CN1324319C - Detecting reagent box for fish virus and its detecting method - Google Patents

Abstract

The present invention relates to a detecting reagent kit for fish viruses and a detecting method thereof. Round holes for installing various bottle bodies filled with solution are arranged on a kit body; spare accessories, such as hybridization membranes, bags, etc., can be put on an extension tongue of the bottom surface of the kit body; three kinds of washing solution, three kinds of buffer solution, two kinds of control solution, two kinds of color developing solution, one kind of closed solution, one kind of antibody solution, one kind of probe solution and one kind of grinding solution are filled in fourteen bottles. The method comprises: firstly, fish tissue to be detected is taken and sufficiently ground in the grinding solution C; then, supernatant solution is taken; after the supernatant solution is denatured, a sample is pointed on a hybridization membrane; after the membrane is crosslinked and prehybridized, the membrane carries out a hybridization reaction with the denatured probe solution K; then, the washing solution D and the washing solution B are respectively used for washing the membrane; the membrane carries out an immune reaction with the antibody solution L; after the membrane is washed by the washing solution A, the membrane carries out a color developing reaction with the color developing solution NBT/BCIP; finally, a color developing result on the hybridization membrane is inspected and recorded; corresponding fish tissue with blue spots in the sample pointing position appears and the fish tissue is infected by viruses. The present invention can early and conveniently detect male fish, female fish, parent fish, fries and adult fish infected by the viruses for avoiding causing heavy loss.

Description

A kind of detection kit of fish virus and detection method thereof

Technical field

(Lymphocystic Disease Virus China, LCDV-cn) a kind of nucleic acid probe dot hybridization kit and detection technique thereof are a kind of detection kit and detection method thereof of fish virus specifically to the present invention relates to lymphocystis disease virus.

Background technology

LCDV-cn virus is a kind of DNA type virus that can infect multiple fish, and the breeding seawater fish of provinces such as China Shandong, Hebei, Guangdong all suffers its evil.It is reported that this disease is found as far back as Europe, the existing so far history that goes up a century.Its infection symptoms is that body surface, lip, the gill and the fin ray etc. fish are located, and the tumour of some likeness in form cauliflowers occurs, sick fish outward appearance ugliness, and mortality ratio rises, and commercial value descends greatly, and the economic loss that causes is very serious.And this virus has the characteristics of subclinical infection, invades in the fish body and can hide more than two months, do not show macroscopic symptom.And in a single day symptom appears, then the state of an illness increases the weight of, and does not also have desirable medicine in addition, so the consequence that causes is serious.Therefore,, remove and carry viral fish, just can accomplish that cut-out infects, minimizing is popular, ensure the healthy fish breed by early detection to parent population and fry.But up to now, the whole day, the ELISA detection method that professor has set up lymphocystis disease virus was kept in this Hokkaido University Jishui, and this method can qualitative, quantitative virus, but detected as early stage, trace, and its sensitivity is then not enough far away.Nucleic acid probe-dot hybridization technology is a kind of gene diagnosis technology efficiently.According to point-like spot on the hybond membrane have or not and strong and weak, can judgement sample whether contaminate, can also the sxemiquantitative cause of disease.Domestic, the application of dot blot technology in the disease of prawn diagnosis is more.(2000) such as Lo etc. (1999), Shi Chengyin etc. (1999) and Xu Hongtao utilize the method to detect prawn white spot syndrome (WSSV).Huanghai Sea aquatic products research institute of Chinese aquatic science institute has developed the disease chamber " the former nucleic acid probe of the explosive epidemic disease of prawn-dot blot diagnostic kit ", but dot blot is applied to the detection of cultured fishes virosis report is not arranged as yet.

Summary of the invention

The detection kit and the detection method thereof that the purpose of this invention is to provide a kind of fish virus are so that detect the fish that LCDV-cn infects in early days and more easily, especially to the early detection of fry and parent population, with the loss of avoiding this virus infections to cause.

The present invention adopts nucleic acid probe-dot hybridization technology, and it is a kind of detection technique on nucleic acid level, has good, the highly sensitive characteristics of accuracy.Its principle is: the DNA that utilizes LCDV-cn be a kind of duplex structure nucleic acid and thermal denaturation and renaturation characteristics (promptly the heating condition under, DNA can become strand by two strands; And under proper condition, also can become two strands by strand), as long as its base sequence is identical, two of separate sources dna single chains can the phase mutual cross after the sex change.According to the principle of this DNA hybridization, adopt the digoxigenin labeled specific dna probe liquid of National Bureau of Oceanography's first ocean research development, carry out dot hybridization and just can detect LCDV-cn.

