CN102943127A - Primers and detection kit for avian leukosis J subgroup virus PCR detection - Google Patents
Primers and detection kit for avian leukosis J subgroup virus PCR detection Download PDFInfo
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Abstract
The present invention provides primers and a detection kit for avian leukosis J subgroup virus PCR detection. According to the present invention, PCR detection primers are designed by analyzing avian leukosis J subgroup virus env gene, wherein nucleotide sequences of the primers are represented by sequence lists SEQ ID No.1 and SEQ ID No.2; and the primers have good specificity, and the detection method has characteristics of rapidness, simpleness, high accuracy and good sensitivity so as to provide an excellent detection method for avian leukosis J subgroup virus identification. The present invention further provides a detection kit for the avian leukosis virus, wherein the detection kit comprises a primer pair represented by the sequence lists SEQ ID No.1-2.
Description
Technical field
The present invention relates to biology field, the PCR that particularly relates to for detection of avian leukosis J subgroup virus detects with primer and detection kit.
Background technology
Along with deepening continuously of aviculture large-scale cultivation, the threat of poultry diease also highlights day by day.Avian leukosis (Avian Leukosis, AL) has high infection rate because of it, the characteristic that all can propagate of (aleopation between the chicken group) and vertical direction (this disease can entail the next generation) in the horizontal direction, this disease can infect all chicken kinds, chicken infectedly be gradual morbidity, the low actual that continues is one of principal disease of harm poultry husbandry, and the annual financial loss that causes because of poultry disease in the whole world is up to tens billion of units.
But human almost also at one's wit's end to the control of avian leukosis so far, both there be not vaccine to utilize, there is not effective medicine to treat yet, the main method of control avian leukosis is to detect by cause of disease, eliminates positive chicken, purifies population.At present, detecting in the world the ideal detection method of avian leukosis virus antigen is the ELISA method, and it has the advantages that susceptibility is high, simple and efficient, be applicable to extensive detection.Since 1979, the ELISA diagnostic kit of avian leukosis has been realized commercialization abroad, especially in recent years, China cultivates successively and has set up several SPF chicken houses, also can constantly increase from now on, the inspection of SPF chicken group AL/SV-gs antigen is mainly relied on the import reagent box at present, not only expensive but also inconvenient.Therefore, the advanced domestic reagent box of development is the problem that our current urgent need will solve.
Avian leukosis is the general designation of the chicken kinds of tumors disease that caused by the virus among the avian leukosis sarcoma virus group.Drop to feature with generation lymph sample tumour and egg productivity in Adult Chicken.Most strains of avian leukosis virus can well be grown in the instar chicken embryo on 11st～12, and abdominal cavity or other approach inoculation 1～14 age in days susceptible chick can cause the chicken morbidity.Facing and examined many forms, mainly is leukemic lymphoblastoid.Avian leukosis virus can be divided into A, B, C, D, E and six subgroups of J at present.Wherein the J subgroup is newfound in broiler chicken after the nineties, is the at present common pathogenic virus subgroup that has.After avian leukosis virus infects chicken, can reduce growth and the production performance of chicken, or cause that serious pathologic reaction appears in chicken, even dead, this virus is except can be by myelocytome broiler chicken causes than high mortality, also can cause chicken body weight quality degradation, can cause in the serious situation that the chicken group breaks out other complication, thereby cause chicken group's Large Scale Death.Five kinds of subgroups (A, B, C, D, E) in addition can detect virus but chicken does not fall ill usually.The J subgroup avian leucosis is to be caused the bone marrow cell carcinoma disease or become myelogenous leukemia by a kind of infectivity that Retroviridae J subgroup avian leucosis virus causes.Since this disease self-discovery, be widely current in countries in the world meat kind chicken, commodity meat type chicken and egg kind chicken with vertical and two kinds of forms of horizontal transmission, cause meat kind chicken death and become thin, serious retarded growth, the immunosuppression of laying hen generation body, reproductive performance continue to reduce, and have brought great harm for the countries in the world poultry husbandry, therefore the chicken group of intensive culture is carried out the detection of J subgroup virus infection, significant to the development of poultry husbandry.
