CN107099585A - Vibrio damsela detection primer pair, kit and detection method - Google Patents
Vibrio damsela detection primer pair, kit and detection method Download PDFInfo
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Abstract
Vibrio damsela detection primer pair, kit and detection method.The primer pair base sequence is SEQ ID NO.1/2.The present invention further discloses the Vibrio damsela detection method that the kit comprising above-mentioned primer pair and the above-mentioned primer pair of application carry out TD PCR detections.The primer specificity of the present invention is strong, and detection kit and method simplicity are easy-to-use, as a result accurately, with very high specificity and sensitivity.
Description
Technical field
The present invention relates to the quick determination method of foodborne bacterial pathogenses, more particularly to using in TD-PCR technology for detection samples
The method of Vibrio damsela.Further relate to detect used specific primer sequence and detection kit.
Background technology
Kinds of pathogenic vibrio is widely present in littoral seawater, deposit and marine organism, and some kinds are found in fresh water,
It is considered as fish, shrimp, a kind of common pathogen of mottle clam, some of which kind is also to cause acute human gastroenteritis
The main pathogenic fungi, have strict restriction index to it in aquatic products sanitary standard.Vibrio damsela (Vibrio
Damsela it is) one of 12 kinds of kinds of pathogenic vibrio, also known as Mermaid luminous bacillus (Photobacterium damselae).
Vibrio damsela is isolated from the maiden's fish disease stove for suffering from skin ulcer first equal to 1981 by Love.Bacterium distribution is wide
It is general, its presence can be found in seawater, marine product and aquatic/marine animals；As Vibrio furnissii, the bacterium is also in recent years
Come a kind of new kinds of pathogenic vibrio generally acknowledged.There is data to show, substantial amounts of cell can be produced after Vibrio damsela intrusion host cell
Dissolving element, from the cytotoxin produced by other vibrios on physicochemical property it is different.Vibrio damsela main infection poikilotherm is outstanding
It is cultured fishes, can also cause the wound infection of the mankind, therefore be referred to as " the common ill substance of mermaid ".
Fast and accurately pathogenic bacteria are the important leverages of effective prevention and control pathogenic bacterial infection in detection food, still
At present both at home and abroad in existing detection method, there was only conventional pathogenicbacteria separation cultivation, this method to the detection of Vibrio damsela
Not only waste time and energy, it is impossible to meet the requirement that fast high-flux detects pathogen；Along with vibrio bacteria biochemical reaction is relative
It is unstable, many difficult and uncertainty is brought to appraisal, false negative and false positive results, influence detection easily occurs
Outcome quality, and larger economic loss can be brought.With the development of molecular biology technology, round pcr is commonly used
In the quick diagnosis of bacterium.Touchdown PCR (touchdown PCR) technology, is mainly used in the optimization of PCR conditions.In many situations
The design of lower primer causes PCR to be difficult to, such as specific not enough fallibility with.Annealing temperature is too high to make PCR efficiency mistakes
It is low, but annealing temperature is too low, non-specific amplification can be made excessive.Although this can be optimized by making repeated attempts, time-consuming to take
Power.Touchdown PCR provides a more easy optimization method.Its principle is：Expand at a higher temperature first, though now
Right amplification efficiency is low, but non-specific amplification does not have substantially, and with the reduction of annealing temperature, non-specific amplification can progressively increase
It is many.But because now specific amplification products have reached certain predominance, therefore non-specific amplification can be produced strong
Strong Competitive assays, so as to greatly improve the specificity and efficiency of PCR amplifications.This round pcr enters medical treatment in large quantities
Clinical practice, the HLA partings of the detection method based on high flux PCR, such as people use this technology mostly.And prior art
In yet there are no for Vibrio damsela detect TD-PCR technologies, therefore the present invention for Vibrio damsela qualitative detection have weight
Want meaning.
The content of the invention
It is an object of the invention to provide one kind can quickly, accurate, delicately ocean fish in detection food and aquatic products sample
The TD-PCR detection methods of vibrios.
