CN1142291C - Reagent kit and method for quickly detecting alga-dissolving vibrio - Google Patents

Reagent kit and method for quickly detecting alga-dissolving vibrio Download PDF

Info

Publication number
CN1142291C
CN1142291C CNB011301279A CN01130127A CN1142291C CN 1142291 C CN1142291 C CN 1142291C CN B011301279 A CNB011301279 A CN B011301279A CN 01130127 A CN01130127 A CN 01130127A CN 1142291 C CN1142291 C CN 1142291C
Authority
CN
China
Prior art keywords
reagent
minute
concentration
vibrio alginolyticus
reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB011301279A
Other languages
Chinese (zh)
Other versions
CN1373358A (en
Inventor
偿 陈
陈偿
胡超群
沈琪
任春华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Sea Institute of Oceanology of CAS
Original Assignee
South China Sea Institute of Oceanology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Sea Institute of Oceanology of CAS filed Critical South China Sea Institute of Oceanology of CAS
Priority to CNB011301279A priority Critical patent/CN1142291C/en
Publication of CN1373358A publication Critical patent/CN1373358A/en
Application granted granted Critical
Publication of CN1142291C publication Critical patent/CN1142291C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Abstract

The present invention provides a kit for the rapid detection of vibrio alginolyticus. The solvent comprises a DNA extraction reagent, a PCR reaction reagent and an electrophoretic and coloring reagent, wherein the DNA extraction reagent contains a buffer reagent A and a cell disruption reagent B; the PCR reaction reagent contains a reaction premixing reagent C and a reaction primer reagent D, the reagent C contains a reaction buffer solution of 10 times, thermopolymerization enzymes and sterilizing double distilled water, and the reagent D contains specific primer pairs of VP<01>5'-ATTGAGAACCCGACAGAAGCGAAG and VP<02>5'-CCTAATGCGGTGATCAGTGTTACTPCR, dNTP and MgCl2. The present invention also provides a method for the rapid detection of vibrio alginolyticus by the kit, and has the advantages of high speed and accuracy.

