CN103834723A - NASBA-microwell plate detection kit and detection method for five vibrios in mariculture - Google Patents
NASBA-microwell plate detection kit and detection method for five vibrios in mariculture Download PDFInfo
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Abstract
The invention relates to an NASBA-microwell plate detection kit and a detection method for five vibrios in mariculture. The kit comprises an enzyme mixed solution, a reaction liquid containing a forward primer and a reverse primer, and five probe solutions. The five probe solutions are a vibrio parahaemolyticus probe solution, a vibrio alginolyticus probe solution, a vibrio harveyi probe solution, a vibrio vulnificus probe solution and a vibrio anguillarum probe solution. The detection method comprises the steps of extracting total RNA by using a cracking liquid, amplifying the total RNA to obtain an NASBA amplification product by using the reaction solution; and at the same time, coating the probes in the microwell plate; subjecting the NASBA amplification product and the probes to hybrid chromogenic reaction; and then detecting absorption value of A405 nm. The detection kit is sensitive and specific, is convenient for operation in detection and can detect five vibrios in the mariculture rapidly.
Description
Technical field
The present invention relates to the detection technique of vibrio pathogen, be specifically related to NASBA-microwell plate detection kit and the detection method of 5 kinds of vibrios in sea farming.
Background technology
Along with developing rapidly of mariculture industry, disease problem appears day by day, and throughout the year sickness rate reaches more than 50%, rate of loss 20% left and right, and the direct economic loss therefore causing is every year up to 10,000,000,000 yuan, and in rising trend.The principal feature that China's sea farming disease occurs is at present: the sea farming of morbidity is wide in variety, as breed varieties such as fish, crustaceans, shellfishes; Kinds of Diseases are many, variation is fast, as the skin ulcer of fish, enteritis disease, tail, rotted gill disease, Flexibacter columnaris disease and class sarcoidosis etc., crustacean red leg disease, bacillary young septicemia, black gill syndromes, blind's disease, crust Peptic Ulcers, enteron aisle Peptic Ulcers, Characters of Flexibacter Strains disease, rotted gill disease, Macrobrachium rosenbergii black spot and filamentous bacterial infection etc., Aeromonas As Pathogen and the vibriosis etc. of shellfish; The speed of disease popularity is accelerated, and region expands, and harm increases; The comprehensive morbidity of many cause of diseases is general, concurrent, secondary disease is common.Wherein bacterial pathogen comprises vibrio alginolyticus (Vibrio alginolyticus), Vibrio parahaemolyticus (V.parahaemolyticus), Vibrio harveyi (V.harveyi), vibrio cholerae (V.cholerae), Vibrio vulnificus (V.vulnificus), Listonella anguillarum (Listonella anguillarum), aeromonas salmonicida (Aeromonas salmonicida), Mermaid luminous bacillus (Photobacterium damsela), blunt tarda (Edwardsiella tarda) and column are bent oar bacillus (Flexibacter columnaris) etc.The detection of vibrio pathogen at present has PCR, ELISA (Enzyme-linked Immunosorbent Assay), LAMP and gene chip etc., as the application for a patent for invention that publication number is CN1012025557, just detects vibrio alginolyticus by triple PCR primer; Publication number is the application for a patent for invention of CN101020926, just by LAMP technology for detection vibrio cholerae; Notification number is that the patent of invention of CN101178363 is just surveyed Vibrio parahaemolyticus with primer and probe constant-temperature amplification, enzyme joint inspection; But it is sensitive, special, simple, quick, stable and be easy to the features such as automated operation that ELISA has, shortcoming is also more obvious, its experimental period is longer, generally need 1-2 days time, and specific monoclonal antibody preparation is more difficult, and what it detected simultaneously is albumen, so for Bacteria Detection, cannot judge that object bacteria is dead bacterium or viable bacteria, easily produces mistake.RT-PCR is quick, responsive, special, it is to detect in real time, online pcr amplification process and without pcr amplification product being carried out to aftertreatment, overcome the shortcoming that conventional round pcr easily pollutes, but RT-PCR needs expensive plant and instrument, operational requirement to technician is high, is not suitable for basic unit and uses.LAMP constant-temperature amplification, extremely quick, but have equally shortcoming, as unstable in design trouble, the reaction system of primer, can not carry out multiplex amplification.Gene chip can be realized by an experimental implementation and draw multiple pathogenic bacteria qualification result accurately, meet the requirement to the multiple background bacterium collimation that coexists detects in complex sample, but need expensive professional plant and instrument, and testing cost is very expensive at present, common laboratory there is no method configuration, says nothing of in basic unit and applies.
