CN106520937A - Shellfish vibrio parahemolyticus LAMP detection kit and application thereof - Google Patents
Shellfish vibrio parahemolyticus LAMP detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses shellfish vibrio parahemolyticus detection primer which is prepared from outside upstream primer F3, outside downstream primer B3, inside upstream primer FIP, inside downstream primer BIP, annular upstream primer LF and annular downstream primer LB. The invention further discloses a shellfish vibrio parahemolyticus LAMP detection kit and application thereof. A detection method of the shellfish vibrio parahemolyticus LAMP detection kit can quickly, conveniently and accurately detect pathogene; furthermore, the detection method is high in sensitivity; the results show that the LAMP detection method is strong in specificity by amplifying 10 strains of vibrio parahemolyticus and 20 strains of other vibrio; compared with a general PCR detection method, the detection method is high in sensitivity, and the sensitivity can reach 88fg/LAMP; in addition, whether byproducts are generated can be directly observed through eyes according to the LAMP detection method, and the reaction results can be judged.
Description
Technical field
The present invention relates to aquaculture shellfish vibrio detection field, and in particular to a kind of shellfish vibrio parahaemolytious LAMP detections
Test kit and its application.
Background technology
Vibrio parahaemolyticus (Vibrio Parahemolyticus), are gram negative bacilli, in arcuation, shaft-like, silk
The various shapes such as shape, anodontia spore.Food of the feed containing the bacterium can cause alimentary toxicosis, also referred to as halophilic bacteria alimentary toxicosis.Clinically
With Acute onset, stomachache, vomiting, diarrhoea and watery stool as cardinal symptom.Vibrio parahaemolyticus are a kind of marine bacterias, main next
Come from the marine products such as fish, shrimp, Eriocheir sinensiss, shellfish and Sargassum.This bacterium acid labile, can kill for 5 minutes in common vinegar；To heat
Resistance is weaker.
At present for the detection method of shellfish vibrio parahaemolyticus has：Traditional cultural method, immunological method, classics
PCR methods, real time fluorescent quantitative method etc..But, what is had in these detection methods time-consuming longer, some complex operations.Dividing so far
Very big progress is had been achieved for the detection method of vibrio in sub- level, each research show by PCR expand based on it is qualitative,
Quantitative detection method is practicable, and shows very big development prospect.But, in order to ensure the peace of shellfish food
Quan Xing, caters to the demand of quick detection shellfish vibrio parahaemolyticus, need to set up more rapidly, it is more accurate, sensitiveer, more special
Detection method.
2000, Japanese Rong Yan chemistry strains formula meeting Notomi T et al. developed a kind of new nucleic acid amplification technologies-ring
Mediated isothermal amplification technology.The amplification technique be able to can enter in the short time (typically 1h) under conditions of isothermal (60-65 DEG C)
The substantial amounts of nucleic acid amplification of row, is a kind of gene amplification for integrating the various advantages such as easy, quick, accurate, cheap.It
Compared with conventional PCR, it is not necessary to the process such as thermal denaturation, cyclic amplification, electrophoresis observation.LAMP detection method is characterized in that pin
Two pairs of primers (inner primer and outer primer) are designed to six regions on target gene, strand displacement type is utilized under conditions of constant temperature
Archaeal dna polymerase is reacted, and just can realize 10 in 15-60min9-1010Amplified reaction again, and it is substantial amounts of to react generation
Whether magnesium pyrophosphate white precipitate, directly carried out to judge to expand by observing white precipitate, and then judge whether target gene is deposited
.Loop-mediated isothermal amplification technique is a kind of method of brand-new nucleic acid amplification, with more sensitivity height, the spy of high specificity
Point, and it is easy to operate, it is independent of any special instrument and equipment and just can realizes that live high flux is quickly detected, detection
Cost is also well below quantitative fluorescent PCR.Therefore, the amplification technique can match in excellence or beauty in Standard PCR in each index, even better than routinely
PCR。
The content of the invention
Goal of the invention：The present invention will solve first technical problem and be to provide a kind of vibrio parahaemolytious detection primer.
