CN101818200A - Wound vibrio quantitative detecting test strip - Google Patents
Wound vibrio quantitative detecting test strip Download PDFInfo
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- CN101818200A CN101818200A CN201010131211A CN201010131211A CN101818200A CN 101818200 A CN101818200 A CN 101818200A CN 201010131211 A CN201010131211 A CN 201010131211A CN 201010131211 A CN201010131211 A CN 201010131211A CN 101818200 A CN101818200 A CN 101818200A
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Abstract
The invention discloses a primer, a probe, a wound vibrio detecting method and a detecting kit for wound vibrio detection in aquatic products. The target gene of the primer and probe combined site is vvhA350 site-800 site of the cytolysin structure gene of the wound vibrio. The wound vibrio in the aquatic products is quantitatively detected by a wound vibrio quantitative detecting test strip, comprising detecting test paper and reaction buffer solution, wherein the detecting test paper consists of a sample absorbing zone, a result scanning window and a terminal indicating window. The invention is characterized in that the marking object is the epitropous phosphor particle, has the advantages of quantitative detection, good sensitivity, stability and safety, and can quantitatively detect the wound vibrio in the aquatic products.
Description
Technical field
The present invention relates to a kind ofly, particularly Vibrio vulnificus specific gene fragment is had specific primer and probe based on vibrio vulnficus gene diagnosis detection by quantitative test strip in the fishery products of up-converting phosphor (UPT) nucleic acid chromatographic technique; Also relate to and use the pcr amplification method to detect the up-converting phosphor nucleic acid chromatography detection method and the detection kit of fishery products Vibrio vulnificus above-mentioned primer and probe.This detection method in order to sensitive, fast, Vibrio vulnificus in the detection by quantitative fishery products.
Background technology
Vibrio vulnificus is a kind of halophilism pathogenic bacteria that extensively distributes in ocean and the salt lake, this bacterium is for diabetes, alcoholic liver disease, liver cirrhosis, hepatitis and agnogenic hepatic insufficiency, acquired immune deficiency syndrome (AIDS) or other severe case's harm is quite serious, particularly to primary septicemia case, mortality ratio surpasses 50%.Most of in the world maritime nation has the case report that the lonely bacterium of wound infects at present.The route of infection of Vibrio vulnificus has two: the one, and peroral infection causes the primary septicemia, this mainly is because edible that give birth to or cause without the shellfish of fully processing; The 2nd, invade by skin wound, form redness, erythema in former skin wound and have an intense pain, development finally causes septicemia rapidly, this mainly is because the wound contact exists the shellfish of Vibrio vulnificus or seawater to cause.European Union has required the fishery products from China's import are detected Vibrio vulnificus, therefore, carries out the pollution condition investigation of Vibrio vulnificus in the fishery products, and the research rapid detection seems particularly important with the method for separating Vibrio vulnificus.
The traditional detection method of Vibrio vulnificus is to increase bacterium 12～16h by the APW that at first uses Vibrio vulnificus enrichment medium basic peptone water (APW) or interpolation PXB, fibres modified disaccharides PXB Polymyxin E agar or cellobiose Polymyxin E agar streak are cultivated 18h～24h, the suspicious bacterium colony of picking is inoculated with 3% sodium-chlor tryptose soya agar, carry out biochemical test after choosing bacterium, or select API 20E diagnostic reagent bar for use.Traditional method needs multiple substratum through a plurality of screening steps, and is consuming time longer; Other has the method for utilizing the vvh monoclonal antibody to detect; this method utilizes immunological method to identify after requiring the single bacterium colony of picking to increase bacterium in a large number; specificity is higher; but two kinds of method detection required times are all longer; in order to reduce human fishery products because of contact or the pollution of edible Vibrio vulnificus as far as possible, it is very important to the protection human health to set up sensitive, special detection method.
The vibrio vulnficus gene detection technique also is an emphasis of following Vibrio vulnificus detection method research for the rapid detection of pathogenic bacteria provides strong instrument.The present invention combines the vibrio vulnficus gene detection technique with nucleic acid amplification product chromatography detection method, set up quick, highly sensitive vibrio vulnficus gene diagnosis detection by quantitative test strip.
Summary of the invention
One object of the present invention is, provides a kind of Vibrio vulnificus specific gene fragment is had specific primer.
Above-mentioned purpose is achieved by the following technical solution.
A kind of Vibrio vulnificus detects and uses primer, it is characterized in that, and energy amplified target gene specific base sequence, described target gene is Vibrio vulnificus cytolysin structure gene vvhA.A part or its complementary strand complementation of 350～800 the Nucleotide of described primer and vvhA.