Description

Test kit of rapid detection vibrio alginolyticus and detection method
Technical field
The present invention relates to the test kit of a kind of rapid detection vibrio alginolyticus, further relate to the method for the test kit rapid detection vibrio alginolyticus of using the rapid detection vibrio alginolyticus.
Technical background
In recent years, China's mariculture industry fast development, sea farming output has increased 5-6 doubly in the period of 10 in the past.But the great development band of last stage sea farming bears the character of much blindness even is destructive, causes this sea farming increase of production in 2 years to begin to slow down, and especially the change of the problem of disease is more and more serious.Wherein vibriosis is one of important cause of disease that endangers multiple sea farming biologies such as culturing fish and shrimp shellfish algae.Vibrios is the native bacterium of ocean origin, widely distributes in seawater and marine organisms; Vibrio pathogen can cause that aquaculture organism is explosive dead, and can cause co-infected with virus, other bacterium or parasite etc.In China's sea farming biology, vibrio alginolyticus is modal vibrio pathogen, and the loss that caused by vibrio alginolyticus every year is very huge.Current vibrios cause of disease detection technique mainly contains following several: 1, the routine biochemistry detection technique, it be with the microbionation behind the purifying in the substratum that contains different biochemical substances, because the material of bacterial metabolism not of the same race is incomplete same, also different with the result of developer reaction in the substratum, with these results and reference culture and Documentary Records result contrast, (the reference: Lou Yongxin Wang Jinliang chief editor " practical clinical bacteriology check and progress " 1992 Tianjin Scientific English Translation publishing company) of just can reaching a conclusion.This methods and results more accurately but very consuming time, and is and very unreliable to the detected result of new kind; 2, quick bacteria identification kits such as API-20E, statistical study drew expert's conclusion after it adopted and detects a few biochemical indicator, relatively save time, but the result was not too reliable.
Summary of the invention
The test kit that the purpose of this invention is to provide a kind of rapid detection vibrio alginolyticus particularly provides a pair of vibrio alginolyticus Auele Specific Primer VP 01And VP 02, and a kind of method of using the test kit rapid detection of rapid detection vibrio alginolyticus and monitoring vibrio alginolyticus in the several samples is provided, thus the foundation of science is provided for healthy aquaculture.
Cardinal principle of the present invention is: reagent A and B carry out cracking with the bacterium in the sample, discharge DNA of bacteria, reagent C and D are polymerase chain reaction technology reagent, by vibrio alginolyticus specific DNA fragment primer contained in the reagent D and the compositions such as hot polymerization synthase in the reagent C, the DNA that sample is discharged carries out specific amplification, because our primer of design is peculiar by vibrio alginolyticus, therefore, if contain vibrio alginolyticus in the sample, the specific fragment of its DNA is after the process amplification so, and concentration will reach 10 8More than the mol (mol), like this, behind electrophoresis and ethidium bromide staining, UV-light detects the purpose band that just can detect certain molecular weight.If there is not vibrio alginolyticus, the purpose band of the same molecular weight that then can not increase.
The test kit of a kind of rapid detection vibrio alginolyticus of the present invention is characterized in that solvent comprises:
(a) DNA extraction reagent: contain buffer reagent A and cell cracking agent B,
Reagent A contains ethylenediamine tetraacetic acid (EDTA) (EDTA) 0.1-10mM of trihydroxy methyl aminomethane (Tris) 1-100mM, pH8.0 of NaCl0.1-0.3M, pH8.0 and Triton (Triton) X-100 that V/V concentration is 0-20%;
It is 1-100mM trihydroxy methyl aminomethane (Tris) that reagent B contains N,O-Diacetylmuramidase and pH8.0, the concentration that concentration is 1-10mg/ml;
(b) polymerase chain reaction (PCR reaction) reagent: comprise reaction premix reagent C and reaction primer reagent D,
Reagent C contains 10 times of polymerase chain reaction damping fluids, hot polymerization synthase (Taq enzyme) and sterilization distilled water (ddH 2O), content is respectively 2.5-5.0 μ l, a 0.5-2.5 unit (5 units of concentration/μ l) and 18-35.5 μ l;