Second technical problem to be solved by this invention there is provided vibrio parahaemolytious detection primer and prepare detection shellfish
Application in the LAMP detection kit of class vibrio parahaemolytious.
3rd technical problem to be solved by this invention there is provided the LAMP detection reagent of shellfish vibrio parahaemolyticus
Application of the box in the LAMP context of detection of shellfish vibrio parahaemolyticus.
4th technical problem to be solved by this invention there is provided the detection method of shellfish vibrio parahaemolyticus.
Technical scheme：In order to solve the problems, such as prior art, the present invention is employed the following technical solutions：A kind of secondary haemolysis
Vibrio detection primer, the detection primer include sequence table SEQ ID NO：Outside forward primer F3 shown in 1, sequence table SEQ
ID NO：Outside downstream primer B3 shown in 2, sequence table SEQ ID NO：Inner side forward primer FIP shown in 3, sequence table SEQ
ID NO：Inner side downstream primer BIP shown in 4, sequence table SEQ ID NO：Ring-type forward primer LF and sequence table SEQ shown in 5
ID NO：Ring-type downstream primer LB shown in 6.
Above-mentioned vibrio parahaemolytious detection primer is in the LAMP detection kit for preparing detection shellfish vibrio parahaemolytious
Using.
A kind of detection kit of the LAMP of shellfish vibrio parahaemolyticus, the test kit include：
1) LAMP reactant liquors：Which includes：Sequence table SEQ ID NO：Outside forward primer F3 shown in 1, sequence table SEQ
ID NO：Outside downstream primer B3 shown in 2, sequence table SEQ ID NO：Inner side forward primer FIP shown in 3, sequence table SEQ
ID NO：Inner side downstream primer BIP shown in 4, sequence table SEQ ID NO：Ring-type forward primer LF and sequence table SEQ shown in 5
ID NO：Ring-type downstream primer LB shown in 6；
2) positive reference substance；
3) negative controls.
Wherein, above-mentioned LAMP reactant liquors also include buffer, MgSO4, dNTPs, Betaine (glycine betaine), Bst DNA gather
Synthase.
Wherein, in above-mentioned detection kit various composition for 2.5 μ L10 × buffer (MgSO4 containing 2mM), 1.5 μ
The MgSO4 of L100mM, 3.5 μ L concentration dNTPs, the outside forward primer F3 that 0.25 μ L concentration is 25 μM and 0.25 μ L for 10mM
It is 25 μM that concentration is 25 μM of outside downstream primer B3, the inner side forward primer FIP that 2 μ L concentration are 25 μM and 2 μ L concentration
Inner side downstream primer BIP, the ring primer LF that 0.5 μ L concentration is 25 μM and ring primer LB, 0.25 μ L that 0.5 μ L concentration is 25 μM are dense
It is 8U/ μ L Bst archaeal dna polymerases to spend Betaine for 1.8M, 1 μ L concentration, uses ddH2O adds to cumulative volume for 25 μ L.
The LAMP detection kit of above-mentioned shellfish vibrio parahaemolyticus is in the LAMP detection sides of shellfish vibrio parahaemolyticus
The application in face.
The detection method of the LAMP detection kit of shellfish vibrio parahaemolyticus is comprised the following steps：
1) extract sample DNA；
2) respectively with step 1) sample DNA and negative control as template, carry out LAMP amplification obtain LAMP amplified productions；
3) LAMP amplified productions are centrifuged, have seen whether magnesium pyrophosphate white precipitate, so as to whether judgment sample is sun
Property；Or product carries out electrophoresis, whether trapezoid-shaped strips are had according to electrophoresis amplified band, come whether judgment sample is parahemolyticas arc
Bacterium.
Beneficial effect：Compared with prior art, it is an advantage of the invention that：
1st, the present invention devises a kind of vibrio parahaemolytious detection primer of uniqueness first.The present invention is tasted when primer is designed
Several genes target has been tried, and multipair primer, and the specificity and susceptiveness to every pair of primers has been devised for different target genes
Detected, it is through lot of experiments screening, optimization, final to determine using the primer of the present invention.
