CN105353124A - Method for fast reaction of P27 protein of meconium ALV virus - Google Patents
Method for fast reaction of P27 protein of meconium ALV virus Download PDFInfo
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- CN105353124A CN105353124A CN201510886247.2A CN201510886247A CN105353124A CN 105353124 A CN105353124 A CN 105353124A CN 201510886247 A CN201510886247 A CN 201510886247A CN 105353124 A CN105353124 A CN 105353124A
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Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a method for fast reaction of P27 protein of a meconium ALV virus. The method comprises the following steps: (1) taking poults hatched by the same maternal poultry as a unit, merging meconium of two poults in each nest of each family number to be used as a detection sample, and adding the detection sample into a buffer solution contained sample collection tube; (2) putting the sample collection tube into a heat preservation box, adding liquid nitrogen, and taking out the sample collection tube after complete freeze; (3) conducting water bath constant temperature melting, and uniformly mixing; (4) repeating the steps (2) to (3) once; (5) at the room temperature, respectively adding a supernatant of the detection sample treated in the step (4) into the sampling end of a detection test strip coated with a group of specific monoclonal antibodies in advance aiming at the P27 protein of the meconium ALV virus for specific reaction; (6) conducting selection by adopting a family selection system. According to the method, the avian leukemia virus antigen in the meconium can be quickly and efficiently detected, and the cost can be lowered.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of to meconium ALV virus P27 albumen rapid-action method,
Background technology
Avian leukosis (avianleukemia, AL) is the poultry neoplastic disease caused by avian leukosis virus (avianleukemiavirus, ALV).ALV, in all kinds of chicken group popularity infection, comprises the yellow chicken in Cold boiled chicken, laying hen and place.To the prevention and control measure of ALV, only have and purified by disease, there is no other better shortcut.ALV both can horizontal transmission, also can vertical transmission.For breeding enterprise, the harmfulness of vertical transmission is larger.In the vertical transmission link of avian leukosis, the highest with the poison amount of carrying of 1 age in days meconium, horizontal infectiousness is the strongest, so the detection of meconium ALV virus and the superseded of positive chick are most important core links in AL purification process.
In enormous quantities, current poultry breeding enterprise both domestic and external identifies that the immunological method of meconium ALV is mainly based on ELISA (enzyme-linked immunosorbent assay), be auxiliary with indirect immunofluorescence (IFA), PCR.Although various method may provide certain help clinically above, because operating process is comparatively loaded down with trivial details, efficiency is lower.Calculate with the scale of 2000 plumage chick, the ELISA that this link is traditional detects and roughly needs 20 hours with method of eliminating, whole meconium ALV detects/and to eliminate link time tediously long, cause primary shoot chicken waiting as long for testing result, dehydration easily appears in high density under leaving state concentratedly, cause other cause of disease (cause of disease of particularly vertical transmission and the cause of disease of 1 age in days horizontal transmission) cross-infection probability between chick to increase simultaneously, production technology personnel are tired out simultaneously, and efficiency is lower.Therefore a kind of method setting up field diagnostic chicken group meconium ALV is rapidly and efficiently necessary, significant to clinical operation.
Summary of the invention
Invention broadly provides a kind of to meconium ALV virus P27 albumen rapid-action method, this detection method can detect avian leukosis fast and efficiently, and cost-saving.Concrete technical scheme is as follows: a kind of to meconium ALV virus P27 albumen rapid-action method, comprises the following steps:
(1) in units of the poult hatched by same maternal instinct poultry, brood is a family, number separately each family, the meconium of 2 poults in every brood merges as a detection sample, adds in the sampling pipe containing damping fluid by detection sample;
(2) sampling pipe is put into incubation chamber, in incubation chamber, add liquid nitrogen, the complete freezing rear taking-up of sample to be detected;
(3) detection sample freezing for step (2) is put into water-bath constant temperature to melt, and mix;
(4) step (2)-(3) are repeated once;
(5), under room temperature, the supernatant of detection sample step (4) processed joins bag in advance respectively and is carried out specific reaction by the application of sample end for the test strip of a group-specific monoclonal antibody of ALV virus P27 albumen;
(6) after reacting completely, take family elimination system, i.e. the poult that hatches of the kind egg of same poultry, if there is one to occur, that meconium detects ALV is positive, then this nest poult is all eliminated.
Preferably, in step (2), the freezing time is 2-4min.
Preferably, in step (3), the constant temperature thawing time is 2-4min.
Preferably, the time reacted completely in step (6) is 5-10min.
Preferably, described poultry is chicken, duck, goose.
