CN102108402B - Kit and detection method for detecting vibrio harveyi - Google Patents
Kit and detection method for detecting vibrio harveyi Download PDFInfo
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- CN102108402B CN102108402B CN 201010586975 CN201010586975A CN102108402B CN 102108402 B CN102108402 B CN 102108402B CN 201010586975 CN201010586975 CN 201010586975 CN 201010586975 A CN201010586975 A CN 201010586975A CN 102108402 B CN102108402 B CN 102108402B
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Abstract
The invention relates to a kit and detection method for detecting vibrio harveyi by using the loop-mediated isothermal amplification technique. The kit comprises a loop-mediated isothermal amplification reaction solution, a Bst DNA (deoxyribonucleic acid) polymerase and a color-developing agent, wherein the reaction solution contains a reaction buffering solution Thernopol, dNTP (deoxyribonucleotide triphosphate), MgSO4, a forward inter primer (Vh-FIP) TTCGCTTTCGCGAGCCATCTGGTTACCAATTGATCGCCCG, a reverse inter primer (Vh-BIP) ACGCAGAATCAAGCAGTGTGCCGATTTATTCGCCACGACA, a forward outer primer (Vh-F3) CAAAACGGTTCCGAAACGC, a reverse outer primer (Vh-B3) TCGATTCCCCAAGTTTGGAG, a betaine, and sterile distilled water. The detection method comprises the following steps: extracting bacterium DNAs, carrying out loop-mediated isothermal amplification, carrying out color-developing detection and the like. By designing the primers according to the vibrio harveyi toxR gene conservation area and detecting by using the LAMP (loop-mediated isothermal amplification) technique, the invention achieves high specificity and high sensitivity; the kit has the advantages of high detection speed, high accuracy, excellent sensitivity, convenient on-site application and the like; and the defects of long cycle, low sensitivity, high cost, difficult on-site application and the like in the prior art are solved.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of test kit that adopts loop-mediated isothermal amplification technique rapid detection aquatic animal Vibrio harveyi, and the method that detects Vibrio harveyi with this test kit.
Background technology
Vibrio harveyi is a kind of Gram-negative halophilic bacterium that is distributed widely in the ocean environment.Vibrio harveyi is the important pathogenic bacteria of the aquatic animal that just is realized nearest more than ten years.It is the important pathogenic bacteria of the countries and regions cultured prawns such as Indonesia, Thailand, India, Australia, Ecuador and China (comprising Taiwan) and shellfish, is again the cause of disease of many cultured fishes, has caused huge financial loss to mariculture industry.
Sensitive, quick, special cause of disease detection technique is significant to the prevention and control of the aquatic animal disease that Vibrio harveyi causes.The method that is used at present the Vibrio harveyi detection comprises pathogen separation and molecular biology method.Based on the Maximum probable number method (Most probable number, MPN) of bacterium separation and Culture although be widely adopted the method time-consuming (4-6 days); Although the nucleic acid probe hybridization technology is special, sensitive, complex steps; Polysaccharase connection formula reaction (polymerase chain reaction, PCR) and real-time fluorescence quantitative PCR (real-time PCR) although technology is sensitive, fast, but two kinds of methods require the laboratory that PCR instrument or quantitative fluorescent PCR equipment are arranged, and high to the operator quality requirement, and need also be a numerous and diverse problem by electrophoresis step acquisition result behind the pcr amplification.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplication, LAMP) is a kind of novel nucleic acid amplification technologies.Amplified reaction is carrying out (60-65 ℃) under constant temperature, be a kind of special, efficiently, the technology of DNA amplification fast.Because the amplification efficiency of LAMP is very high, the amplified reaction result can be by visual inspection behind the fluorescent dyeing; Amplified reaction can produce a large amount of magnesium pyrophosphates and make reaction present muddiness, need not electrophoresis after LAMP amplification finishes directly visual inspection react and whether have turbid phenomenon to judge whether amplification, and do not need expensive PCR instrument, the basic unit that can be widely used in cause of disease is detected.
Summary of the invention
One of purpose of the present invention is to provide a kind of test kit that adopts loop-mediated isothermal amplification technique rapid detection aquatic animal Vibrio harveyi.
Two of purpose of the present invention is to provide a kind of method of using this test kit to detect Vibrio harveyi.