Detection kit of the present invention is the square box body that there is extension tongue (portion) bottom surface, is provided with in the box body to insert interior Sheng circular hole solution, multiple bottle, and is placed in the external packing box.Solution in the bottle comprises each 3 kinds of washing lotion, damping fluids; Each a kind of contrast liquid, colour developing liquid each 2 kinds and confining liquid, antibody liquid, probe liquid, lapping liquid, and be infused in 14 containers (bottle) of inner box.Box bottom extends on the tongue can place standby annex, as hybond membrane, hybridization bag, thin walled tube and grinding rod.

Detection method of the present invention is to get fish tissue to be measured (in order to injure fish less, suggestion clip 0.05-0.1 gram fin ray as far as possible) earlier, fully grinds in lapping liquid C; Get its supernatant then, treat sex change after point sample on a hybond membrane; This film is behind crosslinked and prehybridization, with the probe liquid K hybridization after the sex change; Clean with washing lotion D, washing lotion B respectively then, carry out immune response with antibody liquid L again; After washing lotion A cleaned, NBT/BCIP carried out chromogenic reaction with colour developing liquid again, checked and write down colour developing result on the hybond membrane at last.The corresponding fish tissue of blue spot appears in the point sample place, is considered as the virus infections by LCDV-CN; Otherwise its corresponding fish is organized not by the LCDV-CN virus infections.

Description of drawings:

Fig. 1 is a general structure synoptic diagram of the present invention.

Fig. 2 is a box body vertical view of the present invention.

Wherein 1, box body; 2, bottle; 3, hybridization bag; 4, hybond membrane; 5, grinding rod; 6, centrifugal (thin-walled) pipe; 7, circular hole.

Embodiment:

As shown in Figure 1 and Figure 2, detection kit of the present invention is the square box body (1) that there is extension tongue (portion) bottom surface, contains the circular hole of the multiple bottle (2) of solution in laying above the box body (1).Extend on the tongue box body (1) bottom surface can put standby annex, as hybond membrane (4), hybridization bag (3), thin walled tube (6) and grinding rod (5).Solution in its bottle comprises washing lotion, each 3 kinds of damping fluids, and each a kind of contrast liquid, each 2 kinds of colour developing liquid and confining liquid, antibody liquid, probe liquid, lapping liquid, and be seated in corresponding 14 containers (bottle) of inner box.Following article are housed in the box body of the present invention: the A bottle (washing lotion A, 25ml); The B bottle (washing lotion B, 20ml); The C bottle (sample lapping liquid C, 5ml); The D bottle (washing lotion D, 5ml); The E bottle (damping fluid E, 5ml); The F bottle (damping fluid F, 5ml); The G bottle (damping fluid G, 3ml); The H bottle (confining liquid H, 1ml); The I bottle (positive control solution I, 10ul); The J bottle (negative controls J, 10ul); The K bottle (probe liquid K, 10ul); The L bottle (antibody liquid L, 10ul); The M bottle (colour developing liquid M, 3ul); The N bottle (colour developing liquid N, 4ul).Also have a plurality of annexes of biotech firm's systems such as the biological company limited of Beijing ancient cooking vessel state in addition, comprise many hybond membranes, a plurality of hybridization bags and Duo Gen grinding rod, and some 0.5ml thin walled tubes.

Press solution and the composition such as the following table of lexicographic order sign in the box:

Sequence number	Reagent name	Composition	Purposes
				A	Washing lotion A	PH 7.0 aqueous solution that contain 0.3M (every liter of despatch that) sodium chloride, 0.03M trisodium citrate and 0.1%WN (weight/volume) sodium dodecylsulphonate	The sample washing
B	Washing lotion B	PH 7.0 aqueous solution that contain 0.075M (every liter of despatch that) sodium chloride, 0.0075M trisodium citrate and 0.1%WN (weight/volume) sodium dodecylsulphonate	The sample washing
				C	Lapping liquid C	SEMP sample lapping liquid Trizol (American I NVITROGEN company)	Sample grinds
D	Washing lotion D	PH 7.5 aqueous solution that contain 0.1M maleic acid, 0.15M sodium chloride and 0.3%VN (volume/volume) polysorbas20 (worker's bioengineering company limited is given birth in Shanghai)	The sample washing
				E	Damping fluid E	PH 7.5 aqueous solution that contain 0.1M maleic acid, 0.15M sodium chloride	Buffering
F	Damping fluid F	PH 9.5 aqueous solution that contain 0.1M trishydroxymethylaminomethane (worker's bioengineering company limited is given birth in Shanghai), 0.1M sodium chloride and 0.05M magnesium chloride	Buffering
				G	Damping fluid G	PH 7.0 aqueous solution that contain 0.75M sodium chloride, 0.075M trisodium citrate, 10% (VN) sarcosyl, 1%WN sealer (U.S. sieve third constellations company) and 0.02% (WN) sodium dodecylsulphonate	Hybridization buffer
H	Confining liquid H	The aqueous solution that contains the PH7.5 of 10%WN sealer (U.S. sieve third constellations company), 0.1M maleic acid and 0.15M sodium chloride	In order to reduce nonspecific reaction
				I	Positive control solution I	The grinding supernatant that grinds with sample lapping liquid C, suffer from LCDV-cn lefteye flounder tissue	Positive control
J	Negative controls J	The grinding supernatant of lefteye flounder tissue that grind with sample lapping liquid C, normal	Negative control
				K	Probe liquid K	The specific dna probe (Oceanographic Inst. No.1 of State Bureau of Oceanography) that contains digoxigenin labeled	LCDV-cn is provided hybridization probe
L	Antibody liquid L	PH 7.5 aqueous solution that contain rabbit anti digoxin antibody (U.S. sieve third constellations company), 10%WN sealer (U.S. sieve third constellations company), 0.1M maleic acid and the 0.15M sodium chloride of 0.003 international unit/milliliter alkali phosphatase enzyme mark	Carry out specific immune response with the digoxin in the nucleic acid probe
				M	Colour developing liquid M	The aqueous solution that contains 7.5%W/V tetrazole indigo plant (NBT) (the biological company limited of Beijing ancient cooking vessel state), 68%V/V dimethyl formamide (DMF)	The colour developing effect
N	Colour developing liquid N	Dimethyl formamide (DMF) solution that contains 5-bromo-4-chloro-3-indoles phosphoric acid-toluene amine salt (BCIP) (the biological company limited of Beijing ancient cooking vessel state) of 5%W/V	The colour developing effect

The concrete operations step is as follows:

1, handles sample: cut fish to be measured with sterilization blade (knife blade, razors slice etc. all can) and organize 50-100mg, put into standby thin walled tube, add 100-200ul sample lapping liquid C, it is ground, left standstill about 2 minutes, precipitation is sunk with grinding rod;

2, each 10 microlitre of supernatant of getting in the above-mentioned lapping liquid inject thin walled tube, add positive control solution I and negative controls J again, in 100 ℃ of boiling water water-bath 5-10 minute, place ice-water bath fully to cool off rapidly, the in-house virus of fish is discharged, and utilize the thermal denaturation mode of quenching again, make the dna structure of virus become strand by two strands;

3, point sample: get 1ul (2) supernatant, in the little lattice on the point sample what hybond membrane, dry (noticing that the point sample amount is not excessive, in case adjacent sample room interpenetrates) naturally;

4, crosslinked: as the hybond membrane behind the point sample to be clipped between the two layers of filter paper, in 120 ℃ of baking ovens, to toast half an hour, the single stranded DNA after the sex change is fixed on the hybond membrane;

5, prehybridization: again hybond membrane is placed the prehybridization bag, add 1-2ml damping fluid G, seal, hatch half an hour in the 62-70 ℃ of water-bath with capper;

6, probe liquid sex change: with probe liquid K (together with container), 100 ℃ of boiling water baths 5 minutes place ice-water bath that it is fully cooled off rapidly, with the sex change of probe liquid, make the dna structure in the probe liquid become strand by two strands;

7, hybridization: cut off the prehybridization bag in the 5th step, add the solution K after the 6th step handled, hybridization 6-16 hour is hybridized to guarantee above-mentioned single-stranded probe and the strand sample that is fixed on the hybond membrane of allowing in the water-bath;

8, wash film: hybond membrane is taken out, puts into new hybridization bag, with washing more than the 2ml washing lotion D cleaning once; Hybond membrane is taken out, puts into new hybridization bag again, under 62-70 ℃ of condition, with 10ml washing lotion B, again more than the cleaning once, so that the probe liquid of not hybridizing on the film is washed off;

9, immune response: a new hybridization bag is put in above-mentioned hybond membrane taking-up, added 2ml damping fluid E, add 200ul confining liquid H, placed 30 minutes; Cut off one jiao of above-mentioned hybridization bag, add the antibody liquid L of 1/50 volume, the digoxin generation immune response that made in 30 minutes in antibody liquid and the probe liquid is placed in sealing;

10, wash film: hybond membrane is taken out, puts into a new hybridization bag, wash more than 1 time with 10ml washing lotion A; Embathed 1-2 minute with 2ml damping fluid F again, so that unconjugated antibody is washed off;

11, colour developing: hybond membrane is taken out, puts into a new hybridization bag.Add 1ml damping fluid F, add 3ul colour developing liquid M and 4ul colour developing liquid N again, place 2-12 hour (centre is not stirred) in the what darkroom.The time of colour developing, with on the hybond membrane not the blank space of point sample variable color do not occur and be as the criterion, in a single day variable color appear in blank space, should take next step with stopped reaction immediately;

12, stopped reaction: washed above-mentioned hybond membrane 5-15 minute with big water gaging, end chromogenic reaction;

13, record result: if LCDV-cn virus is arranged in the testing sample, then dark or shallow blue spot can appear in the point sample place on the hybond membrane, and blue spot should appear in positive control solution I point sample place; Negative controls J point sample place blue spot can not occur.In institute's test sample product, not having the sample of colour developing is normal fish, and the sample that blue spot occurs is the fish that is infected by LCDV-cn, and its blueness degree dark more, that explanation is infected is big more.If will preserve hybond membrane, then film can be clipped in and preserve again after making its drying in the filter paper.