Adopt above-mentioned to meconium ALV virus P27 albumen rapid-action method, the present invention has the following advantages:
(1), in reaction method of the present invention, poult is detected by only gathering meconium the meconium merging detection being promoted to two poults, and detection sensitivity is high, saves a large amount of man power and materials;
(2) reaction method of the present invention takes family elimination system, namely the poult that the kind egg of a maternal instinct poultry is hatched is brood, as long as ALV is positive to have one to occur, namely all poults of this nest are eliminated, reduce the harmfulness of vertical transmission, this need carry out same numbering to same family, avoid the drawback that all poults are numbered one by one, reduce sample number duration greatly, avoid the stimulation of existing puncture numbering to poult simultaneously, reduce the input of numbering expense, use manpower and material resources sparingly;
(3) method of fast freeze-thaw is adopted in reaction method of the present invention, traditional freezing-thawing method need consuming time 4 hours, cause leaving concentratedly when primary shoot chicken dewaters for a long time, the young seedling of high density waits for testing result causing other cause of disease easily mutually to infect, and freeze thawing once only needs about 6min in the present invention, greatly reduction detects duration, avoid the running time long impact on test simultaneously, avian leukosis virus antigen in meconium can be detected fast and efficiently.
Embodiment
One, specific embodiment
Embodiment 1
To detect the ALV virus of yellow-feather broiler, following detection method is equally applicable to other bird ALV Viral diagnosis.
1. meconium collection and freeze thawing
(1) preparation of article: the freeze-thawing resistant sample box (containing tail band lid) that sample collection is special, the liquid nitrogen container of filled with liquid nitrogen 2, incubation chamber with cover 3, water-bath, special Sample Dilution lysate;
(2) sample collection: with nest (chick that same hen hatches) for unit, each family is numbered separately.Simultaneously according to the quantitative examination result of this research, the meconium of every 2 chick is merged into one containing in the sampling pipe of special Sample buffer liquid, this than classic method (collecting by only monocyte sample) save half testing cost and manpower and materials follow-up in a large number; (3) fast freeze-thaw sample: removed by the bottom shell of box after receiving sample, only puts into specific incubation chamber by lid and sample part, uses container in foam box, to add liquid nitrogen, the cap covers of rapid incubation chamber.Take out after sample is all freezing, refrigerating process approximately needs 2-4min;
(4) sample is melted rapidly: adjust water-bath temperature in advance to 37 DEG C, above-mentioned freezing sample former state (sample part and lid) is put into water-bath, and put upside down mixing, take out after sample all melts, melting process approximately needs 2-4min;
(5) freeze thawing is repeated: above-mentioned (3)-(4) step;
For freeze thawing one box sample, whole freeze thawing link needs 15min altogether, than the classic method (about 4h) of subzero 70 DEG C of refrigerator freeze thawing, saves time more than three hour.
2. the quick detection of meconium ALV
(1) article prepare: the meconium sample of twice is crossed in freeze thawing, small-sized disposable plastic tube, detect reagent and dull and stereotyped (putting test strips).
(2) by wrap in advance by the test strip of the group-specific monoclonal antibody for ALV virus P27 albumen be arranged side by side in detect dull and stereotyped.Use disposable little plastic suction pipe, put order by sample and drip 200 microlitres (4) sample (supernatant) to the application of sample end of detector bar, generally under room temperature (20-25 degree), react 5-10 minute judged result.If the running time long (> 10 ~ 20min), can non-specific responding be caused.Skilled operation personnel application of sample 50, take time about 5-6min.
(3) technician is arranged to carry out result judgement: every 50 samples can judge (application of sample is about 5min) after application of sample terminates, on recording chart, record the result of this time point, residue sample need be placed 2 ~ 5min and judge (application of sample terminates rear 5min) again simultaneously.
(4) eliminate link: take family to eliminate, i.e. the seedling chicken that hatches of the kind egg of same hen, as long as there is one to occur, that meconium detects ALV is positive, and namely other seedling chickens produced of this hen are all eliminated.
3. caution of operation point
(1) for ensureing the stable of whole detection system, avoid the interference of some non-specific factors, to often criticizing detection reagent, with viral capsid proteins P27 gene expression product as positive control, freshly prepared Sample buffer liquid and pure water (5 parts) are verified as negative control, normal condition P27 albumen in 5-10min is feminine gender for positive, Sample buffer liquid and pure water.Detect the preservation of reagent, room temperature (about 25 DEG C), the time does not exceed 2 months.
(2) note the part not drawing impurity during application of sample, prevent from sticking very much and cause sample protein can not move in test strip.
(3) different family chicken group, notes the replacing of suction pipe, avoids cross pollution.
(4) sample is replaced after adding, and discards after waiting result judgement errorless again.
(5) when judging, illumination is eager to excel, and the too weak result that can affect judges.
(6) positive chicken group mark and process: in the chicken group of each family, has more than one to occur the positive, marks with red pen, must eliminate.