The present invention detects the test kit of aquatic animal Vibrio harveyi, and its reagent comprises following three group reagents:
The first group reagent is for being used for the reagent of preparation isothermal amplification liquid, and this group reagent comprises Themopol reaction buffer, dNTP solution, MgSO
4Solution, upstream inner primer (Vh-FIP), downstream inner primer (Vh-BIP), upstream outer primer (Vh-F3), downstream outer primer (Vh-B3), alkali solution of beet and aseptic double-distilled water;
The moiety of described Themopol reaction buffer is Tris-HCl the solution, (NH of pH8.8
4)
2SO
4, KCl, MgSO
4And Triton X-100;
Described upstream inner primer (Vh-FIP) is TTCGCTTTCGCGAGCCATCTGGTTACCAATTGATCGCCCG, such as sequence＜400〉shown in 3;
Described downstream inner primer (Vh-BIP) is ACGCAGAATCAAGCAGTGTGCTCCGATTTATTCGCCACGACA, such as sequence＜400〉shown in 4;
Described upstream outer primer (Vh-F3) is CAAAACGGTTCCGAAACGC, such as sequence＜400〉shown in 1;
Described downstream outer primer (Vh-B3) is: TCGATTCCCCAAGTTTGGAG, such as sequence＜400〉shown in 2;
And the amount of each reagent in this group reagent and concentration are enough to make the isothermal amplification liquid that each composition has following working concentration:
The second group reagent is the Bst archaeal dna polymerase;
The 3rd group reagent is developer.
Preferably, the concentration of each reagent is as follows in described the first group reagent:
In actual the use, the compositional system of the every pipe 23 μ L of described isothermal amplification liquid is preferably as follows:
The concentration of described Bst archaeal dna polymerase is 8 activity units of every microlitre, and namely concentration is 8U/ μ L.
Described developer is 10wt% fluorescence dye SYBR GREEN I.
The present invention uses the mentioned reagent box to detect the method for aquatic animal Vibrio harveyi, and it comprises the steps:
(1) extraction of vibrios DNA
A. 28 ℃ of single bacterium colonies of cultivating 16-24 hour of picking place the centrifuge tube that contains the 0.1-0.5ml sterilized water, and thalline fully suspends;
B.95-100 take out ice bath 2-5 minute after ℃ boiling water bath 10-15 minute;
C. use desk centrifuge 10000-120000 rev/min centrifugal 5-10 minute, collect in supernatant liquor to the new 1.5mL centrifuge tube, be the plate DNA that touches to be checked;
(2) ring mediated isothermal amplification of Vibrio harveyi
A. prepare isothermal amplification liquid;
B. in the reaction tubes that 23 μ L isothermal amplification liquid are housed, add the 1 μ L plate DNA that touches to be checked, in the electric heating constant temperature water bath, placed 3-5 minute for 95 ℃, placed immediately 1-3 minute on ice;
C. in reaction tubes, add 1 μ L Bst archaeal dna polymerase;
D. in the electric heating constant temperature water bath is bathed 60-65 ℃ placed 30-90 minute;
E. the electric heating constant temperature water-bath is transferred to 80-95 ℃ of termination reaction, is taken out after 3-5 minute;
(3) color developing detection
Add 1 μ L developer in reaction tubes, whether the colour-change that directly detects by an unaided eye is Vibrio harveyi according to colour-change situation judgement sample.
Wherein, the composition of described 23 μ L isothermal amplification liquid is preferably:
The concentration of described Bst archaeal dna polymerase is preferably 8 activity units of every microlitre, and namely concentration is 8U/ μ L.
Described developer is preferably 10wt% fluorescence dye SYBR GREEN I, adopts in the situation of this developer, in color developing detection, if color is yellow, illustrate that bacterium to be detected is not Vibrio harveyi, become green such as color, then interpret sample is Vibrio harveyi.
Advantage of the present invention:
The present invention has set up loop-mediated isothermal amplification detection kit and the detection method of aquatic animal Vibrio harveyi, the primer pair that this test kit adopts is identified the toxR sequences Design according to the vibrios kind, select the toxR conserved sequence of Vibrio harveyi during design of primers, considering simultaneously that primer can not increase carries the vibrios of toxR sequence, such as Vibrio parahaemolyticus and vibrio alginolyticus, to guarantee to detect the reliability of different sources Vibrio harveyi bacterial strain.The present invention adopts the LAMP technology, this technology high specificity, highly sensitive, as not need costliness PCR instrument, only need common constant temperature water bath to get final product, amplification is observed with fluorescence dye and is got final product, and detection method is easily gone, and is easy and simple to handle, and with low cost, can detect quicker, special, delicately Vibrio harveyi.Can be used for the detection of Vibrio harveyi, be particularly suitable for department of basic unit and use.Solved long, the defective such as sensitivity is lower, cost is high, rig-site utilization is difficult of cycle of existing in the prior art.