To achieve the above object, present invention firstly provides a kind of Vibrio damsela detection primer pair, its base sequence is SEQ
ID NO.1/2。
Forward primer (SEQ ID NO.1)：5 '-CTGAACTTTACATTGTGGAGGGTG-3 ',
Reverse primer (SEQ ID NO.2)：5′-AACGCAAGCGAAGTCTGGAA-3′.
On the other hand, the present invention provides a kind of Vibrio damsela detection kit, and the kit is TD-PCR detection examinations
Agent box, including the primer pair that base sequence is SEQ ID NO.1/2.
Preferably, also including being used for the positive control template that TD-PCR is detected, the positive control mould in the kit
Plate is that concentration is 10nmol/LThe DNA extracts of 100nmol/L Vibrio damsela reference culture.The kit should in detection
In, it is negative control template that can use other any non-Vibrio damsela DNA extracts, and the use of distilled water is blank pair
According to.
Another object of the present invention is to provide a kind of detection method of Vibrio damsela, methods described is TD-PCR detection sides
Method, it includes entering the step of performing PCR is expanded using SEQ ID NO.1/2 as primer pair.
The detection object of the detection method of the Vibrio damsela of the invention described above is food and aquatic products sample, it is therefore intended that inspection
Look into and whether there is Vibrio damsela in testing sample.In this method, PCR is expanded with Vibrio damsela gyrB gene (the GenBank numbers of logging in
AJ249850.1 the nucleotide sequence of 941~1370 (SEQ ID NO.3)) is target gene, with shown in SEQ ID NO.1/2
Primer pair, by target gene described in TD-PCR selective amplifications, and detects and is confirmed whether to there are amplified production.
Except the selectivity to primer is used, also include in the detection method of Vibrio damsela of the present invention to TD-PCR
The setting of specific amplification reaction condition, the amplification procedure and condition are：
1. 94 DEG C of pre-degeneration 4min；
2. expand：94 DEG C of 30s, 69 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94℃30s、68℃30S、72℃
1min, 2 circulations；Amplification：94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94℃30S、66℃30S、72℃
1min, 2 circulations；Amplification：94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94℃30S、64℃30S、72℃
1min, 2 circulations；Amplification：94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94℃30S、60℃30S、72℃
1min, 18 circulations；
3. 72 DEG C extend 7min, 4 DEG C of preservations.
More specifically, the invention described above Vibrio damsela detection method, comprise the following steps：
(1) testing sample DNA is extracted, template liquid is obtained；
(2) TD-PCR is expanded：
Reaction system：The μ L of template liquid 2, the μ L of 10 × PCR buffer solutions 2.5, dNTP 2 μ L, the μ L of Taq enzyme 0.125, concentration are 10 μ
The M μ L of primer pair SEQ ID NO.1/2 1, supplement sterilizing distilled water to cumulative volume is 25 μ L；Above each component is added to 0.2mL
In PCR reaction tubes, mix, 5000r/min centrifugations 10s；
Reaction condition：1 zero 94 DEG C of pre-degeneration 4min；2 zero circulations of amplification (94 DEG C of 30s, 69 DEG C of 30S, 72 DEG C of 1min) 2,
(94 DEG C of 30s, 68 DEG C of 30S, 72 DEG C of 1min) 2 circulations are expanded, amplification (94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min) 2 is circulated,
(94 DEG C of 30S, 66 DEG C of 30S, 72 DEG C of 1min) 2 circulations are expanded, amplification (94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min) 2 is circulated,
(94 DEG C of 30S, 64 DEG C of 30S, 72 DEG C of 1min) 2 circulations are expanded, amplification (94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min) 2 is circulated,
Expand (94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 1min) 18 circulations；3 zero 72 DEG C of extension 7min, 4 DEG C of preservations；
(3) TD-PCR amplified productions electrophoresis detection：
2% Ago-Gel is prepared with electrophoretic buffer, step (2) prepared μ L of TD-PCR amplified productions 5 are taken, with 1 μ L
Point sample after sample-loading buffer mixing, reference is done with DNA molecular amount label；3V/cm~5V/cm constant pressure electrophoresis, electrophoresis 20min~
40min, electrophoresis detection result Labworks image acquisition and analysis software is recorded and preserved；
(4) result judgement：Using Vibrio damsela reference culture as positive control, the moon is used as using other non-Vibrio damsela bacterial strains
Property control, using sterilize distilled water for masterplate as blank control progress step (1)~operation (4)；
The positive can be determined as testing sample am-plified fragments size is consistent with positive control, if sample without amplified production or
The cause not of uniform size of person's product fragment can be determined as feminine gender, and positive amplification fragment need to be sequenced and be compared with reference sequences
It is right；
Under testing conditions of the present invention：The TD-PCR amplified productions of positive control are 430bp, negative control and blank control without
Amplified production.
