CN1240847C - Reagent box for determining infectious chicken anemia - Google Patents

Abstract

The present invention relates to an eiasy for rapidly diagnosing avian infectious anaemia by PCR. The present invention comprises tissue DNA extract, PCR reaction liquid, Taq DNA polymerase and CIAV PCR positive control templates. When the present invention is used for detecting avian infectious anaemia, the specificity is strong, the reaction results for other avian normal pathogens present negative, the sensitivity can reach 1 fg/microliter, and the whole detection process can be completed within 5 hours.

Description

The chicken infectious anemia detection kit

Technical field:

The present invention relates to the chicken infectious anemia detection kit, particularly a kind of detection kit with PCR method quick diagnosis chicken infectious anemia.

Background technology:

Chicken infectious anemia is by chicken anaemia virus (Chicken infectious anemia virus, the chicken disease that CIAV) causes.Chicken anaemia virus is the representative of new Viraceae one a PCV-II section, can cause the serious anaemia of chicken, and protoerythrocyte is damaged lymphocyte, particularly atrophy of thymus gland in the subcutaneous hemorrhage of muscle, marrow.At present, chick anemia has been taken as important worldwide poultry diease.

CIAV detection method commonly used at present mainly contains following several:

(1) serology detection method

As fluorescent antibody test and enzyme linked immunosorbent assay, can detect the CIA specific antibody in the serum sample and tire, and the immune effect of CIA vaccine.But because the hysteresis quality that antibody produces, this method can not accurately reflect the instant infection state of chicken group CIA.Need the highly purified CIAV antigen of mass production in enzyme linked immunosorbent assay, cost is higher.

(2) histology method

1, neutralization test: can detect CIAV cause of disease or antibody, susceptibility and specificity are all stronger, but length consuming time generally needs several days time;

2, spot hybridization: can detect the CIAV cause of disease, high specificity, but complex operation;

3, polymerase chain reaction (PCR reaction): can detect the CIAV cause of disease, susceptibility and specificity are very strong (referring to document B.W.Calnek etl, Diesases of Poultry (tenth edition) .749 and document Todd, D, .K.A.Maawhinney, and M, S.McNulty.1992.Detection and differentiantion ofchicken anaemia virus isolates by using the polymerase chain reaction.J ClinMicrobiol 30:1661-1666).But need to use organic solvent in the PCR operation of prior art, as extracting DNA with phenol, chloroform etc., to remove albumen, these organic solvents have toxicity, can cause environmental pollution, and influence operator ' s health.And all ingredients composition needs to preserve separately in the reaction solution, and reaction solution must now be joined in use, carries, stores and operate all comparatively loaded down with trivial details.

Summary of the invention:

The technical problem to be solved in the present invention provides a kind of detection kit with PCR method quick diagnosis chicken infectious anemia, and the detection reagent in this test kit is easy to use, does not cause environmental pollution.The extracting solution and the preparation that particularly prepare extracting sample DNA template comprise at the Auele Specific Primer (primers) of Chicken Anemia Virus (CAV) (chicken infectious anemia virus CIAV) and the reaction solution that can carry out the PCR reaction.

PCR (polymerase chain reaction) technology is utilized the principle of viral DNA sex change and renaturation under differing temps, under appropriate reaction conditions, the copy number of target DNA is almost increased by geometric progression, 20 PCR circulation will produce 1,000,000 times amplified production, make in the sample trace exist virus particle to react through PCR and become macroscopic result.

Chicken infectious anemia detection kit provided by the invention includes tissue DNA extracting solution, PCR reaction solution, TaqDNA polysaccharase and CIAV PCR positive control template.

Described tissue DNA extracting solution contains 20mmol/L Tutofusin tris, 10mmol/L ethylenediamine tetraacetic acid (EDTA), 10% sodium laurylsulfonate, 400 μ g/ml Proteinase Ks and 2 μ g/ml RNA enzymes (RnaseA).

Described PCR reaction solution contains 10mmol/L Tutofusin tris, 50mmol/L Repone K (KCl), 1.5mol/L magnesium chloride (MgCl ₂), 0.1% Octylphenoxy poly-ethoxy ethanol, 0.4mmol/L deoxynucleoside triphosphate and 0.4nmol/L CIAV Auele Specific Primer.