Nucleotide sequence dependent amplification technology (Nucleic acid sequence-based amplification, NASBA) be a rapid isothermal amplification technique taking RNA as template, NASBA amplification system comprises enzyme mixation and reaction solution (containing sense primer, antisense primer, deoxyribonucleotide, ribonucleotide etc.), contains fowl source myeloblastoma reversed transcriptive enzyme (AMV-RT), ribonuclease H (RNase H) and T in enzyme mixation
7rNA polymerase, sense primer (Sense primer) is about 45 Nucleotide, and its 5'-end is with can be by T
7the promoter sequence of RNA polymerase identification, the 3'-end sequence complementation of its 3'-end and template ribonucleic acid; Antisense primer (Antisense primer) is about 20 Nucleotide, consistent with the 5'-end sequence of template ribonucleic acid.Amplified reaction is mainly at AMV-RT, RNase H and T
7under the acting in conjunction of three kinds of enzymes of RNA polymerase, taking single stranded RNA as template, carry out the amplification of nucleotide sequence, it not only has the sensitivity higher than technology such as conventional PCR, accuracy, and easy to operate, do not need special temperature regulating device, be specially adapted to field quick detection.The fields such as diagnosis and monitoring, the disease liability that at present, this technology has been widely used in detection, the tumour of pathogenic agent, gene unconventionality detects, monitoring, the diagnosis of beast disease, monitoring water quality and the medical jurisprudence of food.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of detect sensitive, special, easy to operate, can field quick detection sea farming in NASBA-microwell plate detection kit and the detection method of 5 kinds of vibrios.
The present invention solves the problems of the technologies described above adopted technical scheme: the NASBA-microwell plate detection kit of 5 kinds of vibrios in sea farming, this NASBA-microwell plate test kit comprises enzyme mixation and contains sense primer, the reaction solution of antisense primer, also comprise 5 kinds of probe solutions, described sense primer is primer gyrB-R, described antisense primer is primer gyrB-F1 and primer gyrB-F2, described 5 kinds of probe solutions are respectively the Vibrio parahaemolyticus probe solution that contains biotin labeled probe Vpa-probe, the vibrio alginolyticus probe solution that contains biotin labeled probe Val-probe, the Vibrio harveyi probe solution that contains biotin labeled probe Vha-probe, the Vibrio vulnificus probe solution that contains biotin labeled probe Vvu-probe, the Vibrio anguillarum probe solution that contains biotin labeled probe Van-probe, the nucleotide sequence of each primer and probe is as follows respectively:
Primer gyrB-R:AATTCTAATA CGACTCACTA TAGGGAGA GTCATGATGA TGATGTTGTG48;
Primer gyrB-F1:GACTGTCAGG AAAAAGATCC20;
Primer gyrB-F2:CGTGAAGCGG CGCGTAAAGC20;
Probe Vpa-probe:CAGCTAAGCA GGGTCGTAAT20;
Probe Val-probe:GTACAACCCG GACAAGCTTC20;
Probe Vha-probe:GCTATCTTCT CAAGAAGTAG C21;
Probe Vvu-probe:CTGATCACTG CACTAGGTTG20;
Probe Van-probe:AAAACCAAGC GATTTTGCCA20.