Embodiment
Below the invention will be further described by binding sequence table and embodiment.
Its reagent of test kit in the present embodiment comprises following three group reagents:
The first group reagent is for being used for the reagent of preparation isothermal amplification liquid, and this group reagent comprises Themopol reaction buffer, dNTP solution, MgSO
4Solution, upstream inner primer (Vh-FIP), downstream inner primer (Vh-BIP), upstream outer primer (Vh-F3), downstream outer primer (Vh-B3), alkali solution of beet and aseptic double-distilled water;
The moiety of described Themopol reaction buffer is Tris-HCl the solution, (NH of pH8.8
4)
2SO
4, KCl, MgSO
4And Triton X-100;
Described upstream inner primer (Vh-FIP) is TTCGCTTTCGCGAGCCATCTGGTTACCAATTGATCGCCCG, such as sequence＜400〉shown in 3;
Described downstream inner primer (Vh-BIP) is ACGCAGAATCAAGCAGTGTGCTCCGATTTATTCGCCACGACA, such as sequence＜400〉shown in 4;
Described upstream outer primer (Vh-F3) is CAAAACGGTTCCGAAACGC, such as sequence＜400〉shown in 1;
Described downstream outer primer (Vh-B3) is: TCGATTCCCCAAGTTTGGAG, such as sequence＜400〉shown in 2;
And the amount of each reagent in this group reagent is enough to satisfy the demands, and the concentration of each reagent is as follows:
The second group reagent is that concentration is the Bst archaeal dna polymerase of 8 activity units of every microlitre, and namely concentration is 8U/ μ L;
The 3rd group reagent is developer, and the developer of the present embodiment is 10wt% fluorescence dye SYBR GREEN I.
Below adopt test kit of the present invention and detection method of the present invention to detect to following six vibrios samples:
Sample 1, Vibrio harveyi bacterial strain Zj2008; Sample 2, Vibrio harveyi (ATCC 33842); Sample 3, Vibrio harveyi XS2007; Sample 4, Vibrio harveyi NH2009; Sample 5: Vibrio parahaemolyticus (ATCC 33846); Sample 6: vibrio alginolyticus Zj2008.
Use the mentioned reagent box to detect in the present embodiment and detect as follows Vibrio harveyi:
(1) extraction of vibrios DNA
A. 28 ℃ of single bacterium colonies of cultivating 16 hours of picking place the centrifuge tube that contains the 0.5mL sterilized water, and thalline fully suspends;
B.100 ℃ water-bath was taken out after 10 minutes, ice bath 2 minutes;
C. use 120000 rev/mins of desk centrifuges centrifugal 5 minutes, and collected in the new 1.5mL centrifuge tube of supernatant liquor to, be the plate DNA that touches to be checked.
(2) ring mediated isothermal amplification of Vibrio harveyi
A, select in the test kit reagent to press the row formulated for detection of the loop-mediated isothermal amplification liquid (23 μ L/ pipe) of Vibrio harveyi:
Ring mediated isothermal amplification (LAMP) reaction solution, system is as follows:
Thernopol reaction buffer in the upper table is by 10 times of 1 * Thernopol reaction buffers that form of the dilution of the 10 * Thernopol reaction buffer in the test kit.
B. in the reaction tubes of the ring isothermal amplification liquid A that 23 μ L are housed, add the 1 μ L plate DNA that touches to be checked, in the electric heating constant temperature water bath, placed 3 minutes for 95 ℃, placed immediately 2 minutes on ice;
C. add 1 μ L Bst archaeal dna polymerase B in reaction tubes, the concentration of Bst archaeal dna polymerase B is 8U/ μ L;
D. in the electric heating constant temperature water bath is bathed 65 ℃ placed 60 minutes;
E. the electric heating constant temperature water-bath is transferred to 95 ℃ of termination reactions, is taken out after 3 minutes;
(3) color developing detection
Add 1 μ L developer in reaction tubes, developer is 10wt% fluorescence dye SYBR GREEN I, and colour-change directly detects by an unaided eye.The result shows: after adding fluorescence dye in the reaction tubes of sample 1～4, color becomes green by yellow, and in the sample 5,6 reaction tubes, add fluorescence dye after, the hue preserving yellow is constant.Detected result conforms to practical situation.The present embodiment is prepared into from DNA and develops the color roughly that required time is 80 minutes.