Described CIAV PCR positive control template is the genomic dna through extractive CIAV standard strain CUX-1 strain, can be directly used in PCR reaction and conduct and detect the whether normal mark of PCR reaction.

Described CIAV Auele Specific Primer is according to the CIAV genome sequence design of delivering, the upstream and downstream primer lays respectively at genome 358bp and 1042bp place, primer sequence is respectively: 5 '-ATACTCGAGGCGGTCCGGGTGGATGC-3 ' and 5 '-GGTGCGGCCGCCTCACACTATACGT-3 ', the amplified fragments size is 684bp.

Described Taq archaeal dna polymerase (Thermus aquaticus) is by the genetically engineered enzyme of purifying in the intestinal bacteria, has 5 ' 3 ' the 5 prime excision enzyme activity of the polymerization of depending on, and lacks 3 ' 5 ' 5 prime excision enzyme activities.

Confirm that through susceptibility and specificity test use test kit of the present invention that the pathological material of disease of organizing of doubtful CIAV infected chicken is detected, susceptibility can reach 1fg/ μ l, be about as much as 100 CIAV genome copies.Former for other common diseases of chickens, no matter dna virus or RNA viruses, or bacterium, reaction result is all negative, proves that this test kit specificity is stronger.Whole testing process can be finished in 5 hours.

In detecting the test of artificial challenge's Chicken Anemia Virus (CAV) chick, through comparison shows that: detect with test kit of the present invention with spot hybridization, susceptibility than spot hybridization (dot blot hybridization) is high 100 times, and the reaction times shortens 30 hours.

Embodiment:

Embodiment 1 chicken infectious anemia detection kit

Composition:

30ml tissue DNA extracting solution, 5mlPCR reaction solution, 100 μ l Taq enzymes, 100 μ l positive control dnas.

The tissue DNA extracting solution is stored in 4 ℃, and other component need be kept at-20 ℃.Validity period is 10 months.

The preparation of embodiment 2 chicken infectious anemia detection kit

(1) tissue DNA extracting solution

1. basal liquid:

1mol/L Tutofusin tris (Tris, PH8.0) solution: at 800ml water (H ₂O) dissolving 121.1 gram Tutofusin triss add concentrated hydrochloric acid (HCl) and regulate pH value to 8.0 in.Add water and be settled to 1000ml, high pressure sterilization in 15 pounds, 20 minutes, room temperature preservation is stand-by.

0.5mol/L ethylenediamine tetraacetic acid (EDTA) (ethylene diamine tetraacetic acid EDTA PH8.0) solution: in 800ml water, add 186.1 grams, two water disodium ethylene diamine tetraacetate (EDTA-Na2H ₂O), vigorous stirring on magnetic stirring apparatus, constant volume is in 1000ml then to regulate pH value to 8.0 (needing 20 gram NaOH particles approximately) with sodium hydroxide (NaOH), and high pressure was sterilized in 15 pounds, 20 minutes, and room temperature preservation is stand-by.

20% sodium laurylsulfonate (sodium dodecyl sulfatepolyacrylamide, SDS) solution: dissolving 200 gram electrophoresis level SDS in 900ml water, be heated to 68 ℃ of hydrotropies, the pH value to 7.2 that adds several concentrated hydrochloric acids (HCl) regulator solution, add the water constant volume in 1000ml, room temperature preservation is stand-by.

200mg/ml Proteinase K solution: the 200mg Proteinase K is dissolved in the 10ml water, and-20 ℃ of preservations are stand-by.

10mg/ml RNA enzyme A (RNaseA) solution: 50mg RNA enzyme A (RNaseA) is dissolved in the 5ml RNA enzyme storage buffer, in 100 ℃ of heating 15 minutes, slowly cools to room temperature, it is stand-by in-20 ℃ of preservations to be distributed into aliquot.RNA enzyme storage buffer

At 800ml water (H ₂O) dissolving 1.21 gram Tutofusin triss (10mmol/L) in, (NaCl 15mmol/L) adds concentrated hydrochloric acid (HCl) and regulates pH value to 7.5 8.77 gram sodium-chlor.Add water and be settled to 1000ml, high pressure sterilization in 15 pounds, 20 minutes, room temperature preservation is stand-by.