Described enzyme mixation contains the fowl source myeloblastoma reversed transcriptive enzyme that concentration is 8U, the ribonuclease H that concentration is 0.08U, the T that concentration is 40U
7rNA polymerase; Described reaction solution contains the deoxyribonucleotide that concentration is 0.7mM (dNTP), concentration is the ribonucleotide (NTP) of 12U, concentration is the uridine triphosphate (rUTP) of 0.91mM, concentration is the Triphosaden (rATP) of 1mM, concentration is the cytidine (rCTP) of 1mM, concentration is the GTP (guanosine triphosphate) (rGTP) of 1mM, concentration is the uridine triphosphate (DIG-11-rUTP) of the digoxigenin labeled of 0.09mM, concentration is the KCl of 10mM, concentration is Tutofusin tris-hydrochloric acid (Tris – HCl) of 40mM, the MgCl that concentration is 12mM
2concentration is the bovine serum albumin (BSA) of 0.1 μ g/ μ L, concentration expressed in percentage by volume is 5% dimethyl sulfoxide (DMSO) (DMSO), the dithiothreitol (DTT) (DTT) that concentration is 5mM, concentration be all 0.2mM primer gyrB-R, primer gyrB-F1 and and primer gyrB-F2; The concentration of described Vibrio parahaemolyticus probe solution middle probe Vpa-probe is 0.1 μ M, the concentration of described vibrio alginolyticus probe solution middle probe Val-probe is 0.1 μ M, the concentration of described Vibrio harveyi probe solution middle probe Vha-probe is 0.1 μ M, the concentration of described Vibrio vulnificus probe solution middle probe Vvu-probe is 0.1 μ M, and the concentration of described Vibrio anguillarum probe solution middle probe Van-probe is 0.1 μ M.
Utilize above-mentioned NASBA-microwell plate detection kit to detect the method for 5 kinds of vibrios in sea farming, its step is as follows:
A, get inoculum, add RNAiso reagent (lysate) extracted total RNA, obtain RNA template, specifically operate according to RNAiso reagent (Takara company) specification sheets;
B, get above-mentioned RNA pattern 5 μ L, add described reaction solution 32.5 μ L, incubation 5min at 65 DEG C, then add described enzyme mixation 12.5 μ l, reacts 1h at 42 DEG C, obtains NASBA amplified production;
C, with pH7.2, the micropore of the phosphoric acid buffer cleaning streptavidin enzyme plate of the polysorbas20 (TWEEN20) that contains concentration expressed in percentage by volume 0.05% 3 times, the each 300 μ L in every hole, after cleaning, each hole adds respectively the described Vibrio parahaemolyticus probe solution of 100 μ L, vibrio alginolyticus probe solution, Vibrio harveyi probe solution, Vibrio vulnificus probe solution, Vibrio anguillarum probe solution, soft oscillation incubation 1h under room temperature, blot liquid in micropore, use again pH7.2, the phosphoric acid buffer of the polysorbas20 that contains concentration expressed in percentage by volume 0.05% cleans micropore 3 times, each every hole 300 μ L, obtain the micropore of dressing probe,
D, get 10 μ L NASBA amplified productions, at 65 DEG C, hatch 5min, add pH7.0, concentration is that the phosphoric acid buffer 90 μ L of 50mM mix, then join in the micropore of dressing probe, at 37 DEG C, hatch 1h, blot liquid in micropore, clean micropore 3 times with hybridizing washing lotion, pat dry, every micropore adds sealing under 200 μ L confining liquid room temperatures and hatches 30min, the Trisodium Citrate that described hybridization washing lotion contains concentration 0.0015mol/L, the NaCl of concentration 0.015mol/L, the sodium laurylsulfonate (SDS) of mass percentage concentration 0.1%, described confining liquid is the polysorbas20 that contains concentration expressed in percentage by volume 0.05%, the tris-HCI buffer of the bovine serum albumin (BSA) of mass percentage concentration 1%,
E, sealing are hatched after 30min, in micropore, add the alkali phosphatase enzyme mark biotin antibody 100 μ L of 100 times of dilutions, under room temperature, hatch 30min, add again NBT (NBT)/5-bromo-4-chloro-3-indolylphosphate salt (BCIP) 100 μ L, lucifuge colour developing 15min at 37 DEG C, add sulfuric acid stopped reaction, in microplate reader, measure the absorption value of the micropore A405nm of dressing probe.
Can do sample, blank and negative control bacterial classification (as intestinal bacteria) test according to aforesaid method simultaneously, or can first determine in advance the absorption value of the A405nm of blank and negative control bacterial classification, if the sample of the micropore of above-mentioned dressing probe colour developing, result is that the corresponding vibrios of probe is positive, if the relatively significant difference of absorption value with negative control bacterial classification, be greater than negative control bacterial classification, further confirm as the positive.