Claims (7)
1. test kit that detects Vibrio harveyi, it comprises following three group reagents:
The first group reagent is for being used for the reagent of preparation isothermal amplification liquid, and this group reagent comprises Thernopol reaction buffer, dNTP solution, MgSO
4Solution, upstream inner primer Vh-FIP, downstream inner primer Vh-BIP, upstream outer primer Vh-F3, downstream outer primer Vh-B3, alkali solution of beet and aseptic double-distilled water;
The moiety of described Thernopol reaction buffer is Tris-HCl the solution, (NH of pH8.8
4)
2SO
4, KCl, MgSO
4And Triton X-100;
Described upstream inner primer Vh-FIP is TTCGCTTTCGCGAGCCATCTGGTTACCAATTGATCGCCCG;
Described downstream inner primer Vh-BIP is ACGCAGAATCAAGCAGTGTGCTCCGATTTATTCGCCACGACA;
Described upstream outer primer Vh-F3 is CAAAACGGTTCCGAAACGC;
Described downstream outer primer Vh-B3 is: TCGATTCCCCAAGTTTGGAG;
And the amount of each reagent in this group reagent and concentration are enough to make the isothermal amplification liquid that each composition has following working concentration:
The second group reagent is the Bst archaeal dna polymerase;
The 3rd group reagent is developer.
2. test kit according to claim 1 is characterized in that the concentration of each reagent in described the first group reagent is as follows:
3. test kit according to claim 2 is characterized in that the compositional system of the every pipe 23 μ L of described isothermal amplification liquid is as follows:
4. according to claim 1 and 2 or 3 described test kits, the concentration that it is characterized in that described Bst archaeal dna polymerase is 8 activity units of every microlitre, and namely concentration is 8U/ μ L.
5. according to claim 1 and 2 or 3 described test kits, it is characterized in that described developer is 10wt% fluorescence dye SYBR GREEN I.
6. a right to use requires the described test kit of arbitrary claim in 1～5 to carry out the method for non-medical diagnosis on disease detection aquatic animal Vibrio harveyi, it is characterized in that comprising the steps:
(1) extraction of vibrios DNA
A. 28 ℃ of single bacterium colonies of cultivating 16-24 hour of picking place the centrifuge tube that contains the 0.1-0.5ml sterilized water, and thalline fully suspends;
B.95-100 take out ice bath 2-5 minute after ℃ boiling water bath 10-15 minute;
C. use desk centrifuge 10000-120000 rev/min centrifugal 5-10 minute, collect in supernatant liquor to the new 1.5mL centrifuge tube, be the plate DNA that touches to be checked;
(2) ring mediated isothermal amplification of Vibrio harveyi
A. prepare isothermal amplification liquid;
B. in the reaction tubes that 23 μ L isothermal amplification liquid are housed, add the 1 μ L plate DNA that touches to be checked, in the electric heating constant temperature water bath, placed 3-5 minute for 95 ℃, placed immediately 1-3 minute on ice;
C. in reaction tubes, add 1 μ L Bst archaeal dna polymerase;
D. in the electric heating constant temperature water bath is bathed 60-65 ℃ placed 30-90 minute;
E. the electric heating constant temperature water-bath is transferred to 80-95 ℃ of termination reaction, is taken out after 3-5 minute;
(3) color developing detection
Add 1 μ L developer in reaction tubes, whether the colour-change that directly detects by an unaided eye is Vibrio harveyi according to colour-change situation judgement sample;
Consisting of of described 23 μ L isothermal amplification liquid:
7. method according to claim 6, the concentration that it is characterized in that described Bst archaeal dna polymerase is 8 activity units of every microlitre, namely concentration is 8U/ μ L.
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| CN 201010586975 CN102108402B (en) | 2010-12-14 | 2010-12-14 | Kit and detection method for detecting vibrio harveyi |
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| CN102108402B true CN102108402B (en) | 2013-04-24 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102443656B (en) * | 2011-11-01 | 2013-07-17 | 中国水产科学研究院南海水产研究所 | Primer set, rapid diagnostic kit and detection method used for detecting abalone muscular atrophy associated virus |
| CN104450945A (en) * | 2014-12-30 | 2015-03-25 | 山东恒诚检测科技有限公司 | Kit for detecting vibrio furnissii via loop-mediated isothermal amplification method |
| CN104988220A (en) * | 2015-06-26 | 2015-10-21 | 中国科学院南海海洋研究所 | LAMP primer group for detecting vibrio harveyi and kit thereof |
| CN106636073A (en) * | 2017-01-20 | 2017-05-10 | 青岛农业大学 | Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture |
| CN111455080A (en) * | 2020-06-05 | 2020-07-28 | 青岛大螠海创生物科技有限公司 | Method for rapidly detecting pathogenic vibrios of invertebrate |
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