Described primer is made up of following primer:
Forward primer vvhA1:ccgcacttacattggtcc
Reverse primer vvhA2:tagggttgaacttcgtctta
Especially, vvhA1 forward primer or vvhA2 reverse primer be with vitamin H or digoxigenin labeled, 350～800 the Nucleotide of the vvhA that can increase.
For increasing the specificity of reaction, the present invention also provides a probe, a part or its complementary strand complementation of 350～800 the Nucleotide of this probe and vvhA, and the sequence of this probe is:
vvp：gccgtctttgttcacttccgc
And, this probe mark FITC.
Another object of the present invention is, a kind of detection method and detection kit of utilizing above-mentioned primer and probe to detect Vibrio vulnificus based on the polymerase chain reaction method is provided.
Above-mentioned purpose is achieved by the following technical solution:
A kind of detection method of Vibrio vulnificus: this method is used for detecting the Vibrio vulnificus of fishery products, it is characterized in that, a part or its complementary strand complementation with the Nucleotide of Vibrio vulnificus cytolysin structure gene vvhA350 position～800 are target, with the above-mentioned target region of above-mentioned primer selective amplification, confirm whether to have amplified production by the PCR method.
The test strip of fast quantification detection nucleic acid amplification product has been chosen in this experiment of pcr amplification product, comprise by absorption of sample district, result and scan detection test paper and the reaction buffer that window and terminal point indicating window constitute, fast quantification detects the nucleic acid amplification product test strip and is divided into base plate, glass layer, antibody adsorption film and thieving paper, and the method that fast quantification detects nucleic acid amplification product comprises and is labeled converting phosphor detection on thing antibody, the nucleic acid amplification mark.
Test paper is made up of sample pad, antibody adsorption film, thieving paper, viscosity end liner, test strip shell, wherein vitamin H or DigiTAb are adsorbed in the antibody adsorption film, sample is through last converting phosphor labelled reagent mark, process comprises mark vitamin H or digoxin primer amplified, flag F ITC specific probe and amplified production hybridization, last converting phosphor particle flag F ITC antibodies specific is in conjunction with FITC, and application of sample is in sample application zone, and labelling experiment result is by the interpretation of UPT biosensor analysis.
Concrete detection method is:
(1) sample preparation and template extraction
The water intaking product removes shell and organizes high-speed homogenization to handle, and gets the homogenate enrichment liquid and increases bacterium 12h, abandons supernatant after getting the centrifugal 5min of 1.5Ml enrichment liquid 10000rpm, adds 30 μ L distilled waters and boils 5min, gets supernatant 2 μ L behind the centrifugal 10min of 12000rpm as template DNA to be checked.
(2) the segmental amplification of wound bacillus purpose
Get amplification reaction solution, add reaction buffer, vvhA1, vvhA2, template, enzyme successively, last distilled water, form the reaction system of cumulative volume 20-100 μ L, mixing, instantaneous centrifugal afterreaction program is 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 30 circulations of 72 ℃ of 30s, 72 ℃ of 6min place 100 inactivator 5min at last.
(3) probe hybridization
Add probe in above-mentioned amplified production, final concentration is 1 μ M, and adding the afterreaction program is 98 ℃ of 30s, 58 ℃ of 20min.
(4) will go up step hybridization product and add the reaction buffer mixing, add in the test strip absorption of sample district, combine with the UCP marker.
Behind (5) 5～15min, observe the terminal point indicating window and determine that reaction finishes,, obtain the respective detection result with UPT biosensor scanning result scanning window.
A kind of Vibrio vulnificus detection kit.It is characterized in that test kit comprises: primer vvhA1, vvhA2, probe vvp, the PCR reaction buffer, the probe hybridization damping fluid contains FITC antibody, antibiotin or the DigiTAb of UCP mark, the test strip of positive control antibody.
Compared with prior art, the present invention has outstanding advantage and practicality:
1. detect fast: the method for separating the back biochemical identification with tradition is compared, and shortens in the 24h, detects fast.
2. detect accuracy rate height, high specificity: because the singularity of last converting phosphor technology, the stability that guarantees detection is gone up in its luminous influence that is not subjected to external environment to greatest extent, can specific recognition target dna fragment based on the gene probe of molecular hybridization.
3. highly sensitive, lowest detection is limited to 5pg DNA.
4. the result is the machine automatic interpretation, has reduced personal errors.