Reagent D: comprise that special primer is to VP 01And VP 02, dNTP (mixtures of four kinds of deoxynucleotides) and MgCl 2, content is respectively 0.2-1 μ M and 0.2-1 μ M, 20-200 μ M and 0.5-10mM;
(c) electrophoresis and colouring reagents comprise reagent E and reagent F,
Reagent E: be 50 times of TAE (electrophoretic buffer), contain Tris-acetate and EDTA, concentration is respectively: 0.04M Tris-acetate, 0.001M EDTA;
Reagent F: contain agarose that W/V is 0.8-2% and the ethidium bromide of 0.5-20 μ g/ml;
Special primer in the reagent D is to VP 01And VP 02Be special primer and the essential composition that carries out the PCR reaction, VP 01Refer in particular to 5 '-ATTGAGAACCCGACAGAAGCGAAG; VP 02Refer in particular to 5 '-CCTAATGCGGTGATCAGTGTTCT.
The best group of every tube reaction reagent becomes: reagent C: 5.0 μ l, 10 * PCR Buffer, 0.5 μ l Taq (concentration 5U/ μ l), 35.5 μ lddH 2O, reagent D: 4.0 μ l dNTP (2.5mM each), 3.0 μ l MgCl 2(25mM), 10pM VP 0110pM VP 02
Reagent A is the DNA extraction damping fluid, for the extraction of DNA provides suitable buffer environment and suppresses the degraded of DNA; Reagent B is a cell cracking agent, makes the bacterium cracking and discharges DNA; Reagent C is a PCR reaction premix reagent, is one of main component of PCR reaction reagent, and reagent D is a PCR primer reagent, also is the main component of PCR reaction, includes the most key special primer VP of this test kit 01And VP 02Reagent E is 50 times of TAE (electrophoretic buffer), and the buffer system of suitable pH value, ionic strength etc. is provided for sample electrophoresis; Reagent F is electrophoretic medium agarose+developer ethidium bromide.
Use the method for the test kit rapid detection vibrio alginolyticus of rapid detection vibrio alginolyticus to comprise the following steps: successively
(a). sample preparation: get 0.1-0.5 gram sample, with 100-400 μ l reagent A suspended sample, and with the abundant mixing of sample; The centrifugal 2-5 of 10000-12000g minute, abandon supernatant;
(b) .DNA extracts: with the 100-300 μ l reagent A precipitation that suspends once more, and adding 20-100 μ l reagent B mixing, room temperature was placed more than 2 minutes, was incubated 1-10 minute again in boiling water bath, and cell is broken fully, discharged DNA; The centrifugal 8-10 of 10000-12000g minute, get supernatant and add the dehydrated alcohol deposit D NA of 2 times of supernatant volumes (240-800 μ l); Room temperature was placed 5-10 minute, the centrifugal 3-8 of 10000-12000g minute; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in the 10-50 μ l sterilization distilled water; Be DNA extraction liquid;
(c) .PCR amplification: the DNA that extracts with 0.5-1 μ l is as the template of pcr amplification; Getting 21-42 μ l reagent C mixes with 4-8 μ l reagent D; On thermal cycler, the dna profiling that extracts is increased, unwind 2-5 minute by 94 ℃-98 ℃; Then 93-95 ℃ of sex change 30 seconds-1 minute, 54-58 ℃ of renaturation 30 seconds-1 minute, 72 ℃ were extended 30 seconds-1.5 minutes, so circulated 25-35 time, were incubated 8-12 minute at 72 ℃ at last, and the specific fragment of vibrio alginolyticus is increased to more than the 108mol;
(d). electrophoresis and color developing detection: get the sample after 5-10 μ l increases, with 2-6 μ l V/V is that 0.25% tetrabromophenol sulfonphthalein is mixed, W/V is agarose electrophoresis and the colour developing of 0.8-1.2%, under UV-light, detect the band whether one 300 nucleotide pair is arranged, if the band of one 300 nucleotide pair is arranged, interpret sample has vibrio alginolyticus to infect or pollutes, otherwise does not then have.Advantage of the present invention and positively effect:
Detection technique based on nucleic acid, comprise dna probe and round pcr (polymerase chain reaction technology), relatively other vibrios cause of disease detection technique and other cause of disease detection technique, present method has several significant advantages: at first, this method detects very quick, can learn detected result in 3-4 hour, other method then needs 3 days or one more than week at least; Secondly, this method detection sensitivity is high, and the detection lower limit is low to moderate the bacterium about 10, and other method must have the pure culture propagation of certain hour to reach 10 5After just can detect.The present invention can be applied to the detection of several samples; Can detect the situation that cultivated animals is infected by vibrio alginolyticus, also can monitor cause of disease vibrio alginolyticus in the aquaculture water environment, can also detect feed ingredient and fresh food and polluted by vibrio alginolyticus.
