CN1232653C - Kit for fast test of vibrio harveyi and the test method thereof - Google Patents

Abstract

The present invention relates to a vibrio harveyi detection kit and a detection method thereof. The kit comprises a DNA extraction agent which comprises a buffering reagent A and a cell disruption reagent B and a polymerase chain reaction reagent which comprises a reaction premixing reagent C and a reaction attractor reagent D, wherein the specificity attractor of the reagent D is 5'-GCACCATGGGTTAAAAGCCGCT for VP01 and is 5'-AGAGAACTGACGCACAACGTGGTTC for VP02, and the kit also comprises a cataphoresis and color developing reagent which comprises a reagent E and a reagent F. The detection method comprises sample processing, DNA extracting, PCR amplifying, and cataphoresis and color developing detecting; if a strip with 480 nucleotide pairs occurs in ultraviolet light detection, samples are infected or polluted with the vibrio harveyi, and otherwise the samples are not infected or polluted with the vibrio harveyi. The vibrio harveyi detection kit and the detection method thereof have the advantages of rapid detection and high sensitivity.

Description

The test kit of rapid detection Vibrio harveyi and detection method

Technical field

The present invention relates to a kind of test kit of rapid detection Vibrio harveyi, further relate to the method that the test kit that uses the rapid detection Vibrio harveyi is surveyed the inspection Vibrio harveyi fast.

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 with virus, other bacterium or parasite etc. are caused co-infected together.In China's sea farming biology, Vibrio harveyi is one of modal vibrio pathogen, and every year is very huge by the loss that Vibrio harveyi causes.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, just can reach a conclusion.(reference: Lou Yongxin Wang Jinliang chief editor " practical clinical bacteriology check and progress " 1992 Tianjin Scientific English Translation publishing company), 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 harveyi particularly provides a pair of vibrio harveyi specific primer VP ₀₁And VP ₀₂, and provide a kind of test kit of rapid detection Vibrio harveyi that uses to survey the method for Vibrio harveyi in inspection and the monitoring several samples fast, 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 harveyi 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 harveyi, therefore, if contain Vibrio harveyi 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 harveyi, the purpose band of the same molecular weight that then can not increase.

The object of the present invention is achieved like this: a kind of test kit of rapid detection Vibrio harveyi 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 unit/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;

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 '-GCACCATGGGTTAAAAGCCGCT; VP ₀₂Refer in particular to 5 '-AGAGAACTGACGCACAACGTGGTTC.

(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.

The best group of every tube reaction reagent becomes: reagent C: 5.0 μ l, 10 * PCR Buffer, 0.5 μ l Taq (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.

The method of using the test kit of above-mentioned rapid detection Vibrio harveyi to survey the inspection Vibrio harveyi fast comprises 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 harveyi 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 480 nucleotide pair is arranged, if the band of one 480 nucleotide pair is arranged, interpret sample has Vibrio harveyi 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 harveyi, also can monitor cause of disease Vibrio harveyi in the aquaculture water environment, can also detect feed ingredient and fresh food and polluted by Vibrio harveyi.

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 harveyi 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 Buffer, 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 Vibrio harveyi 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 480 nucleotide pair is arranged, interpret sample has Vibrio harveyi 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 harveyi 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 60 ℃ 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 480 nucleotide pair is arranged, interpret sample has Vibrio harveyi to infect or pollutes; Otherwise then do not have.

Claims (2)

1. the test kit of a rapid detection Vibrio harveyi 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 and MgCl ₂, content is respectively 0.1-1 μ M and 0.1-1 μ M, 20-200 μ M and 0.5-25mM;

VP ₀₁Refer in particular to 5 '-GCACCATGGGTTAAAAGCCGCT; VP ₀₂Refer in particular to 5 '-AGAGAACTGACGCACAACGTGGTTC

(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%.

2. a method of using the test kit rapid detection Vibrio harveyi in the claim 1 comprises 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 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 harveyi 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 480 nucleotide pair is arranged, if the band of one 480 nucleotide pair is arranged, interpret sample has Vibrio harveyi to infect or pollutes, otherwise does not then have.