Above-mentioned three primers are according to the universal amplification design of primers of vibrios 16S RNA and gyrB gene mRNA, and 16SrRNA gene is that all bacteriums are total, is gene the most conservative in bacterium evolutionary process; It is of moderate size, and about 1.5Kb left and right, is made up of conserved regions and variable region, and conserved regions is that all bacteriums are common, there is no difference between bacterium; There is difference in various degree in variable region, have the specificity that belongs to or plant between different bacterium.The specificity of each vibrios is screened probe according to the specific fragment design of each vibrios, its middle probe Vpa-probe is Vibrio parahaemolyticus, probe Val-probe is vibrio alginolyticus, probe Vha-probe is Vibrio harveyi, probe Vvu-probe is Vibrio vulnificus, probe Van-probe is Vibrio anguillarum, and application Primer primer5.0 software design screening obtain, and primer is synthetic by the raw work in Shanghai.
Compared with prior art, the invention has the advantages that NASBA-microwell plate detection kit and the detection method of 5 kinds of vibrios in sea farming, this NASBA-microwell plate test kit comprises enzyme mixation and the reaction solution containing sense primer and antisense primer, also comprise 5 kinds of probe solutions, sense primer is primer gyrB-R, antisense primer is primer gyrB-F1 and primer gyrB-F2, 5 kinds of probe solutions are respectively the Vibrio parahaemolyticus probe solution that contains biotin labeled probe Vpa-probe, the vibrio alginolyticus probe solution that contains biotin labeled probe Val-probe, the Vibrio harveyi probe solution that contains biotin labeled probe Vha-probe, the Vibrio vulnificus probe solution that contains biotin labeled probe Vvu-probe, the Vibrio anguillarum probe solution that contains biotin labeled probe Van-probe, detection method is lysate extracted total RNA, obtain NASBA amplified production with reaction solution total RNA that increases, carry out each probe at microwell plate is coated with simultaneously, NASBA amplified production and probe hybridization color reaction, then detect the absorption value of A405nm, this NASBA-microwell plate detection kit is sensitive, special, easy to operate when detection, can field quick detection go out 5 kinds of vibrios in sea farming.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
The NASBA-microwell plate detection kit of 5 kinds of vibrios in sea farming, comprises and contains the fowl source myeloblastoma reversed transcriptive enzyme that concentration is 8U, the ribonuclease H that concentration is 0.08U, the T that concentration is 40U
7the enzyme mixation of RNA polymerase; Contain the dNTP that concentration is 0.7mM, concentration is the NTP of 12U, concentration is the rUTP of 0.91mM, concentration is the rATP of 1mM, the rCTP that concentration is 1mM, the rGTP that concentration is 1mM, concentration is the DIG-11-rUTP of 0.09mM, concentration is the KCl of 10mM, the Tris – HCl that concentration is 40mM, the MgCl that concentration is 12mM
2, concentration is the BSA of 0.1 μ g/ μ L, the DMSO that concentration expressed in percentage by volume is 5%, the DTT that concentration is 5mM, concentration be all 0.2mM primer gyrB-R, primer gyrB-F1 and and the reaction solution of primer gyrB-F2; The concentration of probe Vpa-probe is the Vibrio parahaemolyticus probe solution of 0.1 μ M; The concentration of probe Val-probe is the vibrio alginolyticus probe solution of 0.1 μ M; The concentration of probe Vha-probe is the Vibrio harveyi probe solution of 0.1 μ M; The concentration of probe Vvu-probe is the Vibrio vulnificus probe solution of 0.1 μ M; The concentration of probe Van-probe is the Vibrio anguillarum probe solution of 0.1 μ M.