2nd, the inventive method can detect the sample of infection vibrio parahaemolyticus, particularly for detecting shellfish parahemolyticas
Vibrio, detection method energy is quick, easy for this, accurately detect pathogen, and detection, and detection spirit can be completed in 70min
Sensitivity is very high, by being expanded to 10 plants of vibrio parahaemolyticus and 20 plants of other vibrios, as a result shows LAMP detection method
Specificity is very strong；Compare with Standard PCR detection method, also show that very high sensitivity, sensitivity is up to 88fg/LAMP；
3rd, in addition, LAMP detection method can be by the presence or absence of generated by-product of direct visual perception reaction, it is not necessary to
By developer so as to judging reaction result.
Description of the drawings
Fig. 1：Vibrio parahaemolyticus LAMP product direct results：PCR pipe is centrifuged, pipe 1：The positive (has white magnesium pyrophosphate
Precipitation), show white precipitate in pipe 1 at arrow indication, therefore can be determined that and there occurs for LAMP reactions；2：Negative control：
Then do not occur white precipitate in pipe 2, therefore can be determined that to be the no generation of LAMP reactions.
Fig. 2：The vibrio parahaemolyticus LAMP product electrophoresis results of embodiment 3；M：2000marker 1-10 swimming lanes：It is secondary molten
Courageous and upright vibrio (totally 10 plants), 10 swimming lanes：Positive control, 11 swimming lanes：Negative control；After by LAMP product electrophoresis, if occurring
Trapezoid-shaped strips, then explanation reaction occur, and are positive；If there are not trapezoid-shaped strips, show to react and do not occur, be negative.
Added in 1-10 swimming lanes in figure is all LAMP product with each vibrio parahaemolyticus DNA as template, and 11 swimming lanes are the positive
Comparison DNA, and 12 swimming lanes add be with ddH2LAMP product of the O for template.As seen from the figure, 1-10 swimming lanes and 11 swimming
There are trapezoid-shaped strips in road, and 12 swimming lanes then do not occur.
Fig. 3：Vibrio parahaemolyticus and other vibrio LAMP product electrophoresis results；1 swimming lane sample-adding is with positive parahemolyticas
LAMP product of the vibrio DNA for template；It is anti-with LAMPs of the vibrio parahaemolyticus DNA as template that sample that 2 swimming lanes sample-adding is
Answer product；And the addition of 3 swimming lanes is with ddH2LAMP product blanks of the O for template；4～23 swimming lanes：Other vibrios
LAMP product (totally 20 plant) of the DNA for template；
Fig. 4：LAMP detection method sensitivity test result；With spectrophotometer to the positive vibrio parahaemolyticus extracted
DNA measures concentration, and surveyed concentration is 441.6ng/ μ L, and in each LAMP reaction system, template addition is 2 μ L, therefore, it is not dilute
It is 883.2ng that template amount is added equivalent to a LAMP system if releasing, equivalent to the template amount for adding after 10 times of doubling dilutions
It is respectively：88ng/LAMP、8.8ng/LAMP、880pg/LAMP、88pg/LAMP、8.8pg/LAMP、880fg/LAMP、88fg/
LAMP, 8.8fg/LAMP, 0.88fg/LAMP and 0.088fg/LAMP.In figure, 1-10 swimming lanes are 10 times to 10 respectively10Dilute again,
11 swimming lanes are negative controls, and 12 swimming lanes are positive controls, M：2000marker.LAMP detection method sensitivity may determine that by figure
88fg/LAMP can be reached.
Specific embodiment
The present invention is described in further details with reference to specific embodiment.
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not also without limitation on sheet described in detail in claims
Invention.Following examples are the not restrictions to inventing in order to be described in further detail to the present invention.The experiment side of the present invention
The equal reference of method《Fine works molecular biology experiment guide》Proposed by (F.M. Ao Sibai etc. are edited, and Science Press publishes for 2005)
Experiment condition.