Embodiment
Following example is further to explanation of the present invention, should not be used as limitation of the present invention.
Embodiment 1:
Make the test kit of rapid detection vibrio alginolyticus by following prescription:
Reagent A: 0.1M NaCl, 10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0), 5% Triton X-100
Reagent B:10mg/ml N,O-Diacetylmuramidase, 10mM Tris.Cl (pH8.0)
Reagent C: 5.0 μ l, 10 * PCR Bufffer, 0.5 μ l Taq (5U/ μ l), 35.5 μ l ddH 2O
Reagent D: 4.0 μ l dNTP (2.5mM each), 3.0 μ l MgCl 2(25mM),
20pM?VP 01?20pM?VP 02
Reagent E: 50 * TAE
Reagent F: agarose (1%)+ethidium bromide (0.5 μ g/ml)
Detect according to following program:
1. the extraction of sample preparation and DNA: get the about 0.1-0.5 of fish tissue gram, add 400 μ l reagent A in sample, the sterilization toothpick is smashed to pieces, and centrifugal 2 minutes of 12000g abandons supernatant, with 350 μ l reagent A and the 50 μ l reagent B precipitation that suspends again, mixing.Room temperature was placed 5 minutes, 100 ℃ of water-baths 5 minutes.Be cooled to room temperature, centrifugal 10 minutes of 12000g gets supernatant, adds 800 μ l dehydrated alcohols, room temperature was placed 5 minutes, and centrifugal 5 minutes of 12000g thoroughly removes supernatant, and 75% washing with alcohol once, allow ethanol volatilize, precipitation is dissolved in 30 μ l sterilization distilled water, be DNA extraction liquid.
2. the specific fragment of marine alga vibrios amplification
In a pipe reagent C, add 1 μ l DNA extraction liquid, add 8 μ l reagent D again, centrifugal 30 seconds of 10000g.On PCR thermal cycle reaction instrument, carry out following reaction:
94 ℃ of heat denatured 4 minutes; Unwind 1 minute at 95 ℃ then, 58 ℃ of renaturation 1 minute, 72 ℃ were extended 1 minute, and circulated altogether 35 times; 72 ℃ the insulation 10 minutes after stopped reaction.
3. electrophoresis and color developing detection: reaction is got 5-10 μ l reaction solution after finishing, and is that 0.25% tetrabromophenol sulfonphthalein is mixed with 4 μ l V/V, with W/V is 1% agarose gel electrophoresis, detect under the UV-light, if the band of one 300 nucleotide pair is arranged, interpret sample has vibrio alginolyticus to infect or pollutes; Otherwise then do not have.
Embodiment 2:
Press the various solution of following formulated:
Reagent A: 0.3M NaCl, 10mM Tris.Cl (pH8.0), 1mM EDTA (pH8.0),
Reagent B:5mg/ml N,O-Diacetylmuramidase, 10mM Tris.Cl (pH8.0)
Reagent C: 2.5 μ l, 10 * PCR Buffer, 0.25 μ l Taq (5U/ul), 35.5 μ l ddH 2O
Reagent D: 2.0 μ l dNTP (2.5mM each), 1.5 μ l MgCl 2(25mM),
10pM?VP 01(0.5μl)，10pM?VP 02(0.5μl)
Reagent E:: 50 * TAE
Reagent F: agarose (1.2%)+ethidium bromide (0.8 μ g/ml)
Operate according to following program:
1. the extraction of sample preparation and DNA
Get the about 0.1-0.5 of fish tissue gram, add 200 μ l reagent A in sample, the sterilization toothpick is smashed to pieces, and centrifugal 2 minutes of 12000g abandons supernatant, with 150 μ l reagent A and the 50 μ l reagent B precipitation that suspends again, mixing.Room temperature was placed 5 minutes, 100 ℃ of water-baths 1 minute.Be cooled to room temperature, centrifugal 10 minutes of 12000g.Get supernatant, add 400 μ l dehydrated alcohols, room temperature was placed 5 minutes, and centrifugal 5 minutes of 12000g thoroughly removes supernatant, and 75% washing with alcohol once allows ethanol volatilize, and precipitation is dissolved in 30 μ l sterilization distilled water, was DNA extraction liquid.
2. the specific fragment of vibrio alginolyticus amplification
In a pipe reagent C, add 1 μ l DNA extraction liquid, add 8 μ l reagent D again, centrifugal 30 seconds of 10000g.On PCR thermal cycle reaction instrument, carry out following reaction:
94 ℃ of heat denatured 3 minutes; Unwind for 45 seconds at 95 ℃ then, in 58 ℃ of 30 seconds of renaturation, 72 ℃ were extended for 30 seconds, circulate altogether 30 times; 72 ℃ the insulation 10 minutes after stopped reaction.
3. electrophoresis and color developing detection: reaction is got 5-10 μ l reaction solution after finishing, and is that 0.25% tetrabromophenol sulfonphthalein is mixed with 4 μ l V/V, with W/V is 1% agarose gel electrophoresis, detect under the UV-light, if the band of one 300 nucleotide pair is arranged, interpret sample has vibrio alginolyticus to infect or pollutes; Otherwise then do not have.