Embodiment 2
In sea farming, the NASBA-microwell plate detection method of 5 kinds of vibrios, gets inoculum, adds RNAiso reagent extracted total RNA, obtains RNA template, specifically operates according to RNAiso reagent specification sheets.Get RNA template 5 μ L, add the reaction solution 32.5 μ L of embodiment 1, incubation 5min at 65 DEG C, then add the enzyme mixation 12.5 μ l of embodiment 1, reacts 1h at 42 DEG C, obtains NASBA amplified production.With pH7.2, the micropore of the phosphoric acid buffer cleaning streptavidin enzyme plate of the polysorbas20 that contains concentration expressed in percentage by volume 0.05% 3 times, the each 300 μ L in every hole, after cleaning, each hole adds respectively the Vibrio parahaemolyticus probe solution of the embodiment 1 of 100 μ L, vibrio alginolyticus probe solution, Vibrio harveyi probe solution, Vibrio vulnificus probe solution, Vibrio anguillarum probe solution, soft oscillation incubation 1h under room temperature, blot liquid in micropore, use again pH7.2, the phosphoric acid buffer of the polysorbas20 that contains concentration expressed in percentage by volume 0.05% cleans micropore 3 times, each every hole 300 μ L, obtain the micropore of dressing probe.Get 10 μ L NASBA amplified productions, at 65 DEG C, hatch 5min, add pH7.0, concentration is that the phosphoric acid buffer 90 μ L of 50mM mix, then join in the micropore of dressing probe, at 37 DEG C, hatch 1h, blot liquid in micropore, clean micropore 3 times with hybridizing washing lotion, pat dry, every micropore adds sealing under 200 μ L confining liquid room temperatures and hatches 30min, the Trisodium Citrate that hybridization washing lotion contains concentration 0.0015mol/L, the NaCl of concentration 0.015mol/L, the SDS of mass percentage concentration 0.1%, confining liquid is the polysorbas20 that contains concentration expressed in percentage by volume 0.05%, the Tris-hydrochloride buffer of the BSA of mass percentage concentration 1%, sealing is hatched after 30min, in micropore, add the alkali phosphatase enzyme mark biotin antibody 100 μ L of 100 times of dilutions, under room temperature, hatch 30min, add again NBT/BCIP100 μ L, lucifuge colour developing 15min at 37 DEG C, if develop the color in testing sample, the corresponding vibrios of probe is positive, add sulfuric acid stopped reaction, in microplate reader, measure the absorption value of micropore A405nm.Sample experiment arranges blank and negative control (intestinal bacteria etc. are not the bacterial classifications of above-mentioned 5 kinds of vibrios) simultaneously, if if the absorption value of sample is significantly greater than negative control in correspondent probe, result is that this vibrios is positive.
Embodiment 3
Vibrio alginolyticus sample, Vibrio parahaemolyticus sample, Vibrio harveyi sample, vibrio cholerae sample, Vibrio vulnificus sample, Vibrio anguillarum sample, vibrio fluvialis sample, Aeromonas hydrophila sample, blunt tarda sample, carry out NASBA-microwell plate by the method for embodiment 2 and carry out specific detection, if micropore is all that Vibrio parahaemolyticus probe is coated, result shows, in Vibrio parahaemolyticus probe mark micropore, Vibrio parahaemolyticus sample has color reaction, the absorption value of A405nm is 1.50, be significantly higher than the absorption value of other each bacterium at the A405nm of Vibrio parahaemolyticus probe mark micropore, therefore, the be decided to be feminine gender of other each bacterium.The in like manner positive result of Vibrio harveyi sample in Vibrio harveyi probe solution, the positive result of Vibrio vulnificus sample in Vibrio vulnificus probe solution, the positive result of vibrio alginolyticus sample in vibrio alginolyticus probe solution, the positive result of Vibrio anguillarum sample in Vibrio anguillarum probe solution.And vibrio fluvialis sample, Aeromonas hydrophila sample, Listeria monocytogenes, Yellowtail fish Nocardia bacteria, Streptococcus iniae, blunt tarda sample are all negative in each probe solution.
Embodiment 4
Taking Vibrio parahaemolyticus sample as example, nutrient solution 10
5after 10 times of gradient dilutions of sample of CFU/mL, carrying out NASBA-microwell plate susceptibility by the method for embodiment 2 respectively detects, result lowest detection is limited to 100CFU/mL, be equivalent to 5-10 cell, the detection of same vibrio alginolyticus, Vibrio harveyi, vibrio cholerae and Vibrio vulnificus is rolled off the production line and is 100CFU/mL.