Embodiment 1
1st, experiment material and reagent
Material：The present invention the vibrio parahaemolytious for being used all be isolated from Shandong Province Main Economic shellfish, including Scapharca subcrenata,
Tegillarca granosa, Conch Meretricis seu Cyclinae, Mactra quadrangularis, Solen strictuss, basket whelk, red spiral shell etc..Wherein 10 plants of vibrio parahaemolyticus (GenBank serial numbers are respectively：
KU197939、KU198017、KU197924、KU197923、KU197921、KU197906、KU197904、KU197903、
KU197899, KU197898, KU197893), (GenBank serial numbers are 20 plants of other vibrios respectively：KU197862、
KU197863、KU197864、KU197865、KU197866、KU197867、KU197868、KU197869、KU197947、
KU197948、KU197949、KU197950、KU197951、KU197952、KU197953、KU197954、KU197955、
KU197957、KU197958、KU197959。
Reagent：Bst archaeal dna polymerases (8000U/ml), dNTP (10mM), glycine betaine, MgSO4Solution (100mM) and routine
The LAMP related reagents such as PCR buffer (10 ×), purchased from New England Biolab companies；DNTP (4mM), buffer (10
×) etc. PCR related reagents, buy in Quan Shi King Companies；SanPrepDNA glue reclaim test kits, buying has in Shanghai biological engineering
Limit company；PMD18-T carriers, purchased from TaKaRa companies；DNA Marker2k are bought in Dalian TaKaRa companies；TCBS solids are trained
Foster base, purchased from Beijing Luqiao Technology Co., Ltd.；Agar, high-quality protein peptone, yeast invade profit powder, sea water crude salt etc..
2nd, vibrio parahaemolytious detection primer：Thermo-labile hemotoxin gene tlh base of the present invention for vibrio parahaemolyticus
Because of (serial number：KU197898 three pairs of specific primers) are devised：
The sequence of F3 is 5 '-TGCGAAAGTGCTTGAGATGA-3 ', SEQ ID NO：Shown in 1；
The sequence of B3 is 5 '-CGCAATGCGTGGGTGTAC-3 '；SEQ ID NO：Shown in 2；
The sequence of FIP is 5 '-CGTCTCGAACAAGGCGTGAGT-CAAGGCACAAGCGATGTACT-3 ', SEQ ID
NO：Shown in 3；
The sequence of BIP is 5-CTAACTTCTGCGCCCGAAGAGC-GACGATGAGCGGTTGATGTC-3 '；SEQ ID
NO：Shown in 4
The sequence of LF is 5 '-ATGTTGTAACCTT GCGCTTTGT-3 ', SEQ ID NO：Shown in 5；
The sequence of LB is 5 '-ACGGTTTCGTGAACGCGAG-3 '；SEQ ID NO：Shown in 6；
The detection kit of the LAMP of 2 shellfish vibrio parahaemolyticus of embodiment
1) LAMP reactant liquors：2 μ L sample DNA in 25 μ L reaction systems, the 10 × buffer (MgSO4 containing 2mM) of 2.5 μ L,
1.5 μ L concentration are the MgSO4 of 100mM, 3.5 μ L concentration for 10mM dNTPs, the outside forward primer that 0.25 μ L concentration is 25 μM
F3, the outside downstream primer B3 that 0.25 μ L concentration is 25 μM, the inner side forward primer FIP that 2 μ L concentration are 25 μM, 2 μ L concentration are
25 μM of inner side downstream primer BIP, the ring primer LF that 0.5 μ L concentration is 25 μM, the ring primer LB that 0.5 μ L concentration is 25 μM, 1 μ L
Bst archaeal dna polymerase, Betaine, the ddH of 8.75 μ Ls that 0.25 μ L concentration be 1.8M of the concentration for 5U/ μ L2O。
2) positive reference substance：Vibrio parahaemolyticus gene group DNA；
3) negative controls：ddH2O.