The avian leukosis of high infection rate is seriously restricting the development of aviculture, and is threatening the popularization of large-scale farming.Therefore, a kind of energy fast, low-cost, detect the method whether extensive chicken infects avian leukosis virus accurately, significant to the development of aquaculture.
At present often adopt the ELISA test kit to detect for the detection of avian leukosis, its accuracy is higher, but two large shortcomings are arranged: 1) price is very expensive, is not suitable for the full group of plant and detects; 2) ELISA reagent can only detect ectogenic avian leukosis virus, and can not distinguish each subgroup of virus.
Summary of the invention
The object of the present invention is to provide a kind of PCR detection primer of avian leukosis.
Another object of the present invention is to provide the test kit that detects avian leukosis.
The invention provides a kind of avian leukosis virus PCR detection and use primer, it can increase to the env gene of avian leukosis virus, generates avian leukosis virus specific amplification fragment.
Preferably, described primer nucleotide sequence is shown in SEQ ID No.1 and SEQ ID No.2.
The invention provides the diagnostic reagent that contains above-mentioned primer.
The invention provides the detection kit that contains above-mentioned primer.
The invention provides the application of above-mentioned primer in preparation avian leukosis J subgroup virus diagnose reagent case detection kit.
The test kit of detection avian leukosis J subgroup virus provided by the invention, its detecting step is:
Detecting step is:
1) extracts respectively 10 ~ 50 fowl blood, be mixed to get hybrid dna after the individual DNA extracting;
2) hybrid dna of step 1) is used primer and carry out pcr amplification, judge according to amplification purpose band; If the fragment of 525bp appears in the sample amplification band, then sample is avian leukosis J subgroup virus infection positive; If amplified band does not have the 525bp fragment, then negative; Described primer sequence is shown in SEQ ID No.1 and SEQ ID No.2.
The PCR primer of detection avian leukosis J subgroup virus provided by the invention can detect quickly and accurately fowl and whether suffer from leukemia J subgroup virus, the detection kit cost that contains this primer is low, easy to use, can solve the problem of present avian leukosis high detection cost.The present invention is a kind of technology that detects avian leukosis based on molecular level; The detection kit of using primer structure of the present invention is easy and simple to handle fast, highly sensitive, high specificity, and good reproducibility greatly reduces time and cost that full group detects avian leukosis, and full group's rapid detection avian leukosis is achieved, even universal.For example, the ELISA method detects 100,000 chicken groups, and the testing cost of average every chicken is about 30 yuan, and the cost of test kit of the present invention is less than 1 yuan.In addition, on the working hour, the ELISA method, on average an instrument can detect about 400 for each person every day, and this test kit then can detect nearly thousand.Therefore, adopt test kit of the present invention can shorten in time half, save 29 times on the cost.
Description of drawings
Fig. 1 is detection system PCR product electrophorogram, and wherein, Marker is DL2000, and 1,2,3,4,5,6, No. 7 swimming lane is respectively PCR result corresponding to chicken individuals who infects avian leukosis J subgroup virus.
Fig. 2 is the electrophorogram of PCR method minimal detectable concentration of the present invention, uses the dna profiling of 8 gradients to carry out PCR detection, wherein 1:20 μ g/ml; 2:10 μ g/ml; 3:5 μ g/ml; 4:2.5 μ g/ml; 5:1.25 μ g/ml; 6:0.625 μ g/ml; 7:0.313 μ g/ml; 8:0.157 μ g/ml, the rightest swimming lane are Marker DL2000.。
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The design of embodiment 1 PCR primer
The two ends of avian leukosis virus rna gene group are non-coding region, and middle portion is the coding region, and there are 4 structure genes the coding region, and from 5 ' to 3 ' is followed successively by gag/pro-pol-env.The present invention is directed to the special env gene order of J subgroup avian leucosis virus, the design primer.Through multi-turns screen, find that following primer can carry out qualitative detection to the J subgroup avian leucosis virus quickly and accurately.By pcr amplification, the individuality that can obtain this env gene order sheet degree namely represents this individuality and contains the J subgroup avian leucosis virus.