2. tissue DNA extracting solution

Get 2ml 1mol/L Tutofusin tris (Tris, PH8.0) solution (final concentration 20mmol/L); 2ml 0.5mol/L ethylenediamine tetraacetic acid (EDTA) (ethylene diamine tetraacetic acid EDTA PH8.0) solution (final concentration 10mmol/L); 50ml 20% sodium laurylsulfonate (sodium dodecyl sulfatepolyacrylamide, SDS) solution (final concentration 10%); 0.2ml 200mg/ml Proteinase K solution (final concentration 400 μ g/ml); 20 μ l 10mg/ml RNA enzyme A (RNaseA) solution (final concentration 2 μ g/ml) add water and are settled to 100ml, and 4 ℃ of preservations are stand-by.Each sample is with 300 μ l tissue DNA extracting solutions.

(2) PCR positive template

1. material and method

Virus; CIAV virus Cux-1 type strain is through chicken horse Garrick lymphoma cell line (MDCC-MSB ₁) after the propagation, by this laboratory-40 ℃ preservation.

Experimental chicken: 1 age in days SPF chick is provided by China Veterinary Drugs Supervisory Inst.'s Experimental Animal Center.

2. virus is returned the chicken experiment

Virus is prepared: the chicken horse Garrick lymphoma cell culture that will infect CIAV virus Cux-1 strain, freeze thawing three times, add equal amounts of chloroform shake well mixing, after 3000rpm, 20 minutes are centrifugal, get supernatant liquor in 70 ℃ of water-baths 5 minutes, 3000rpm, 20 minutes centrifugal back supernatant liquors are through 0.22 μ m membrane filtration, and be frozen standby after the packing.

Return the chicken experiment: get 10 of 1 age in days SPF chick,, every femoribus internus intramuscular injection CIAV virus liquid 0.2ml raises in shield retaining.Attack to cut open to get extremely in malicious back the 16th day and stay liver.

3. sample preparation: institute's liver of getting grinds back adding PBS through tissue grinder and makes 20% emulsion, 10000rpm after 3 freeze thawing repeatedly, and 5 minutes are centrifugal, get supernatant liquor and are used for extracting DNA.

4.DNA extract

Above-mentioned sample is got 0.25ml, add isopyknic extraction buffer (20mM Tris-Cl (pH7.4), 200mM EDTA, 40mM RNase, 1%SDS), 37 ℃ of effect 1hr, add 200 μ g/ml Proteinase Ks then, 56 ℃ of digestion 2hr, use phenol, phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1), each extracting of chloroform once are dissolved in behind ethanol sedimentation among the 10 μ l10mM TE (pH8.0).Each PCR reaction is got 1 μ l as positive template.

(3) PCR reaction solution:

1.10 doubly Tutofusin tris (Tris, PH9.0) buffer salt solution:

At 80ml water (H ₂O) (Tris 0.1mol/L), adds concentrated hydrochloric acid (HCl) and regulates pH value to 9.0 dissolving 1.211 gram Tutofusin triss in.Dissolving 3.728 gram Repone K (KCl, 0.5mol/L); 304.95 gram magnesium chloride (MgCl ₂6H ₂O, 15mol/L), 1ml TritonX-100 (1%) adds water and is settled to 100ml, and room temperature preservation is stand-by.

2.CIAV Auele Specific Primer:

Design a pair of primer according to the CIAV genome sequence of delivering, the upstream and downstream primer lays respectively at genome 358bp and 1042bp place, gives birth to worker bio-engineering corporation by Shanghai and synthesizes, and the amplified fragments size is 684bp.

Primer sequence is respectively:

5 '-ATACTCGAGGCGGTCCGGGTGGATGC-3 ' molecular weight=8437 mole numbers=19.5nmol is dissolved in the 1950 μ l water (final concentration 10nmol/ml)-20 ℃ of preservations.

