Product and health, ensure food safety, and sets up more complete food safety Regulation system.
Claims (6)
1. the primer sets detecting vibrio parahaemolyticus Vibrio parahaemolyticus, it is characterised in that described draws
In thing group, the sequence information of primer is as follows:
The nucleotides sequence of forward primer is classified as SEQ ID NO:1, and the nucleotides sequence of reverse primer is classified as SEQ ID NO:2.
2. one kind is used for the probe coordinating primer sets described in claim 1 to use, it is characterised in that the sequence of described probe is
SEQ ID NO:3。
3. probe as claimed in claim 2, it is characterised in that the 30th bit base T of described probe carries out CF 5(6)-Carboxyfluorescein
FAM labelling, the 33rd bit base T carries out BHQ labelling, and the 31st bit base C abasic site analog THF substitutes.
4. primer sets described in claim 1 and the probe described in Claims 2 or 3 are at preparation detection vibrio parahaemolyticus Vibrio
Application in the goods of parahaemolyticus.
Apply the most as claimed in claim 4, it is characterised in that described goods are detection kit.
6. a detection vibrio parahaemolyticus Vibrio parahaemolyticus method, it is characterised in that described method bag
Include the steps:
1) preparation RPA reaction system:
Each primer final concentration of: the forward primer F-RPA described in claim and reverse primer each 420nmol/ μ L；Right is wanted
Seek the probe 120nmol/ μ L described in 2；Buffer forms and concentration is: Tris-HCl50mmol/ μ L, KAc 100mmol/ μ L,
DTT 2mmol/ μ L, 5%PEG 20mol/ μ L, dNTPs 200 μm ol/ μ L, ATP 3mmol/ μ L, pc 50mmol/ μ L, CK
100ng/ μ L, archaeal dna polymerase Bsu 30ng/ μ L, strand binding Protein G p32 300ng/ μ L, recombinase UvsX 240ng/ μ L,
Auxiliary enzymes UvsY 60ng/ μ L, exonuclease exo 200ng/ μ L；Sample DNA templates is appropriate, adds distilled water and makes unreacted
Mixed system volume is 23.75 μ L；280mmol/ μ L MgAc 1.25 μ L, making reaction system cumulative volume is 25 μ L；
2) RPA reaction system amplification:
Aseptic 1.5mL centrifuge tube is sequentially added into all reactive components in addition to magnesium acetate and template DNA, centrifugal after vibration；
Add appropriate template DNA, centrifugal after vibration；Reaction mix micropipettor is transferred to that aseptic fluorescent quantitation is special to be added
In sample pipe, add 280mmol/ μ L MgAc 1.25 μ L on the supporting transparent cover of dedicated pipe, close the lid, centrifugal；Rock
10 times, centrifugal；
3) fluorescence-causing substance detection: the fluorescent quantitation instrument of selection is Roche company of Germany96System, aobvious
Response procedures is pre-set, including reaction temperature, reaction volume, the fluorescein that probe luminous signal is corresponding in display screen；Reaction
The every 30s of process gathers a signal, is denoted as a circulation of reaction；After completing sample-adding, rapidly sample is joined the 96 of instrument
On the position that orifice plate is suitable for, and clicking on start button controlling main interface, reaction starts, and instrument is by automatically by arranging collection fluorescence
Signal.
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| CN107227378A (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 mimicus |
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