Embodiment 3LAMP atopic is tested
In order to verify the specificity of the inventive method, with the method for ordinary hot thermal cracking to 10 plants of vibrio parahaemolyticus
(GenBank serial numbers are respectively：KU197939、KU198017、KU197924、KU197923、KU197921、KU197906、
KU197904, KU197903, KU197899, KU197898, KU197893) and 20 plants of other vibrios (GenBank serial numbers difference
It is：KU197862、KU197863、KU197864、KU197865、KU197866、KU197867、KU197868、KU197869、
KU197947、KU197948、KU197949、KU197950、KU197951、KU197952、KU197953、KU197954、
KU197955、KU197957、KU197958、KU197959.) DNA extraction is carried out, 30 sample DNAs are obtained, above-mentioned sample is taken
DNA is template, according to the LAMP reaction systems in embodiment 2 according to loop-mediated isothermal amplification technique (Loop mediated
Isothermal amplification, LAMP) principle expanded, and reaction condition is：65 DEG C of incubation 60min；80 DEG C of degeneration
5min；Amplified production is detected and analysis：Centrifugation observation precipitation or 2% (W/V) agarose gel electrophoresiies testing goal band are simultaneously
Imaging analysis；Result judgement：As shown in figure 1, PCR pipe is centrifuged, white precipitate in pipe 1, at arrow indication, is shown, therefore can
To be judged to that LAMP reactions there occurs.Then do not occur white precipitate in pipe 2, therefore can be determined that as no of LAMP reactions
It is raw.As shown in Fig. 2 1-10 swimming lanes are that (GenBank serial numbers are vibrio parahaemolytious respectively in Fig. 2：KU197939、KU198017、
KU197924、KU197923、KU197921、KU197906、KU197904、KU197903、KU197899、KU197898、
KU197893), 11 is positive control, and 12 is negative control；As shown in figure 3,1,2 swimming lanes are vibrio parahaemolytious in Fig. 3
KU197898、KU197939；3 swimming lanes are blanks；4～23 swimming lanes be other vibrio KU197862, KU197863,
KU197864、KU197865、KU197866、KU197867、KU197868、KU197869、KU197947、KU197948、
KU197949、KU197950、KU197951、KU197952、KU197953、KU197954、KU197955、KU197957、
KU197958、KU197959.After by LAMP product electrophoresis, if there are trapezoid-shaped strips, illustrate that reaction occurs, be positive；
If there are not trapezoid-shaped strips, show to react and do not occur, be negative.
The sensitivity experiment of embodiment 4LAMP reaction
The DNA sample of the vibrio parahaemolyticus extracted by embodiment 3 carries out 10 times of doubling dilutions respectively successively, uses light splitting light
Degree instrument to extract positive vibrio parahaemolyticus DNA measurement concentration, surveyed concentration be 441.6ng/ μ l, each LAMP reactant
In system, template addition is 2 μ l, therefore, it is 883.2ng that undiluted words add template amount equivalent to a LAMP system；10 times
After doubling dilution equivalent to the template amount for adding it is respectively：88ng/LAMP、8.8ng/LAMP、880pg/LAMP、88pg/LAMP、
8.8pg/LAMP, 880fg/LAMP, 88fg/LAMP, 8.8fg/LAMP, 0.88fg/LAMP and 0.088fg/LAMP.As a result as schemed
Shown in 4, LAMP detection method sensitivity can reach 88fg/LAMP.
Embodiment 5：The Performance Evaluation of detection kit
Be that the performance to test kit of the present invention is estimated, product sample is repeatedly made according to the technique after products perfection
Sensitivity, specificity, degree of accuracy, stability to test kit and clinical trial is carried out with the product of trial production, to examine or check product
The performance of product.
1st, the range of linearity of test kit detection and sensitivity test
The DNA sample of the vibrio parahaemolyticus to extracting carries out 10 times of doubling dilutions respectively successively, after 10 times of doubling dilutions
Equivalent to the template amount for adding it is respectively：88ng/LAMP、8.8ng/LAMP、880pg/LAMP、88pg/LAMP、8.8pg/
LAMP, 880fg/LAMP, 88fg/LAMP, 8.8fg/LAMP, 0.88fg/LAMP and 0.088fg/LAMP.As a result show, LAMP
Detection method sensitivity can reach 88fg/LAMP.