5 '-GGTGCGGCCGCCTCACACTATACGT-3 ' molecular weight=8112.5 mole numbers=20.25nmol is dissolved in (final concentration 10nmol/ml)-20 ℃ of preservations in the 2025 μ l water.

3.10mM deoxynucleoside triphosphate (depxunucleoside triphosphate, dNTP):

Commercially available 10mM deoxynucleoside triphosphate mixed solution, comprising dATP, dCTP, dGTP, each 2.5mM. of dTTP is available from the sincere biotechnology of Beijing Xi Er company limited.-20 ℃ of preservations are stand-by.

4.PCR reaction solution:

Get 10 times of Tutofusin triss of 2ml (Tris, PH9.0) buffer salt solution, each 0.8ml of CIAV Auele Specific Primer,

(depxunucleoside triphosphate, dNTP) 0.8ml add water and are settled to the 10mM deoxynucleoside triphosphate

20ml ,-20 ℃ of preservations are stand-by, and each sample is with 50 μ l PCR reaction solutions.

(4) Taq archaeal dna polymerase (Thermus aquaticus):

Be commercially available homemade commercialization enzyme, available from the sincere biotechnology of Beijing Xi Er company limited.-20 ℃ of preservations are stand-by.

Embodiment 3 chicken infectious anemia PCR diagnostic kit working method:

1. getting 2 gram (soya bean size) pathological material of diseases to be checked (liver, spleen) puts into the 1.5ml centrifuge tube and fully studies mill back and dilute with an amount of PBS, 5000rpm * 5 are minute centrifugal, get 300 μ l supernatant liquors and add equivalent DNA extraction liquid, 56 ℃ digested 2 hours, 95 ℃ of deactivations coolings rapidly after 10 minutes;

2.10000rpm * 5 minutes centrifugal, gets supernatant liquor, changes another 1.5ml centrifuge tube over to, adds dehydrated alcohol (the conventional reagent in laboratory ,-20 ℃ frozen) post precipitation of two volumes precooling, 10000rpm * 5 are minute centrifugal;

3. the removal supernatant liquor adds 50 μ l PCR reaction solutions and 1 μ l Taq enzyme, flicks to add 50 μ l (about two) whiteruss behind the tube wall mixing.Place and carry out amplified reaction on the PCR instrument;

Each reaction need be provided with positive control and negative control.This test kit provides the positive control template, and each reaction is got 1 μ l and added 50 μ l PCR reaction solutions and 1 μ l Taq enzyme; Negative control is 50 μ l PCR reaction solutions and 1 μ l Taq enzyme;

Response procedures is: behind 94 ℃, 5min sex change, carry out 94 ℃, 1min → 55 ℃; 1min → 72 ℃, 2min totally 30 circulations, after 72 ℃, 5min extend the back and finish;

4. with 1% sepharose, 40v voltage, 20min electrophoresis detection.Positive should present the DNA band of a 0.68Kb, and is consistent with positive control sample, and the negative control sample does not have band.

Embodiment 4 chicken infectious anemia PCR diagnostic kit susceptibility and specificity test

1. materials and methods

1.1 viral CIAV virus Cux-1 type strain is in MDCC-MSB ₁The propagation back is standby on the cell.

1.2SPF test chicken 1 age in days SPF breeding chickling is in shield retaining, top subcutaneous vaccination CIAV virus Cux-1 type strain, when classical symptom appears in 16 ages in days, cut open and kill, get thymus gland, marrow, spleen, liver, the fabricius bursa, kidney, blood clotting white corpuscle and ight soil respectively and use for detecting.

1.4 template DNA extraction infected chicken different tissues takes a morsel respectively and fully dilutes with an amount of PBS behind the research mill, 5000rpm * 5 are minute centrifugal, get 300 μ l supernatant liquors and add equivalent DNA extraction liquid, 56 ℃ of digestion 2 hours, 95 ℃ of deactivations 10 minutes, rapidly after the cooling, 10000rpm * 5 are minute centrifugal, get supernatant liquor, dehydrated alcohol post precipitation with the two volumes precooling, 10000rpm * 5 are minute centrifugal, remove supernatant liquor, and precipitation is dissolved in the 50 μ l reaction solutions.The CIAV cells infected is by extracting cell toxicant DNA with quadrat method.