Claims (3)

1. the test kit of a rapid detection vibrio alginolyticus comprises:
(a) DNA extraction reagent: contain buffer reagent A and cell cracking agent B,
Ethylenediamine tetraacetic acid (EDTA) 0.1-10mM and V/V concentration that reagent A contains trihydroxy methyl aminomethane 1-100mM, the pH8.0 of NaCl 0.1-0.3M, pH8.0 are the triton x-100 of 0-20%;
It is 1-100mM trihydroxy methyl aminomethane that reagent B contains N,O-Diacetylmuramidase and pH8.0, the concentration that concentration is 1-10mg/ml;
(b) polymerase chain reaction reagent: comprise reaction premix reagent C and reaction primer reagent D,
Reagent C contains 10 times of polymerase chain reaction damping fluids, hot polymerization synthase and sterilization distilled water, and content is respectively 2.5-5.0 μ l, a 0.5-2.5 unit and 18-35.5 μ l;
Reagent D: comprise that special primer is to VP 01And VP 02, dNTP (mixtures of four kinds of deoxynucleotides) and MgCl 2, content is respectively 0.2-1 μ M and 0.2-1 μ M, 20-200 μ M and 0.5-10mM;
(c) electrophoresis and colouring reagents comprise reagent E and reagent F,
Reagent E: be 50 times of electrophoretic buffers, contain trihydroxy methyl aminomethane-acetate and ethylenediamine tetraacetic acid (EDTA), concentration is respectively 0.04M and 0.001M;
Reagent F: contain agarose and 0.5-20 μ g/ml ethidium bromide that W/V is 0.8-2%;
It is characterized in that VP 01Refer in particular to 5 '-ATTGAGAACCCGACAGAAGCGAAG; VP 02Refer in particular to 5 '-CCTAATGCGGTGATCAGTGTTACT.
2. according to the test kit of a kind of rapid detection vibrio alginolyticus described in the claim 1, the prescription that it is characterized in that every tube reaction reagent is 10 times of polymerase chain reaction damping fluids of 5.0 μ l, 2.5 unit hot polymerization synthase, the 35.5 μ l distilled water of sterilizing, 4.0 μ l2.5mM dNTP, 3.0 μ l 25mM MgCl 2, 10pM VP 01, 10pM VP 02
3. one kind is used the method for the test kit rapid detection vibrio alginolyticus described in the claim 1 to comprise the following steps: successively
(1). according to following formulated reagent:
Ethylenediamine tetraacetic acid (EDTA) 0.1-10mM and V/V concentration that reagent A contains trihydroxy methyl aminomethane 1-100mM, the pH8.0 of NaCl 0.1-0.3M, pH8.0 are the triton x-100 of 0-20%;
It is 1-100mM trihydroxy methyl aminomethane that reagent B contains N,O-Diacetylmuramidase and pH8.0, the concentration that concentration is 1-10mg/ml;
Reagent C contains 10 times of polymerase chain reaction damping fluids, hot polymerization synthase and sterilization distilled water, and content is respectively 2.5-5.0 μ l, a 0.5-2.5 unit and 18-35.5 μ l;
Reagent D: comprise that special primer is to VP 015 '-ATTGAGAACCCGACAGAAGCGAAG and VP 025 '-CCTAATGCGGTGATCAGTGTTACT, dNTP (mixtures of four kinds of deoxynucleotides) and MgCl 2, content is respectively 0.2-1 μ M and 0.2-1 μ M, 20-200 μ M and 0.5-10mM;
Reagent E: be 50 times of electrophoretic buffers, contain trihydroxy methyl aminomethane-acetate and ethylenediamine tetraacetic acid (EDTA), concentration is respectively 0.04M and 0.001M;
Reagent F: contain agarose and 0.5-20 μ g/ml ethidium bromide that W/V is 0.8-2%;
(2). detect successively according to the following step:
(a). sample preparation: get 0.1-0.5 gram sample, with 100-400 μ l reagent A suspended sample, and with the abundant mixing of sample; The centrifugal 2-5 of 10000-12000g minute, abandon supernatant;
(b) .DNA extracts: with the 100-300 μ l reagent A precipitation that suspends once more, and adding 20-100 μ l reagent B mixing, room temperature was placed more than 2 minutes, was incubated 1-10 minute again in boiling water bath, and cell is broken fully, discharged DNA; The centrifugal 8-10 of 10000-12000g minute, get supernatant and add the dehydrated alcohol deposit D NA that 2 times of supernatant volumes are 240-800 μ l; Room temperature was placed 5-10 minute, the centrifugal 3-8 of 10000-12000g minute; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in the 10-50 μ l sterilization distilled water; Be DNA extraction liquid;
(c). polymerase chain reaction (PCR) amplification: the DNA that extracts with 0.5-1 μ l is as the template of polymerase chain reaction (PCR) amplification; Getting 21-42 μ l reagent C mixes with 4-8 μ l reagent D; On thermal cycler, the dna profiling that extracts is increased, unwind 2-5 minute by 94 ℃-98 ℃; Used 93-95 ℃ of sex change then 30 seconds-1 minute, 54-58 ℃ of renaturation 30 seconds-1 minute, 72 ℃ were extended 30 seconds-1.5 minutes, so circulated 25-35 time, were incubated 8-12 minute at 72 ℃ at last, and the specific fragment of vibrio alginolyticus is increased to 10 8More than the mol;
(d). electrophoresis and color developing detection: get the sample after 5-10 μ l increases, with 2-6 μ l V/V is that 0.25% tetrabromophenol sulfonphthalein is mixed, W/V is agarose electrophoresis and the colour developing of 0.8-1.2%, under UV-light, detect the band whether one 300 nucleotide pair is arranged, if the band of one 300 nucleotide pair is arranged, interpret sample has vibrio alginolyticus to infect or pollutes, otherwise does not then have.
CNB011301279A 2001-12-27 2001-12-27 Reagent kit and method for quickly detecting alga-dissolving vibrio Expired - Fee Related CN1142291C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB011301279A CN1142291C (en) 2001-12-27 2001-12-27 Reagent kit and method for quickly detecting alga-dissolving vibrio