The test result of the 1 test kit range of linearity of the present invention of table
| Dilution gradient | 10-4 | 10-5 | 10-6 | 10-7 | 10-8 | 10-9 |
| Ct values | 20.4 | 23.3 | 26.7 | 29.9 | 33.7 | No Ct |
2nd, the specificity analyses of test kit detection
Specific detection is carried out to 5 plants of vibrio parahaemolytious and 5 plants of non-vibrio parahaemolytious, has as a result been shown, vibrio parahaemolytious
Equal test positive, non-vibrio parahaemolytious testing result are feminine gender, and specificity reaches 100%.
The test result of 2 test kit specificity of the present invention of table
| Sample | C1 | C2 | C3 | C4 | C5 | N1 | N2 | N3 | N4 | N5 |
| Ct values | 20.3 | 21.6 | 22.9 | 20.8 | 22.1 | No Ct | No Ct | No Ct | No Ct | No Ct |
3rd, the elaboration detected in test kit batch
According to test kit operation instruction, same sample is detected with 10 batch products, as a result shown, between different batches
Difference is not notable, and the coefficient of variation is 2.17%.
The precision test result of 3 test kit of the present invention of table
| Sample | 1 | 2 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | CV% |
| Ct values | 23.5 | 23.9 | 22.8 | 22.9 | 23.2 | 23.6 | 23.2 | 22.6 | 24.1 | 2.17 |
4th, the accuracy of test kit is determined
Carry out confirming the accuracy of test kit of the present invention using sequence measurement.Amplified production is sequenced, sequence with it is pre-
Phase result is completely the same, it was demonstrated that the testing result of test kit of the present invention is accurate.
5th, the Stability Determination of test kit
The stability of 5.1 test kits
The stability of product depends on the stability of each composition.LAMP reactant liquors, Taq DNA are prepared in the present invention
Polymerase, positive control, preserve at -20 DEG C, are preserved always to 4 DEG C of refrigerators after taking-up, and most long preservation has not yet to see under performance for one week
Drop.
In the Product transport prepared for clinical trial, a complete set of product (includes LAMP reactant liquors, Taq archaeal dna polymerases, examination
Agent box positive control), formerly after Jing go through 3 days by a definite date -20 DEG C of freezings, 4 DEG C of long-distance transports, -20 DEG C freeze, melt again etc. it is a series of
After two-way process, detected using quality-control product, testing result has no that there were significant differences.Show that each component of test kit of the present invention is suitable
It is stable.
The stability of 5.2 reference substances
Analysis of the stability of reference substance to result of the test judges have a significant impact, and the reference substance of this test kit is mainly right
Reaction system carries out quality control.This test kit has used a robust positive control and a negative control, carries out freeze thawing to which
Detection.Experimental result as shown in table 4, as a result shows that the reference substance in test kit of the present invention also has good stability.
The freezing-thawing test result of 4 positive reference substance of table
| Number of freezing and thawing | The CT values of positive control | The CT values of negative control |
| 1 | 22.9 | No Ct |
| 2 | 23.2 | No Ct |
| 3 | 23.6 | No Ct |
| 4 | 23.5 | No Ct |
| 5 | 23.9 | No Ct |
| 6 | 22.8 | No Ct |
| 7 | 23.2 | No Ct |
| 8 | 22.6 | No Ct |
Finally it should be noted that above example is only to illustrate technical scheme rather than the present invention is protected
The restriction of scope, although being explained in detail to the present invention with reference to preferred embodiment, one of ordinary skill in the art should manage
Solution, technical scheme can be modified or equivalent, without deviating from technical solution of the present invention essence and
Scope.