The present invention has specific primer sets and probe by providing a kind of to Vibrio vulnificus characteristics gene fragment, the detection examination that detects nucleic acid amplification product in conjunction with fast quantification detects Vibrio vulnificus specific gene fragment in the fishery products, detection reagent of the present invention and detection method have the susceptibility height, fast, advantage that specificity is high, can solve the problem of Vibrio vulnificus rapid detection in the fishery products.
Description of drawings
Accompanying drawing is a nucleic acid amplification product detection by quantitative test strip outward appearance;
Above-mentioned figure number is: 1, absorption of sample district, 2, the UCP marker, 3, the result scans window, 4, detection line, 5, nature controlling line, 6, the terminal point indicating window.
Embodiment
Embodiment describes in further detail the utility model below in conjunction with accompanying drawing:
(1) sample preparation and template extraction
The water intaking product removes shell and organizes high-speed homogenization to handle, and gets the homogenate enrichment liquid and increases bacterium 12h, abandons supernatant after getting the centrifugal 5min of 1.5Ml enrichment liquid 10000rpm, adds 30 μ L distilled waters and boils 5min, gets supernatant 2 μ L behind the centrifugal 10min of 12000rpm as template DNA to be checked.
(2) the segmental amplification of wound bacillus purpose
Get amplification reaction solution, add reaction buffer, vvhA1, vvhA2, template, enzyme successively, last distilled water, form the reaction system of cumulative volume 20-100 μ L, mixing, instantaneous centrifugal afterreaction program is 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 30 circulations of 72 ℃ of 30s, 72 ℃ of 6min place 100 inactivator 5min at last.
(3) probe hybridization
Add probe in above-mentioned amplified production, final concentration is 1 μ M, and adding the afterreaction program is 98 ℃ of 30s, 58 ℃ of 20min.
(4) 100 μ l reaction buffers and 10 μ l detected samples (hybridization product) are mixed; 100 μ l mixed solutions are added in the test strip absorption of sample district 1, react with UCP marker 2
Behind (5) 5～15min, observe terminal point indicating window 6 and determine that reaction finishes,, obtain the respective detection result with UPT biosensor scanning result scanning window 3.
Claims (10)
1. vibrio vulnficus gene diagnosis quantitative detecting method in the fishery products is characterized in that:
(1) design of the Auele Specific Primer of bacterial strain to be measured, screening and preparation;
(2) with specific amplification products can specificity bonded probe sequence design, screening and preparation;
(3) utilize the up-converting phosphor technology to carry out the nucleic acid chromatography detecting test paper strip of detection by quantitative behind the nucleic acid amplification.
2. the Auele Specific Primer of Vibrio vulnificus described in the claim 1 is characterized in that, the target gene of this primer is Vibrio vulnificus cytolysin structure gene vvhA.A part or its complementary strand complementation of 350～800 the Nucleotide of described primer and vvhA.
3. the composition of primer described in the claim 1:
Forward primer vvhA1:ccgcacttacattggtcc
Reverse primer vvhA2:tagggttgaacttcgtctta, and vvhA1 forward primer or vvhA2 reverse primer be with vitamin H or digoxigenin labeled, 350～800 the Nucleotide of the vvhA that can increase.
4. probe described in the claim 1 is characterized in that, this probe and the part or its complementary strand complementation that utilize 350～800 the Nucleotide of vvhA in the claim 2, and the sequence of this probe is: vvp:gccgtctttgttcacttccgc
5. probe described in the claim 4, mark FITC.
6. the detection by quantitative of nucleic acid amplification product described in the claim 1 test strip comprises and detects test paper and reaction buffer, it is characterized in that described marker is a last converting phosphor particle (UCP).
7. nucleic acid amplification product detection by quantitative test strip according to claim 6 is characterized in that having fixed on the test strip detection line vitamin H or DigiTAb.
8. nucleic acid amplification product detection by quantitative test strip according to claim 6, what it is characterized in that the UCP mark is FITC antibody.
9. Vibrio vulnificus detection method according to claim 1 is characterized in that, its concrete detection method is:
(1) sample preparation and template extraction
The water intaking product removes shell and organizes high-speed homogenization to handle, and gets the homogenate enrichment liquid and increases bacterium 12h, abandons supernatant after getting the centrifugal 5min of 1.5Ml enrichment liquid 10000rpm, adds 30 μ L distilled waters and boils 5min, gets supernatant 2 μ L behind the centrifugal 10min of 12000rpm as template DNA to be checked.