Embodiment 5
Sea farming sample detection: 1 sample of conger pile, 1 sample of spotted maigre, 5 samples of large yellow croaker, 3 samples of Portunus trituberculatus Miers totally 10 samples, extract respectively each sample 0.5mL, then carry out total RNA extracting according to the method for embodiment 2, detect with NASBA-microwell plate, result conger pile, spotted maigre, the large yellow croaker of a sample, the Portunus trituberculatus Miers of a sample does not detect above-mentioned 5 kinds of vibrios, the large yellow croaker of two samples detects Vibrio harveyi and Vibrio parahaemolyticus, the large yellow croaker of a sample detects vibrio alginolyticus, the large yellow croaker of a sample detects Vibrio harveyi, the Portunus trituberculatus Miers of a sample detects Vibrio parahaemolyticus, the Portunus trituberculatus Miers of a sample detects vibrio alginolyticus.
<110> University Of Ningbo
NASBA-microwell plate detection kit and the detection method of 5 kinds of vibrios in <120> sea farming
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 48
<212> DNA
<213> artificial sequence
<400> 1
AATTCTAATA CGACTCACTA TAGGGAGAGT CATGATGATG ATGTTGTG 48
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
GACTGTCAGG AAAAAGATCC 20
<210>3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
CGTGAAGCGG CGCGTAAAGC 20
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
CAGCTAAGCA GGGTCGTAAT 20
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
GTACAACCCG GACAAGCTTC 20
<210> 6
<211> 21
<212> DNA
<213> artificial sequence
<400> 6
GCTATCTTCT CAAGAAGTAG C 21
<210> 7
<211> 20
<212> DNA
<213> artificial sequence
<400> 7
CTGATCACTG CACTAGGTTG 20
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
AAAACCAAGC GATTTTGCCA 20
Claims (3)
1. the NASBA-microwell plate detection kit of 5 kinds of vibrios in sea farming, this NASBA-microwell plate test kit comprises enzyme mixation and contains sense primer, the reaction solution of antisense primer, characterized by further comprising 5 kinds of probe solutions, described sense primer is primer gyrB-R, described antisense primer is primer gyrB-F1 and primer gyrB-F2, described 5 kinds of probe solutions are respectively the Vibrio parahaemolyticus probe solution that contains biotin labeled probe Vpa-probe, the vibrio alginolyticus probe solution that contains biotin labeled probe Val-probe, the Vibrio harveyi probe solution that contains biotin labeled probe Vha-probe, the Vibrio vulnificus probe solution that contains biotin labeled probe Vvu-probe, the Vibrio anguillarum probe solution that contains biotin labeled probe Van-probe, the nucleotide sequence of each primer and probe is as follows respectively:
Primer gyrB-R:AATTCTAATA CGACTCACTA TAGGGAGA GTCATGATGA TGATGTTGTG;
Primer gyrB-F1:GACTGTCAGG AAAAAGATCC;
Primer gyrB-F2:CGTGAAGCGG CGCGTAAAGC;
Probe Vpa-probe:CAGCTAAGCA GGGTCGTAAT;
Probe Val-probe:GTACAACCCG GACAAGCTTC;
Probe Vha-probe:GCTATCTTCT CAAGAAGTAG C;
Probe Vvu-probe:CTGATCACTG CACTAGGTTG;
Probe Van-probe:AAAACCAAGC GATTTTGCCA.