1.5PCR reaction conditions and program will be dissolved with 50 μ l PCR reaction solutions of sample DNA and be transferred in the PCR reaction tubes, add Taq enzyme 1 μ l, cover 2 paraffin body oil on behind the mixing, put on the PCR instrument and react.Specific procedure is: after sex change in 94 ℃, 5 minutes, carry out 94 ℃, 1 minute → 55 ℃; Totally 30 circulations in 1 minute → 72 ℃, 2 minutes are after extended the back end in 72 ℃, 5 minutes.Each group is participated in the test sample of PCR reaction, must include a positive control (1 μ l positive template+50 μ l PCR reaction solutions) and a negative control (50 μ l PCR reaction solution).

1.6 electrophoresis: 1.0% sepharose, 40V voltage, 10 μ l are got in each reaction, electrophoresis 25 minutes, 294nm wavelength UV-lamp observations.When positive control amplifies specific band, and negative control illustrates this group pcr amplification credible result when not amplifying any band; When test sample amplifies identical with positive control size and nothing but during specific band, illustrates that this test sample is infected by CIAV.Otherwise illustrate not infected.

1.6 susceptibility is carried out 10 times of serial dilutions with Cux-1 type strain cells infected extracting nucleic acid water, different extent of dilution nucleic acid samples carry out PCR reaction as template respectively, judge susceptibility with the high dilution that can amplify specific band.

1.7 specificity is recombinant plasmid, Cux-1 strain cell toxicant, SR43 strain cell toxicant, the MDCC-MSB1 cell to contain amplified fragments respectively, reaching other cause of disease newcastle disease virus (NDV), chicken Marek's disease virus (MDV), chicken infectivity bursa of Fabricius virus (IBDV), infectious bronchitis virus (IBV), egg-decreasing syndrome virus (EDSV) and Escherichia coli nucleic acid is that template is carried out the PCR reaction, detects the specificity of amplified fragments.

1.7 CIAV experimental infection chicken different tissues sample is got in the detection of experimental infection chicken different tissues, wipes away etc. as liver, spleen, thymus gland, the fabricius bursa, marrow, kidney, white corpuscle and cloaca cotton, extracts template DNA respectively, carries out PCR and detects.

2. result

2.1 the dilution of the susceptibility CIAV Cux-1 type strain cells infected of PCR reaction poison extracting nucleic acid initial concentration is 1ng/ μ l, through 10 * serial dilution to 0.01fg/ μ l.The result shows that the minimum concentration of naked eyes bands visible is 1fg/ μ l.

2.2 the specific PCR result of PCR reaction shows, the recombinant plasmid that contains amplified fragments, Cux-1 type strain and SR43 strain isolated cells infected poison nucleic acid all can amplify tangible specific band, and MDCC-MSB1 nucleus, other fowl encountered pathogenic NDV, MDV, IBDV, IBV, EDSV and E.coli nucleic acid amplification result are all negative.Illustrate that this PCR method specificity is stronger.

2.3 experimental infection chicken different tissues sample detection PCR detected result shows, it is that template all can amplify specific band that liver, spleen, thymus gland, marrow, the fabricius bursa, kidney, white corpuscle and cloaca cotton such as wipe away at each tissue sample, and the brightness power of amplified band can reflect the CIAV content in the different tissues sample.

The main virulent separation and Culture of diagnostic method, serological method and the molecular biology method of present CIA.In view of viral separation and Culture wastes time and energy, serological method is usually used in detecting antibody, thus the investigator to be more prone to use time saving and energy saving, accuracy and susceptibility all higher and organize the height of viral level.Wherein the brightness of liver and spleen amplified band is the highest.And can directly detect antigenic molecular biology method.Nucleic acid hybridization and PCR method report in the CIAV diagnosis is more, compares with nucleic acid hybridization, and PCR method adds time saving and energy saving, and susceptibility raising 10-100 doubly, thereby is widely used in the CIAV diagnosis.