In embodiment, the preparation method of step (1) template liquid can be obtained from the prior art.Art technology
Personnel can determine in template liquid DNA concentration according to prior art and ensure to detect samples met standard.DNA is used in the present invention
Concentration is 10nmol/L100nmol/L template liquid enters performing PCR amplification.
Specifically, following methods can be used by template liquid being prepared by raw material of preservation strain：3% chlorination of testing sample
After sodium basic peptone water Zengjing Granule, take 1.0mL nutrient solutions to centrifuge 5min in 13000rpm, abandon supernatant, extracted and tried with DNA
The template liquid of agent box processing；Or take 1.0mL nutrient solutions to centrifuge 5min in 13000rpm, supernatant is abandoned, 100 μ L is then added and goes out
The vibration of bacterium distilled water boils 10min after mixing, and is cooled to 13000rpm after room temperature and centrifuges 2min, takes 2 μ L of supernatant liquid to do template liquid.
When the above-mentioned Zengjing Granule time generally requires 8h~18h to about 0.5 Maxwell turbidity of bacteria concentration, the template hydroful of gained enough survey by reaction
The requirement of amount.
TD-PCR augmentation detections, the bacterium with classical way are carried out using the method for the present invention, especially specific primer
Culture, separation, authentication method are compared, and method of the invention is more rapidly, accurately.And compared with conventional method high specificity, sensitivity is high,
It is 100% to Vibrio damsela detection specificity, detection sensitivity is 180CFU/mL.
Brief description of the drawings
The width of accompanying drawing 6 of the present invention：
Fig. 1 be Vibrio damsela primer pair 1 to specific assay result, wherein：1、24:Marker DL2000；2、3、4、5、
6、7：Vibrio damsela DNA TD-PCR products；8：Water is compareed；9、10：Comma bacillus DNA TD-PCR products；11、12：Secondary haemolysis
Property vibrios DNA TD-PCR products；13、14：Vibrio alginolyticus DNA TD-PCR products；15、16：Vibrio vulnificus DNA TD-PCR are produced
Thing；17、18：Vibrio mimicus DNA TD-PCR products；19、20：Vibrio furnissii DNA TD-PCR products；21、22：Vibrio campbellii
DNA TD-PCR products；23：Water is compareed.
Fig. 2 is the specific assay result of primer pair 3, wherein：1、25：Marker DL2000；2、3、4、5、6、7：Ocean fish arc
Bacterium DNA TD-PCR products；8、9：Vibrio hollisae DNA TD-PCR products；10、11：Shark vibrio DNA TD-PCR products；
12、13：Aeromonas hydrophila DNA TD-PCR products；14、15：E. coli dna TD-PCR products；16、17：Shigella
DNA TD-PCR products；18、19：Vibrio flurialis DNA TD-PCR products；20、21：Maxwell vibrios DNA TD-PCR products；TD-PCR
Product；22、23：Vibrio cincinnatiensis DNA TD-PCR products；24：Water is compareed.