Claims (4)

1, a kind of detection method of fish virus is characterized in that getting earlier fish tissue to be measured, fully grinds in lapping liquid C; Get its supernatant then, treat sex change after point sample on a hybond membrane; This film carries out hybridization reaction with the probe liquid K after the sex change behind crosslinked and prehybridization; Use washing lotion D then respectively, washing lotion B cleans, and carries out immune response with antibody liquid L again; After washing lotion A cleaned, NBT/BCIP carried out chromogenic reaction with colour developing liquid again, checked and write down colour developing result on the hybond membrane at last.The corresponding fish tissue of blue spot appears in the point sample place, and then by the LCDV-CN virus infections, otherwise its corresponding fish is organized not by the LCDV-CN virus infections.

2, a kind of concrete operations step of fish virus is: (1) handles sample: cut fish to be measured with the sterilization blade and organize 50-100mg, put into standby thin walled tube, add 100-200ul sample lapping liquid C, it is ground with grinding rod, left standstill 2 minutes, and made and avale; (2) each 10 microlitre of supernatant of getting in the above-mentioned lapping liquid inject thin walled tube, add positive control solution I and negative controls J again, at 100 ℃ of boiling water bath 5-10 minutes, place ice-water bath fully to cool off rapidly; (3) point sample: get the 1ul supernatant, in the little lattice of point sample on hybond membrane, dry naturally; (4) crosslinked: as the hybond membrane behind the point sample to be clipped between the two layers of filter paper, in 120 ℃ of baking ovens, to toast half an hour; (5) prehybridization: again hybond membrane is placed the prehybridization bag, add 1-2ml damping fluid G, seal, hatch half an hour in the 62-70 ℃ of water-bath with capper; (6) probe liquid sex change: with probe liquid K, 100 ℃ of boiling water baths 5 minutes place ice-water bath that it is fully cooled off rapidly; (7) hybridization: cut off the prehybridization bag in the 5th step, add the solution K after the 6th step handled, hybridization is 6-16 hour in the water-bath; (8) wash film: hybond membrane is taken out, puts into new hybridization bag, with washing more than the 2ml washing lotion D cleaning once; Hybond membrane is taken out, puts into new hybridization bag again, under 62-70 ℃ of condition, with 10ml washing lotion B, again more than the cleaning once, so that the probe liquid of not hybridizing on the film is washed off; (9) immune response: a new hybridization bag is put in above-mentioned hybond membrane taking-up, added 2ml damping fluid E, add 200ul confining liquid H, placed 30 minutes.Cut off one jiao of above-mentioned hybridization bag, add the antibody liquid L of 1/50 volume, sealing, placement 30 minutes; (10) wash film: hybond membrane is taken out, puts into a new hybridization bag, wash once with 10ml washing lotion A more than; Embathed 1-2 minute with 2ml damping fluid F again; (11) colour developing: hybond membrane is taken out, puts into a new hybridization bag, add 1ml damping fluid F, add 3ul colour developing liquid M and 4ul colour developing liquid N again, placed 2-12 hour in the what darkroom; (12) stopped reaction: washed above-mentioned hybond membrane 5-15 minute with big water gaging, end chromogenic reaction and write down the result.

3, a kind of detection kit of fish virus, it is characterized in that detection kit is that the bottom surface has the square box body (1) that extends tongue, the circular hole that multiple bottle (2) body of containing solution in laying is arranged above the box body (1), extend on the tongue box body (1) bottom surface can put standby annex, comprise hybond membrane (4), hybridization bag (3); thin walled tube (6) and grinding rod (5); the solution in its bottle comprises washing lotion A; B; three kinds of D; damping fluid E; F; three kinds of G and positive control solution I and negative controls J; liquid M develops the color; two kinds of N and confining liquid H; antibody liquid L; probe liquid K; each is a kind of for lapping liquid C, and is seated in corresponding 14 bottles (2) of inner box (1).

4, the detection kit of a kind of fish virus as claimed in claim 3 is characterized in that above-mentioned probe liquid contains the LCDV-cn specific dna probe of digoxigenin labeled.

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