In this PCR method, we select the purpose fragment of CIAV nucleocapsid protein VP1 gene as amplification for use, because of this gene is a CIAV primary structure protein gene, sequence is conservative relatively between the different strains of CIAV, thereby this fragment that increases helps to detect the different strains of CIAV, we also can utilize the gene of amplification to carry out structural analysis and protein expression simultaneously, and control also has meaning to CIAV.

The susceptibility of PCR reaction can reach 1fg/ μ l in this test kit, be about as much as 100 CIAV genome copies, this also is higher in the PCR method of all visible reports, thereby not only can be used for detecting CIAV infection MDCC-MSB1 cell culture, and can be used for detecting the infected chicken tissue sample.In this PCR method specificity test, we select for use common diseases of chickens former, and existing dna virus has RNA viruses again, also has bacterium, and reaction result is all negative, proves that this method specificity is stronger, can be used for the detection of open-air polyinfection tissue sample fully.

The comparison in detecting the test of artificial challenge's Chicken Anemia Virus (CAV) chick of embodiment 5 test kit PCR method of the present invention and spot hybridization

1. material and method

1.1 viral CIAV virus Cux-1 type strain is for preserving this chamber.

1.2 test chicken 1 age in days SPF chick is provided by China Veterinary Drugs Supervisory Inst.'s Experimental Animal Center.

1.3 challenge test

1.3.1 preparing that liver that malicious chicken is adopted is attacked in known CIAV virus Cux-1 strain, virus shreds, add the liver emulsion that 0.01MPBS (pH7.4) makes 20% (W/V) after fully grinding with tissue grinder, freeze thawing three times, add equal amounts of chloroform shake well mixing, after 3000rpm, 20 minutes are centrifugal, get supernatant liquor in 70 ℃ of water-baths 5 minutes, 3000rpm, 20 minutes centrifugal back supernatant liquors are through 0.22 μ m membrane filtration, and be frozen standby after the packing.

1.3.2 challenge test is got 75 of 1 age in days SPF chick, is divided into 2 groups, 50 of experimental group, and every femoribus internus intramuscular injection CIAV virus liquid 0.2ml raises in a shield retaining; 25 of control groups, every intramuscular injection PBS 0.2ml raises in another shield retaining.Attack poison back and cutd open the chicken once sampling every 4 days, after 1 month every 7 days once sampling.The experimental group chicken is cutd open at every turn and kills 4, gets blood, thymus gland, spleen, liver, kidney, the fabricius bursa, marrow and cloaca cotton swab respectively; Control group cuts open at every turn and kills 2, takes tissue sample equally.

1.4 sample preparation chick heart blood sampling 5ml adds antithrombotics, 1000rpm, and 1 minute is centrifugal, draws leukocytic cream and is used for extracting DNA; Respectively get fritter and organize internal organs to grind the back respectively to add PBS and make 20% emulsion, after 3 freeze thawing repeatedly 10000rpm*5 minute centrifugal, get supernatant liquor and be used for extracting DNA; Scrape with cotton swab and to get a small amount of secretory product of cloaca, be dissolved among the PBS, be used for extracting DNA.

1.5PCR detect by above-mentioned PCR test kit method of operation in operation.

1.6 dot hybridization

Get 0.25ml respectively 1.6.1DNA extract above-mentioned sample, add isopyknic extraction buffer (20mM Tris-Cl (pH7.4), 200mM EDTA, 40mM RNase, 1%SDS), 37 ℃ of effect 1hr, add 200 μ g/ml Proteinase Ks then, 56 ℃ of digestion 2hr, use phenol, phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1), each extracting of chloroform once are dissolved in behind ethanol sedimentation among the 10mM TE (pH8.0).

1.6.2 nucleic acid probe preparation with PCR positive template amplified production through phenol: chloroform: the primary isoamyl alcohol extracting, be dissolved in behind the ethanol sedimentation in the pure water, be directly used in digoxigenin labeled.Carry out with reference to Boehringer Manheim company labelling kit schedule of operation.