Fig. 3 is the primer pair specific assay result of primer pair 3, wherein：1、25:Marker DL2000；2、3、4、5、6、7：
Vibrio damsela DNA TD-PCR products；8：Water is compareed；9、10：Comma bacillus DNA TD-PCR products；11、12：Parahemolyticas arc
Bacterium DNA TD-PCR products；13、14：Vibrio alginolyticus DNA TD-PCR products；15、16：Vibrio vulnificus DNA TD-PCR products；
17、18：Vibrio mimicus DNA TD-PCR products；19、20：Vibrio furnissii DNA TD-PCR products；21、22：Vibrio campbellii DNA
TD-PCR products；23、24：Water is compareed.
Fig. 4 is the specific assay result of primer pair 3, wherein：1、25：Marker DL2000；2、3、4、5、6、7：Ocean fish arc
Bacterium DNA TD-PCR products；8、9：Vibrio hollisae DNA TD-PCR products；10、11：Shark vibrio DNA TD-PCR products；
12、13：Aeromonas hydrophila DNA TD-PCR products；14、15：E. coli dna TD-PCR products；16、17：Shigella
DNA TD-PCR products；18、19：Vibrio flurialis DNA TD-PCR products；20、21：Maxwell vibrios DNA TD-PCR products；TD-PCR
Product；22、23：Vibrio cincinnatiensis DNA TD-PCR products；24：Water is compareed.
Fig. 5 is the sensitivity test result of primer pair 1, wherein：1、22:Marker DL2000；2、3：Vibrio damsela (1.8 ×
106CFU/mL) DNA PCR primers；4th, 5 Vibrio damsela (1.8 × 105CFU/mL) DNA PCR primers；6、7：Vibrio damsela (1.8
×104CFU/mL) DNA PCR primers；8、9：Vibrio damsela (1.8 × 103CFU/mL) DNA PCR primers；10、11：Vibrio damsela
(1.8×102CFU/mL) DNA PCR primers；12、13：Vibrio damsela (1.8 × 101CFU/mL) DNA PCR primers；14、15：Sea
Vibrio piscium (1.8CFU/mL) DNA PCR primers；16、17、18、19：Negative control；20、21：Water is compareed.
Fig. 6 is the sensitivity test result of primer pair 3, wherein：1、22:Marker DL2000；2、3：Vibrio damsela (1.8 ×
106CFU/mL) DNA PCR primers；4th, 5 Vibrio damsela (1.8 × 105CFU/mL) DNA PCR primers；6、7：Vibrio damsela (1.8
×104CFU/mL) DNA PCR primers；8、9：Vibrio damsela (1.8 × 103CFU/mL) DNA PCR primers；10、11：Vibrio damsela
(1.8×102CFU/mL) DNA PCR primers；12、13：Vibrio damsela (1.8 × 101CFU/mL) DNA PCR primers；14、15：Sea
Vibrio piscium (1.8CFU/mL) DNA PCR primers；16、17、18、19：Negative control；20、21：Water is compareed.
Embodiment
It is below the specific embodiment of the present invention, its foundation to technical solution of the present invention and its application are made further
It is bright, but present disclosure is not limited in any form.
If without specified otherwise, biological sample used in our department's separating tests comes from the Experiment on Microbiology of Dandong Entry-Exit Inspection and Quarantine Bureau
Room, the aquatic products and animals and plants sample of Dalian Microbiological Lab of Entry-Exit Inspection and Quarantine Bureau.
DNA extraction kit：TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit
Ver.3.0 (article No. 9763) is purchased from precious bioengineering (Dalian) Co., Ltd；
PCR expands enzymatic reagent：TaKaRa Ex Taq (article No. RR001A) are purchased from precious bioengineering (Dalian) Co., Ltd；
ABI Veriti PCR instruments (ABI companies, the U.S.).
Embodiment 1, design of primers
First, the design of primer
The gyrB gene orders of Vibrio damsela are downloaded in NCBI, and Multiple Sequence Alignment is carried out using MEGA6 softwares.Choose sequence
The highly conserved region of row, designs primer, altogether simultaneously using the softwares of Primer 5 and the online design of primers instruments of primer 3
6 pairs of primer sequences are obtained, 1 is shown in Table.