(2) the segmental amplification of wound bacillus purpose
Get amplification reaction solution, add reaction buffer, vvhA1, vvhA2, template, enzyme successively, last distilled water, form the reaction system of cumulative volume 20-100 μ L, mixing, instantaneous centrifugal afterreaction program is 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 30 circulations of 72 ℃ of 30s, 72 ℃ of 6min place 100 inactivator 5min at last.
(3) probe hybridization
Add probe in above-mentioned amplified production, final concentration is 1 μ M, and adding the afterreaction program is 98 ℃ of 30s, 58 ℃ of 20min.
(4) will go up step hybridization product and add the reaction buffer mixing, add in the test strip absorption of sample district, combine with the UCP marker.
Behind (5) 5～15min, observe the terminal point indicating window and determine that reaction finishes,, obtain the respective detection result with UPT biosensor scanning result scanning window.
10. one kind is detected Wound vibrio detection by quantitative test kit, and test kit comprises: primer vvhA1, vvhA2, probe vvp, the PCR reaction buffer, the probe hybridization damping fluid contains FITC antibody, antibiotin or the DigiTAb of UCP mark, the test strip of positive control antibody.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605046A (en) * | 2011-01-25 | 2012-07-25 | 福建省农业科学院生物技术研究所 | Kit and method for detecting streptococcus suis type 2 |
| CN103160601A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for Vibrio vulnificus and detection method using same |
| CN103773848A (en) * | 2013-12-10 | 2014-05-07 | 天津出入境检验检疫局动植物与食品检测中心 | Preparation method and application of nucleic acid lateral flow test strip for detecting listeria monocytogenes |
| CN104498524A (en) * | 2014-12-31 | 2015-04-08 | 山东农业大学 | Edwardsiella tarda ghost of recombinant vibrio vulnificus gene vvhA and construction |
| CN105198992A (en) * | 2015-10-16 | 2015-12-30 | 中国人民解放军海军总医院 | Preparation method and application for human-derived vibrio vulnificus hemolysin (VVH) resistant antibody |
| CN107384769A (en) * | 2017-08-24 | 2017-11-24 | 李峰 | Nucleic acid detection apparatus and its application based on paper |
| CN107922908A (en) * | 2015-08-26 | 2018-04-17 | 株式会社钟化 | Detection of nucleic acids equipment and nucleic acid detection method |
| CN110592201A (en) * | 2019-10-30 | 2019-12-20 | 上海千履基因科技有限公司 | PCR amplification method of one-tube type high-specificity nucleic acid product, rapid nucleic acid detection method and nucleic acid detection test strip |
-
2010
- 2010-03-04 CN CN201010131211A patent/CN101818200A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605046A (en) * | 2011-01-25 | 2012-07-25 | 福建省农业科学院生物技术研究所 | Kit and method for detecting streptococcus suis type 2 |
| CN102605046B (en) * | 2011-01-25 | 2013-09-11 | 福建省农业科学院生物技术研究所 | Kit and method for detecting streptococcus suis type 2 |
| CN103160601A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for Vibrio vulnificus and detection method using same |
| CN103160601B (en) * | 2013-04-08 | 2014-11-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for Vibrio vulnificus and detection method using same |
| CN103773848A (en) * | 2013-12-10 | 2014-05-07 | 天津出入境检验检疫局动植物与食品检测中心 | Preparation method and application of nucleic acid lateral flow test strip for detecting listeria monocytogenes |
| CN104498524A (en) * | 2014-12-31 | 2015-04-08 | 山东农业大学 | Edwardsiella tarda ghost of recombinant vibrio vulnificus gene vvhA and construction |
| CN107922908A (en) * | 2015-08-26 | 2018-04-17 | 株式会社钟化 | Detection of nucleic acids equipment and nucleic acid detection method |
| CN105198992A (en) * | 2015-10-16 | 2015-12-30 | 中国人民解放军海军总医院 | Preparation method and application for human-derived vibrio vulnificus hemolysin (VVH) resistant antibody |
| CN107384769A (en) * | 2017-08-24 | 2017-11-24 | 李峰 | Nucleic acid detection apparatus and its application based on paper |
| CN107384769B (en) * | 2017-08-24 | 2021-04-09 | 李峰 | Paper-based nucleic acid detection device and application thereof |
| CN110592201A (en) * | 2019-10-30 | 2019-12-20 | 上海千履基因科技有限公司 | PCR amplification method of one-tube type high-specificity nucleic acid product, rapid nucleic acid detection method and nucleic acid detection test strip |
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Application publication date: 20100901 |