SEQUENCE LISTING
<110>Nanjing Fu Hai bio tech ltd
<120>A kind of shellfish vibrio parahaemolytious LAMP detection kit and its application
<130> SG2016001
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Outside forward primer F3
<400> 1
tgcgaaagtg cttgagatga 20
<210> 2
<211> 18
<212> DNA
<213>Outside downstream primer B3
<400> 2
cgcaatgcgt gggtgtac 18
<210> 3
<211> 41
<212> DNA
<213>Inner side forward primer FIP
<400> 3
cgtctcgaac aaggcgtgag tcaaggcaca agcgatgtac t 41
<210> 4
<211> 42
<212> DNA
<213>Inner side downstream primer BIP
<400> 4
ctaacttctg cgcccgaaga gcgacgatga gcggttgatg tc 42
<210> 5
<211> 22
<212> DNA
<213>Ring-type forward primer LF
<400> 5
atgttgtaac cttgcgcttt gt 22
<210> 6
<211> 19
<212> DNA
<213>Ring-type downstream primer LB
<400> 6
acggtttcgt gaacgcgag 19
Claims (7)
1. a kind of vibrio parahaemolytious detection primer, it is characterised in that the detection primer includes sequence table SEQ ID NO：Shown in 1
Outside forward primer F3, sequence table SEQ ID NO：Outside downstream primer B3 shown in 2, sequence table SEQ ID NO：Shown in 3
Inner side forward primer FIP, sequence table SEQ ID NO：Inner side downstream primer BIP shown in 4, sequence table SEQ ID NO：5 institutes
The ring-type forward primer LF for showing and sequence table SEQ ID NO：Ring-type downstream primer LB shown in 6.
2. the vibrio parahaemolytious detection primer described in claim 1 is preparing the LAMP detection reagent of detection shellfish vibrio parahaemolytious
Application in box.
3. a kind of detection kit of the LAMP of shellfish vibrio parahaemolyticus, it is characterised in that the test kit includes
1）LAMP reactant liquors：Which includes：Sequence table SEQ ID NO：Outside forward primer F3 shown in 1, sequence table SEQ ID
NO：Outside downstream primer B3 shown in 2, sequence table SEQ ID NO：Inner side forward primer FIP shown in 3, sequence table SEQ ID
NO：Inner side downstream primer BIP shown in 4, sequence table SEQ ID NO：Ring-type forward primer LF and sequence table SEQ ID shown in 5
NO：Ring-type downstream primer LB shown in 6；
2）Positive reference substance；
3）Negative controls.
4. the LAMP detection kit of shellfish vibrio parahaemolyticus according to claim 3, it is characterised in that the LAMP
Reactant liquor mainly also includes buffer, MgSO4, dNTPs, Betaine, Bst archaeal dna polymerase.
5. the LAMP detection kit of shellfish vibrio parahaemolyticus according to claim 4, it is characterised in that the detection
In test kit, the various composition 10 × buffer containing 2mM MgSO4 that is 2.5 μ L, the MgSO4 of 1.5 μ L100mM, 3.5 μ L concentration are
The dNTPs of 10 mM, the outside forward primer F3 that 0.25 μ L concentration is 25 μM and the outside downstream that 0.25 μ L concentration is 25 μM are drawn
Thing B3, the inner side forward primer FIP that 2 μ L concentration are 25 μM and inner side downstream primer BIP, 0.5 μ L that 2 μ L concentration are 25 μM are dense
Spend ring primer LF and ring primer LB that 0.5 μ L concentration is 25 μM for 25 μM, 0.25 μ L concentration dense for Betaine, the 1 μ L of 1.8M
Spend for 8U/ μ L Bst archaeal dna polymerases, use ddH2O adds to cumulative volume for 25 μ L.
6. the LAMP detection kit of the shellfish vibrio parahaemolyticus described in any one of claim 3 ~ 5 is in shellfish parahemolyticas arc
The application of the LAMP context of detection of bacterium.
7. the detection method of the LAMP detection kit of the shellfish vibrio parahaemolyticus described in any one of claim 3 ~ 5, which is special
Levy and be, comprise the following steps：
1）Extract sample DNA；
2）Respectively with step 1）Sample DNA and negative control be template, carry out LAMP amplification obtain LAMP amplified productions；
3）LAMP amplified productions are centrifuged, magnesium pyrophosphate white precipitate has been seen whether, so as to whether judgment sample is positive；Or
Person's product carries out electrophoresis, whether has trapezoid-shaped strips according to electrophoresis amplified band, comes whether judgment sample is vibrio parahaemolyticus.
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