Table 1
2nd, the screening of primer
The primer of design is screened using the Primer-BLAST in NCBI, the length of each primer of Integrated comparative, Tm
The parameters such as value, G/C content, primer self-complementarity, specificity, filter out (the i.e. SEQ ID of Vibrio damsela detection primer pair 1
NO.1/2) tested with primer pair 3.
Embodiment 2, set up detection method
First, primer
Two pairs of specific primers of screening are designed from embodiment 1：
Primer pair 1：
Forward primer：5 '-CTGAACTTTACATTGTGGAGGGTG-3 ',
Reverse primer：5′-AACGCAAGCGAAGTCTGGAA-3′；
Primer pair 2：
Forward primer：5′-AGAAAATGCTGCGCTGTTCA-3′
Reverse primer：5′-TAAGCGCTTCAATCACTGGC-3′.
2nd, the foundation of Vibrio damsela PCR detection method：
1st, template DNA sample solution is prepared；
The DNA extracts reagents that the extracting of the present embodiment genomic DNA is produced using precious bioengineering (Dalian) Co., Ltd
Box (article No. 9763) is simultaneously stripped sample DNA by its operating instruction, obtains template liquid.
The measure of template liquid DNA concentration and purity：Take DNA solution plus ddH made from 5 μ L2O gradient dilutions are used to 1mL
The OD value that nucleic acid-protein analyzer or ultraviolet specrophotometer are surveyed at 260nm and 280nm.DNA concentration is according to following public affairs
Formula, which is calculated, to be obtained：
C=A × N × 50/1000,
In formula：
C is DNA concentration (μ g/ μ L)；
A is the light absorption value at 260nm；
N is nucleic acid extension rate；
1OD260nm=50 μ g/mL double-stranded DNAs；
Work as OD260/OD280Ratio is suitable for PCR amplifications when between 1.7~1.9.
2nd, TD-PCR is detected
(1) reaction system：The μ L of template liquid 2, the μ L of 10 × PCR buffer solutions 2.5, dNTP 2 μ L, the μ L of Taq enzyme 0.125, concentration are
10 μM primer pair 1 (SEQ ID NO.1/2) 1 μ L, supplement sterilizing distilled water to cumulative volume are 25 μ L；Above each component add to
In 0.2mL PCR reaction tubes, mix, 5000r/min centrifugations 10s；
(2) reaction condition
1) the CD-PCR reaction conditions of Vibrio damsela are detected using primer pair 1：1 zero 94 DEG C of pre-degeneration 4min；2 zero amplifications
(94 DEG C of 30s, 69 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30s, 68 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30S, 66 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30S, 64 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 18 circulations of amplification (94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 1min)；3○
72 DEG C of extension 7min, 4 DEG C of preservations；
2) the CD-PCR reaction conditions of Vibrio damsela are detected using primer pair 3：1 zero 94 DEG C of pre-degeneration 4min；2 zero amplifications
(94 DEG C of 30s, 68 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 66 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 64 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 2 circulations of amplification (94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min), amplification
(94 DEG C of 30S, 62 DEG C of 30S, 72 DEG C of 1min) 2 circulations, 18 circulations of amplification (94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 1min)；3○
72 DEG C of extension 7min, 4 DEG C of preservations；
(3) step (2) TD-PCR amplified productions electrophoresis detection：
2% Ago-Gel is prepared with electrophoretic buffer, 5 μ L pcr amplification products are taken, it is mixed with 1 μ L sample-loading buffers respectively
Close, carry out point sample, reference is done with DNA molecular amount label.3V/cm~5V/cm constant pressure electrophoresis, electrophoresis 20min~40min, electricity
Swimming testing result Labworks image acquisition and analysis software is recorded and preserved.The Vibrio damsela TD-PCR amplified productions of positive control are
430bp or 230bp, negative control and blank control are without amplified production.Sample amplification segment size is consistent with positive control to be
The positive, if sample is feminine gender without the cause not of uniform size of amplified production or product fragment.Positive amplification fragment need to be sequenced simultaneously
It is compared with reference sequences.