Two, the assessment of Detection results
Due to the uncertainty of the virus of type place of china yellow-feather broiler ALV at a slow speed toxin expelling time point, seldom do the system evaluation that meconium detects and eliminates effect.This technical system, then according to early-stage Study result, establishes meconium and detects the assessment technology eliminating effect.
Concrete ins and outs are as follows:
(1) not detecting meconium group and detecting meconium group of yellow-feather broiler breeder flock is at a slow speed chosen, following the tracks of 4 week age, 6 week age, 8 week age respectively, sampling for detecting intestinal mucosa toxin expelling (intestines poison) and blood lymphocyte virus (blood poison) positive rate;
(2) blood poison and intestines poison detect: blood poison detection method, by direct for blood lymphocyte inoculated into chick embryo fibroblast DF1 cell, infects after 1 hour, supernatant discarded, Continuous Cultivation 8 days, freeze thawing DF1 cell, then adopts ELISA method to detect ALV virus P27 albumen; Intestines poison detection method directly intestinal mucosa cotton swab is collected specific sample buffer, after multigelation 2 times, directly detects viral P27 antigen by ELISA method.The sample that S/P is greater than 0.2 is judged to be that virus infections is positive.
Table 1 testing result
Site technology assessment typical consequence is as follows:
From intestines poison and the blood poison positive findings of each time period, " detecting meconium group " has obvious reduction than " not detecting meconium group " positive rate.Prove that accuracy and the actual effect of the detection of whole meconium and filtering technique system are high.And do not detect meconium group, along with growing up of chick, its infection rate also increases gradually, finally also increases the mortality of chick or one-tenth chicken, wastes more cost.
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (5)
1., to a meconium ALV virus P27 albumen rapid-action method, it is characterized in that: comprise the following steps:
(1) in units of the poult hatched by same maternal instinct poultry, brood is a family, number separately each family, the meconium of 2 poults in every brood merges as a detection sample, adds in the sampling pipe containing damping fluid by detection sample;
(2) sampling pipe is put into incubation chamber, in incubation chamber, add liquid nitrogen, the complete freezing rear taking-up of sample to be detected;
(3) detection sample freezing for step (2) is put into water-bath constant temperature to melt, and mix;
(4) step (2)-(3) are repeated once;
(5), under room temperature, the supernatant of detection sample step (4) processed joins bag in advance respectively and is carried out specific reaction by the application of sample end for the test strip of a group-specific monoclonal antibody of ALV virus P27 albumen;
(6) after reacting completely, take family elimination system, i.e. the poult that hatches of the kind egg of same poultry, if there is one to occur, that meconium detects ALV is positive, then this nest poult is all eliminated.
2. one according to claim 1 is to meconium ALV virus P27 albumen rapid-action method, it is characterized in that: in step (2), the freezing time is 2-4min.
3. one according to claim 1 is to meconium ALV virus P27 albumen rapid-action method, it is characterized in that: in step (3), the constant temperature thawing time is 2-4min.
4. one according to claim 1 is to meconium ALV virus P27 albumen rapid-action method, it is characterized in that: the time reacted completely in step (6) is 5-10min.
5. the one according to any one of claim 1-4, to meconium ALV virus P27 albumen rapid-action method, is characterized in that: described poultry is chicken, duck, goose.
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| CN201510886247.2A CN105353124A (en) | 2015-12-04 | 2015-12-04 | Method for fast reaction of P27 protein of meconium ALV virus |
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| CN201510886247.2A CN105353124A (en) | 2015-12-04 | 2015-12-04 | Method for fast reaction of P27 protein of meconium ALV virus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108196049A (en) * | 2017-12-28 | 2018-06-22 | 中国农业科学院哈尔滨兽医研究所 | The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus |
| CN114397449A (en) * | 2022-01-05 | 2022-04-26 | 中科国创(南平)生物科技有限公司 | Sample diluent for ELISA detection of avian leukosis virus antigen in meconium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108196049A (en) * | 2017-12-28 | 2018-06-22 | 中国农业科学院哈尔滨兽医研究所 | The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus |
| CN114397449A (en) * | 2022-01-05 | 2022-04-26 | 中科国创(南平)生物科技有限公司 | Sample diluent for ELISA detection of avian leukosis virus antigen in meconium |
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Effective date of registration: 20170322 Address after: 213000 Changzhou City, Wujin Province town, West Village, West Village, the town of cattle cattle, No. 500 Applicant after: JIANGSU LIHUA ANIMAL HUSBANDRY STOCK CO., LTD. Applicant after: Jiangsu Xing Xing Agriculture Technology Co., Ltd. Address before: 213000 Changzhou City, Wujin Province town, West Village, West Village, the town of cattle cattle, No. 500 Applicant before: JIANGSU LIHUA ANIMAL HUSBANDRY STOCK CO., LTD. |
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Application publication date: 20160224 |