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB011301279A CN1142291C (en) 2001-12-27 2001-12-27 Reagent kit and method for quickly detecting alga-dissolving vibrio

Publications (2)

Publication Number Publication Date
CN1373358A CN1373358A (en) 2002-10-09
CN1142291C true CN1142291C (en) 2004-03-17

Family

ID=4669754

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB011301279A Expired - Fee Related CN1142291C (en) 2001-12-27 2001-12-27 Reagent kit and method for quickly detecting alga-dissolving vibrio

Country Status (1)

Country Link
CN (1) CN1142291C (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100495271B1 (en) * 2003-05-09 2005-06-14 한국해양연구원 The algicidal effect of red pigment produced by Hahella chejuensis 96CJ10356 strain and a method for production of red pigment having algalcidal effect therefrom
CN100419087C (en) * 2006-10-19 2008-09-17 天津市水产研究所 Primer sequence and detecting reagent kit for detecting vibrio alginolyticus
CN101928769B (en) * 2009-09-29 2012-07-04 大连水产学院 PCR specific amplification primer for quick detection of vibrio alginnolyficus based on SCAR molecular marker and detection kit
CN108384869A (en) * 2018-05-18 2018-08-10 福州大学 The multiple PCR primer group and detection method and kit of four kinds of morbid vibrios of detection simultaneously

Also Published As

Publication number Publication date
CN1373358A (en) 2002-10-09

Similar Documents

Publication Publication Date Title
Bernhard et al. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes
Dhar et al. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry
Hiney et al. Validation of polymerase chain reaction-based techniques for proxy detection of bacterial fish pathogens: framework, problems and possible solutions for environmental applications
CN103898108A (en) Nucleotide specific to Vibrio fluvialis O2, O4, O13, O15 and O18 and application thereof
CN101225440B (en) Detection method of leptospira
CN1232653C (en) Kit for fast test of vibrio harveyi and the test method thereof
CN1142291C (en) Reagent kit and method for quickly detecting alga-dissolving vibrio
CN107988405B (en) PCR detection kit for Salmonella indiana and non-diagnostic detection method thereof
CN1232654C (en) Reagent kit for quick test of vibrio vulnificus and the test method thereof
CN1506468A (en) PCR test kit for hygrophilous aeromonad and its test method
Carlsgart et al. Multiplex PCR on single unembryonated Ascaris (roundworm) eggs
CN1166783C (en) Reagent kit and process for quickly detecting schwann&#39;s bacterial of alga
CN1286988C (en) Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof
CN102154469A (en) General detection method for pathogenic vibrios and nucleic acid isothermal amplification detection kit used in same
Fernandez‐Senac et al. A comparison of the use of different swab materials for optimal diagnosis of amoebic gill disease (AGD) in Atlantic salmon (Salmo salar L.)
CN104450930A (en) Molecular detection method of vibrio parahaemolyticus and application thereof
Mackie et al. Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples
Sahar et al. Molecular and biochemical diagnosis of Salmonella in wastewater
RU2698651C1 (en) Method for determination of hydrobionts saprobity for assessment of ecological state of water bodies
CN100487134C (en) Reagent kit and detection method for quickly detecting shark vibrio
CN1277124C (en) PCR rapid detection kit for pathogenic Vibrio anguillarum and method for detection thereof
CN101805788B (en) PCR specific amplified primer for fast detection of vibrio parahaemolyticus based on SCAR molecular markers and detection kit
CN101200757A (en) Rapid detection reagent kit for human great pathogen-comma bacillus and detection method thereof
CN1279180C (en) PCR detection method for eel vibrio
CN101691613B (en) Primer group for detecting vibrio coralliilyticus by using LAMP, quick diagnosis kit and detecting method

Legal Events

Date Code Title Description
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20040317

Termination date: 20151227

EXPY Termination of patent right or utility model