Embodiment 3, specific test
The detection method that the Vibrio damsela detection primer that Application Example 1 is screened is set up to 1 and primer pair 3 and embodiment 2,
The DNA profiling of Vibrio damsela reference culture is detected.Detection contains Maxwell vibrios, vibrio alginolyticus, thermophilic aqueous vapor unit cell simultaneously
Bacterium, vibrio mimicus, Vibrio vulnificus, comma bacillus, vibrio parahemolyticus, Vibrio hollisae, shark vibrio, Vibrio flurialis, ocean fish arc
Other DNA of pathogenic samples such as bacterium, Vibrio campbellii, Escherichia coli, staphylococcus aureus, are commented with the specificity to method
Estimate, testing result is as Figure 1-4：
Vibrio damsela detection primer shows (Fig. 1 and Fig. 2) to 1 electrophoresis detection result, and negative control and blank control are without expansion
Increase production thing, illustrate that reaction result is normal, reaction system is pollution-free；It is big that Vibrio damsela gyrB genetic test results amplify 430bp
Small genetic fragment, testing result is the positive；The gyrB genetic test results of other pathogenic bacteria show no amplified production, detection knot
Fruit is feminine gender.
Vibrio damsela detection primer shows (Fig. 3 and Fig. 4) to 3 electrophoresis detection results, and negative control and blank control are without expansion
Increase production thing, illustrate that reaction result is normal, reaction system is pollution-free；It is big that Vibrio damsela gyrB genetic test results amplify 230bp
Small genetic fragment, testing result is the positive；The gyrB genetic test results of other pathogenic bacteria show no amplified production, detection knot
Fruit is feminine gender.
From the above experiments, it was found that Vibrio damsela primer pair 1 and primer pair 3 are respectively provided with good specificity.
Embodiment 3, sensitivity test
First, reference culture pure culture detection sensitivity
The detection method that the Vibrio damsela primer pair 1 and primer pair 3 and embodiment 2 that Application Example 1 is screened are set up, to sea
Vibrio piscium reference culture pure culture carries out the research of detection sensitivity.
Take above-mentioned standard bacterial strain to be cultivated in 3% sodium chloride basic peptone water after 18h or so, survey its Maxwell turbidity, estimate
Its bacterium number is counted, then gradient dilution is incrementally carried out to 10 by ten times-7, take the bacterium solution of last four dilutions to carry out plate count,
Each dilution factor repeats 3, is averaged according to progress colony counting after corresponding method culture and determines its real strain density.
Each dilution respectively takes 1mL bacterium solutions in 2mL centrifuge tubes and makees 3 pipes and repeats simultaneously, for extracting DNA and applying the inventive method
TD-PCR detections are carried out, to determine the detection sensitivity of the inventive method.
The electrophoresis detection result of Vibrio damsela primer pair 1 is shown in Fig. 5, it can be seen that negative control and blank control without
Amplified production, illustrates that reaction result is normal, reaction system is pollution-free；Positive control amplified production is the gene piece of 430bp sizes
Disconnected, reaction result is normal；Reference culture pure culture sensitivity technique result shows that Vibrio damsela reference culture concentration is respectively
It can be detected very well when 1800CFU/mL, 180CFU/mL.As a result show that the standard method detects spirit to reference culture pure culture
Sensitivity 180CFU/mL.
The electrophoresis detection result of Vibrio damsela primer pair 3 is shown in Fig. 6, it can be seen that negative control and blank control without
Amplified production, illustrates that reaction result is normal, reaction system is pollution-free；Positive control amplified production is the gene piece of 230bp sizes
Disconnected, reaction result is normal；Reference culture pure culture sensitivity technique result shows that Vibrio damsela reference culture concentration is respectively
It can be detected very well when 18000CFU/mL, 1800CFU/mL.As a result show that the standard method is detected to reference culture pure culture
Sensitivity 1800CFU/mL.
From the above experiments, it was found that Vibrio damsela primer pair 1 is higher than the sensitivity of Vibrio damsela primer pair 3, difference
10 times, therefore the present invention chooses the progress follow-up test of Vibrio damsela primer pair 1.