2. the NASBA-microwell plate detection kit of 5 kinds of vibrios in sea farming as claimed in claim 1, it is characterized in that described enzyme mixation contains the fowl source myeloblastoma reversed transcriptive enzyme that concentration is 8U, concentration is the ribonuclease H of 0.08U, the t7 rna polymerase that concentration is 40U, described reaction solution contains the deoxyribonucleotide that concentration is 0.7mM, concentration is the ribonucleotide of 12U, concentration is the uridine triphosphate of 0.91mM, concentration is the Triphosaden of 1mM, concentration is the cytidine of 1mM, concentration is the GTP (guanosine triphosphate) of 1mM, concentration is the uridine triphosphate of the digoxigenin labeled of 0.09mM, concentration is the KCl of 10mM, concentration is Tutofusin tris-hydrochloric acid of 40mM, concentration is the MgCl2 of 12mM, concentration is the bovine serum albumin of 0.1 μ g/ μ L, concentration expressed in percentage by volume is 5% dimethyl sulfoxide (DMSO), concentration is the dithiothreitol (DTT) of 5mM, concentration is all the primer gyrB-R of 0.2mM, primer gyrB-F1 and and primer gyrB-F2, the concentration of described Vibrio parahaemolyticus probe solution middle probe Vpa-probe is 0.1 μ M, the concentration of described vibrio alginolyticus probe solution middle probe Val-probe is 0.1 μ M, the concentration of described Vibrio harveyi probe solution middle probe Vha-probe is 0.1 μ M, the concentration of described Vibrio vulnificus probe solution middle probe Vvu-probe is 0.1 μ M, and the concentration of described Vibrio anguillarum probe solution middle probe Van-probe is 0.1 μ M.
3. utilize the NASBA-microwell plate detection kit of 5 kinds of vibrios in the sea farming described in claim 1 to detect the method for vibrios in sea farming, it is characterized in that step is as follows:
A, get inoculum, add RNAiso reagent extracted total RNA, obtain RNA template, specifically operate according to RNAiso reagent specification sheets;
B, get above-mentioned RNA pattern 5 μ L, add described reaction solution 32.5 μ L, incubation 5min at 65 DEG C, then add described enzyme mixation 12.5 μ l, reacts 1h at 42 DEG C, obtains NASBA amplified production;
C, with pH7.2, the micropore of the phosphoric acid buffer cleaning streptavidin enzyme plate of the polysorbas20 that contains concentration expressed in percentage by volume 0.05% 3 times, the each 300 μ L in every hole, after cleaning, each hole adds respectively the described Vibrio parahaemolyticus probe solution of 100 μ L, vibrio alginolyticus probe solution, Vibrio harveyi probe solution, Vibrio vulnificus probe solution, Vibrio anguillarum probe solution, soft oscillation incubation 1h under room temperature, blot liquid in micropore, use again pH7.2, the phosphoric acid buffer of the polysorbas20 that contains concentration expressed in percentage by volume 0.05% cleans micropore 3 times, each every hole 300 μ L, obtain the micropore of dressing probe,
D, get 10 μ L NASBA amplified productions, at 65 DEG C, hatch 5min, add pH7.0, concentration is that the phosphoric acid buffer 90 μ L of 50mM mix, then join in the micropore of dressing probe, at 37 DEG C, hatch 1h, blot liquid in micropore, clean micropore 3 times with hybridizing washing lotion, pat dry, every micropore adds sealing under 200 μ L confining liquid room temperatures and hatches 30min, the Trisodium Citrate that described hybridization washing lotion contains concentration 0.0015mol/L, the NaCl of concentration 0.015mol/L, the sodium laurylsulfonate of mass percentage concentration 0.1%, described confining liquid is the polysorbas20 that contains concentration expressed in percentage by volume 0.05%, the tris-HCI buffer of the bovine serum albumin of mass percentage concentration 1%,
E, sealing are hatched after 30min, in micropore, add the alkali phosphatase enzyme mark biotin antibody 100 μ L of 100 times of dilutions, under room temperature, hatch 30min, add again NBT/5-bromo-4-chloro-3-indolylphosphate salt 100 μ L, lucifuge colour developing 15min at 37 DEG C, add sulfuric acid stopped reaction, in microplate reader, measure the absorption value of the micropore A405nm of dressing probe.
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| CN105039507A (en) * | 2015-04-24 | 2015-11-11 | 胡成进 | Kit for quickly detecting marine vibrio vulnificus |
| CN109846968A (en) * | 2019-01-17 | 2019-06-07 | 中国水产科学研究院黄海水产研究所 | Composition and the application for preventing the bacillary intestines problem of far-reaching extra large cultured fishes |
| CN116732204A (en) * | 2023-05-29 | 2023-09-12 | 海南大学 | Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit |
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| CN116732204A (en) * | 2023-05-29 | 2023-09-12 | 海南大学 | Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit |
| CN116732204B (en) * | 2023-05-29 | 2024-02-13 | 海南大学 | Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit |
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