1.6.2.1 probe specificity is respectively with the PCR positive products, Cux-1 strain tissue is malicious, and other cause of disease NDV, MDV, IBDV, IBV, EDSV and Escherichia coli nucleic acid are put on cellulose acetate (NC) film and hybridized the specificity of detection probes with probe.

1.6.2.2 probe susceptibility organizes malicious extracting nucleic acid to carry out 10 times of serial dilutions with the TE damping fluid PCR positive products and Cux-1 strain, different extent of dilution nucleic acid samples are put on the NC film respectively and are hybridized the susceptibility of detection probes with probe.

1.6.3 experimental infection chicken different tissues sample detection is got CIAV experimental infection chicken liver, spleen, thymus gland, the fabricius bursa, marrow, kidney, white corpuscle and cloaca cotton and the extractive DNA of different tissues such as is wiped away, put respectively on the NC film and carry out dot blotting with label probe, test experience infects the DYNAMIC DISTRIBUTION of chick CIAV.

2. result

2.1 attacking the poison back, histopathology variation experiment began to occur tangible histopathology variation on the 12nd day, atrophy appears in thymus gland, marrow begins flavescence, and other internal organs change not obvious, pathological change and remarkable in the time of the 16th day, the whole atrophys of thymus gland, marrow is yellowing also, and atrophy a little also appears in the spleen and the fabricius bursa simultaneously, and the 24th day pathology in poison back is not obvious gradually to attacking, visual inspection recovers normally fully after 28 days, and this conforms to the result (1) of bibliographical information.

2.2PCR attacking the poison back, detected result can from different internal organs, detect CIAV-DNA on the 4th day, to the 16th day recall rate peak (90%), recall rate drops to 50% in the time of 24 days, recall rate increases to closely 80% again in the time of 32 days then, and recall rate reduces gradually subsequently, and recall rate is increased to nearly 80%. recall rates from different internal organs once more 60 days the time, thymus gland and spleen are the highest, next is kidney, marrow, and liver, white corpuscle, the fabricius bursa take second place, and it is minimum that the cloaca cotton is wiped away recall rate.The results are shown in Table 1.

2.3 dot hybridization detected result

2.3.1 specific probe only is obvious positive spots with PCR positive products and the hybridization of Cux-1 strain tissue poison, and spot all do not occur with other cause of disease nucleic acid hybridization, proves that probe has specificity.

2.3.2 the susceptibility probe can reach 1pg/ μ l to the susceptibility of PCR positive products, to organize malicious nucleic acid susceptibility be 100pg/ μ l in strain to Cux-1.

2.3.3 can from thymus gland, spleen and marrow, detect CIAV-DNA on the 8th day behind the experimental infection chicken different tissues sample detection self-infection, recall rate the highest (45%) during to 20 days, rise to 22% when dropping to 10%, 28 day then, descend gradually again then, reduce to 0 in the time of 39 days, rising gradually again subsequently, is 31%. from different internal organs during to 53 days, and thymus gland and spleen recall rate are the highest, secondly be marrow and liver, it is lower that kidney, the fabricius bursa, white corpuscle and cloaca cotton are wiped away recall rate.The results are shown in Table 2.The detected result basically identical of this and PCR, just recall rate is starkly lower than PCR method.

Two kinds of detection methods show that all immune organ still is the main target organ of CIAV, and thymus gland and spleen recall rate in various tissues is the highest, thereby should be the first-selected sample in the clinical detection.The cloaca cotton is wiped away in clinical application relatively simple, can generally investigate raising chicken, yet because of its only when infecting the peak recall rate higher, thereby still can not replace the detection of tissue sample.

From PCR and two kinds of detection methods of dot hybridization relatively, the obvious susceptibility height of PCR method, easy and simple to handle saving time, but within a short period of time qualification result: dot hybridization method high specificity, but operation is more loaded down with trivial details.