2nd, the sensitivity experiment of Vibrio damsela reference culture is added in sample
The detection method that the Vibrio damsela primer pair 1 and embodiment 2 that Application Example 3 is screened are set up, to being added in sample
Vibrio damsela reference culture carries out the research of detection sensitivity.
The sample negative from Vibrio damsela is had determined, takes 10g samples to add in 100mL enrichment liquids and adds simultaneously respectively
Take 1mL homogenizing fluids to extract DNA after the Vibrio damsela of known various concentrations gradient, homogeneous, for sensitivity test, determine in sample
Add the detection sensitivity after Vibrio damsela.Negative sample homogenizing fluid 1mL is extracted simultaneously extracts DNA for TD-PCR detections.
Testing result：Negative control and blank control illustrate that reaction result is normal, reaction system is without dirt without amplified production
Dye；Positive control amplified production is the gene segment of 430bp sizes, and reaction result is normal；Reference culture pure culture sensitivity
Testing result shows that Vibrio damsela reference culture concentration can be detected very well when being respectively 1800CFU/mL, 180CFU/mL.Knot
Fruit shows the standard method to reference culture pure culture detection sensitivity 180CFU/mL.
Embodiment 4, applied to actual sample detection
The detection method that the Vibrio damsela primer pair 1 and embodiment 2 that Application Example 3 is screened are set up, with traditional detection
Method is control (ISO/TS 21872-2Microbiology of food and animal feeding stuffs-
Horizontal method for the detection of potentially enteropathogenic Vibrio
spp.–Part 2:Detection of species other than Vibrio parahaemolyticus and
Vibrio cholerae.).Actually detected sample includes shrimp, fish, chicken in jelly meat and freezes totally 400 parts, testing result such as table 2 such as cauliflower
It is shown：
Table 2
As a result show, detect totally 400 parts of actual sample, wherein：
300 parts of aquatic products are detected, detect that Vibrio damsela is positive 5 parts using TD-PCR methods, using conventional biochemical culture side
Method detection Vibrio damsela is positive 5 parts；
50 parts of animal product is detected, is not detected using TD-PCR methods detection Vibrio damsela, using conventional biochemical culture
Method detection Vibrio damsela does not detect；
50 parts of plant product is detected, is not detected using TD-PCR methods detection Vibrio damsela, using conventional biochemical culture
Method detection Vibrio damsela does not detect.
From substantial amounts of result of practical application, Vibrio damsela is detected using the inventive method, with preferable application knot
Really.
SEQUENCE LISTING
<110>Complex art service centre of Dandong Entry-Exit Inspection and Quarantine Bureau
<120>Vibrio damsela detection primer pair, kit and detection method
<130> N/A
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence
<400> 1
ctgaacttta cattgtggag ggtg 24
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
aacgcaagcg aagtctggaa 20
<210> 3
<211> 430
<212> DNA
<213> Vibrio damsela
<400> 3
ctgaacttta cattgtggag ggtgactctg cgggcggtag tgctaagcag ggtcgtaatc 60
gtaaaaacca agctatcttg ccacttaaag gtaagatcct aaacgttgaa aaagcacgct 120
ttgataagat gctatcttca caagaagtgg caaccctgat cactgcgatg ggttgtggta 180
tcggccgcga tgaatacaac ccagataaat tacgttatca cagcatcatt atcatgactg 240
atgccgacgt cgatggttcg cacattcgta cgctactgtt gaccttcttc tatcgtcaaa 300
tgccagaatt gattgaacgt ggtcatatct atattgctca gccaccgctt tacaaagtga 360
aaaaaggcaa acaagagcag tacatcaaag atgatgaaga tatgcagcag ttccagactt 420
cgcttgcgtt 430
Claims (8)
1. Vibrio damsela detection primer pair, it is characterised in that base sequence is SEQ ID NO.1/2.
2. Vibrio damsela detection kit, including the primer pair that base sequence is SEQ ID NO.1/2.
3. Vibrio damsela detection kit according to claim 2, it is characterised in that the kit also includes concentration
For the DNA extracts of 10~100nmol/L Vibrio damsela reference culture.