The PCR detected result of CAV-DNA in the different number of days tissue behind table 1 artificial challenge

Tissue	4	8	12	16	20	24	28	32	39	46	53	60	Add up to	Positive rate
															Liver spleen thymus gland kidney bursa of farbricius myeloplast cloaca adds up to positive rate	2/4 2/4 2/4 1/4 1/4 3/4 2/4 1/4 14/32 44％	3/4 3/4 4/4 2/4 3/4 4/4 4/4 2/4 25/32 78％	4/4 4/4 4/4 4/4 4/4 4/4 4/4 3/4 31/32 97％	5/5 5/5 5/5 5/5 5/5 5/5 5/5 3/5 38/40 95％	3/5 5/5 5/5 4/5 4/5 5/5 5/5 1/5 32/40 80％	2/4 2/4 2/4 2/4 2/4 1/4 3/4 1/4 15/32 47％	2/4 3/4 3/4 3/4 2/4 2/4 2/4 0/4 17/32 53％	2/4 4/4 4/4 4/4 2/4 4/4 4/4 1/4 25/32 78％	3/4 3/4 3/4 2/4 2/4 2/4 2/4 2/4 19/32 59％	4/4 4/4 4/4 4/4 3/4 1/4 0/4 2/4 22/32 69％	2/4 4/4 4/4 3/4 1/4 2/4 2/4 1/4 19/32 59％	3/4 4/4 4/4 2/4 2/4 4/4 3/4 2/4 24/32 75％	35/50 43/50 44/50 36/50 31/50 37/50 36/50 19/50	70％ 86％ 88％ 72％ 62％ 74％ 72％ 38％

The dot hybridization detected result of CAV-DNA in the different number of days tissue behind table 2 artificial challenge

Tissue	4	8	12	16	20	24	28	32	39	46	53	60	Add up to	Positive rate
															Liver spleen thymus gland kidney bursa of farbricius myeloplast cloaca adds up to positive rate	0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/32 0	0/4 3/4 3/4 0/4 0/4 2/4 1/4 0/4 9/32 28％	1/4 2/4 2/4 0/4 0/4 2/4 0/4 0/4 7/32 22％	0/5 4/5 5/5 0/5 0/5 4/5 1/5 0/5 14/40 35％	4/5 5/5 4/5 0/5 0/5 4/5 1/5 0/5 18/40 45％	1/4 1/4 1/4 0/4 0/4 0/4 0/4 0/4 3/32 9％	1/4 3/4 3/4 0/4 0/4 0/4 0/4 0/4 7/32 22％	0/4 1/4 2/4 1/4 0/4 0/4 0/4 0/4 4/32 13％	0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/32 0	1/4 1/4 1/4 1/4 0/4 1/4 0/4 0/4 5/32 16％	1/4 1/4 1/4 3/4 2/4 1/4 0/4 1/4 10/32 31％	2/4 2/4 3/4 1/4 0/4 0/4 0/4 0/4 8/32 25％	11/50 23/50 25/50 6/50 2/50 14/50 3/50 1/50	22％ 46％ 50％ 12％ 4％ 28％ 6％ 2％

Claims (2)

1, a kind of chicken infectious anemia detection kit includes tissue DNA extracting solution, PCR reaction solution, Taq archaeal dna polymerase and CIAV PCR positive control template, it is characterized in that

Described tissue DNA extracting solution contains 20mmol/L Tutofusin tris, 10mmol/L ethylenediamine tetraacetic acid (EDTA), 10% sodium laurylsulfonate, 400 μ/ml Proteinase K and 2 μ g/mlRNA enzymes,

Described PCR reaction solution contains 10mmol/L Tutofusin tris, 50mmol/L Repone K, 1.5mol/L magnesium chloride, 0.1% Octylphenoxy poly-ethoxy ethanol, 0.4mmol/L deoxynucleoside triphosphate and 0.4nmol/LCIAV Auele Specific Primer,

Described CIAV Auele Specific Primer is the primer that lays respectively at genome 358bp and 1042bp place upstream and downstream, and its sequence is respectively:

5′-ATACTCGAGGCGGTCCGGGTGGATGC-3′，

5′-GGTGCGGCCGCCTCACACTATACGT-3′。

2, detection kit according to claim 1, described CIAV PCR positive control template are the genomic dnas through extractive CIAV standard strain CUX-1 strain.