4. a kind of detection method of Vibrio damsela, it is characterised in that methods described includes as primer pair entering using SEQ ID NO.1/2
The step of row TD-PCR is expanded.
5. the detection method of the Vibrio damsela described in claim 4, it is characterised in that the TD-PCR amplifications are with Vibrio damsela
The nucleotide sequence that gyrB genes are 941~1370 is target gene.
6. detection method according to claim 5, it is characterised in that described TD-PCR amplification reaction conditions are
1. 94 DEG C of pre-degeneration 4min；
2. expand：94 DEG C of 30s, 69 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94 DEG C of 30s, 68 DEG C of 30S, 72 DEG C of 1min, 2
Circulation；Amplification：94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94 DEG C of 30S, 66 DEG C of 30S, 72 DEG C of 1min, 2
Circulation；Amplification：94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94 DEG C of 30S, 64 DEG C of 30S, 72 DEG C of 1min, 2
Circulation；Amplification：94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 1min, 18
Circulation；
3. 72 DEG C extend 7min, 4 DEG C of preservations.
7. the Vibrio damsela detection method described in claim 4, comprises the following steps：
(1) testing sample DNA is extracted, template liquid is obtained；
(2) TD-PCR is expanded：
Reaction system：The μ L of template liquid 2, the μ L of 10 × PCR buffer solutions 2.5, dNTP 2 μ L, the μ L of Taq enzyme 0.125, concentration are 10 μM
The μ L of primer pair SEQ ID NO.1/2 1, supplement sterilizing distilled water to cumulative volume are 25 μ L；Above each component is added to 0.2mL
In PCR reaction tubes, mix, 5000r/min centrifugations 10s；
Reaction condition：1. 94 DEG C of pre-degeneration 4min；2. expand：94 DEG C of 30s, 69 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94
DEG C 30s, 68 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94 DEG C of 30S, 67 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94
DEG C 30S, 66 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification:94
DEG C 30S, 64 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94 DEG C of 30S, 63 DEG C of 30S, 72 DEG C of 1min, 2 circulations；Amplification：94
DEG C 30S, 60 DEG C of 30S, 72 DEG C of 1min, 18 circulations；3. 72 DEG C extend 7min, 4 DEG C of preservations；
(3) TD-PCR amplified productions electrophoresis detection：
2% Ago-Gel is prepared with electrophoretic buffer；Step (2) prepared μ L of TD-PCR amplified productions 5 are taken, with 1 μ L loadings
Point sample after buffer solution mixing, reference is done with DNA molecular amount label；3V/cm~5V/cm constant pressure electrophoresis, electrophoresis 20min~
40min, electrophoresis detection result Labworks image acquisition and analysis software is recorded and preserved；
(4) result judgement：It is right using other non-Vibrio damsela bacterial strains as feminine gender using Vibrio damsela reference culture as positive control
According to, using sterilize distilled water as masterplate as blank control carry out step (1)~operation (4)；
The positive can be determined as testing sample am-plified fragments size is consistent with positive control, if sample is without amplified production or production
Thing clip size is inconsistent can be determined as feminine gender, and positive amplification fragment need to be sequenced and is compared with reference sequences.
8. the Vibrio damsela detection method described in claim 7, it is characterised in that the preparation method of described step (1) template liquid
It is：When testing sample is with 3% sodium chloride basic peptone water Zengjing Granule to about 0.5 Maxwell turbidity of bacteria concentration, 1.0mL is taken to cultivate
Liquid in 13000rpm centrifuge 5min, abandon supernatant, the template liquid handled with DNA extraction kit, or take 1.0mL nutrient solutions in
13000rpm centrifuges 5min, abandons supernatant, then adds after 100 μ L sterilizing distilled water vibrations are mixed and boils 10min, is cooled to room
13000rpm centrifuges 2min after temperature, takes 2 μ L of supernatant liquid to do template liquid.
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