The nucleotide sequence of the primer of detection J subgroup avian leucosis virus provided by the invention:
Upstream primer: 5 ' TTAAACCGGACATCACCCAAAAGGA 3 ' (SEQID No.1)
Downstream primer: 5 ' AGCACACCGAACCAAAGGTAACACA 3 ' (SEQID No.2).
The extraction of embodiment 2 total DNA
(1) extraction of chicken blood DNA.Get the blood sample 10 μ L of citric acid anti-freezing in the 1.5mLEppendorf pipe, add 400 μ L damping fluids and shake up lightly; Then the Guanidinium hydrochloride liquid 100 μ L and the 20 μ l Proteinase Ks that add 47.765g/100mL, vortex oscillation mixing are placed on 56 ℃ of metal bath digestion 5 hours; Add 600 μ L chloroforms, vibrate centrifugal 4 ℃, the centrifugal 15min of 12000rpm gets in another Eppendorf pipe of supernatant liquor transposition; Get supernatant liquor, add the equal-volume Virahol, be placed in the ice chest precipitation after 2 hours centrifugal 4 ℃, 15min, 12000rpm topples over supernatant; Add dehydrated alcohol 777 μ L, centrifugal 4 ℃, 12000rpm, 15min abandons supernatant liquor; Add 48 μ L(an amount of) ultrapure water dissolution precipitation (and obtaining dna solution); Sepharose with 0.8% detects total DNA, and with DNA quantitative instrument quantitatively its concentration and purity.Require DNA purity (OD value) more than 1.300; Packing is preserved, and places-20 ℃ of refrigerators.
(2) with the DNA of chicken, get 3 μ L for every, per 10 ~ 50 are mixed in the EP pipe, whirlpool concussion, mixing, 9000rpm, centrifugal 30s.(every 10-50 only is mixed in a pipe, can both guarantee to have good effect, and particular case can be decided according to this chicken house infection rate, and the infection rate that avian leukosis is general is 5%-30%).
The foundation of embodiment 3 pcr amplification methods
1, PCR reaction system
Take mixed total DNA of embodiment 2 as template, carry out the PCR reaction take the primer of embodiment 1 as primer, 20 μ L reaction systems comprise that PCR-Mix(is purchased from a day root company) 10 μ L, ddH
2O 7 μ L, 10pmol/ μ L upstream and downstream primer each 1 μ L and the total DNA 1 μ L of chicken.
2, PCR reaction conditions
By thermograde PCR instrument, the primer temperature has been carried out condition optimizing, between 52 ℃-62 ℃, carried out the screening of 8 thermogrades, the PCR product detects with 1.5 ﹪ agarose gel electrophoresis, finally selected the purpose band the most single, bright 55 ℃, the PCR annealing temperature condition of the best when using as this test kit.
After the PCR pipe that sample is housed put into the PCR instrument, following condition is set reacts: 95 ℃ of denaturations, 4min; Again through 95 ℃ of sex change 45s, 55 ℃ of annealing 35s, 72 ℃ are extended 1min, 28 circulations; Last 72 ℃ are extended 10min.
3, get the PCR product and carry out the agarose electrophoresis detection.If amplified production purpose clip size is 525bp, then sample corresponding to explanation infected J subgroup ALV.Get pcr amplification product 5 μ L, detect with 1.5 ﹪ agarose gel electrophoresis.The as a result figure that agarose detects PCR sees Fig. 1.After amplified production reclaimed purifying, send biotech firm's order-checking, the purpose band of sequencing result explanation amplification is consistent with the env gene.
The specificity of embodiment 4 PCR detection system, sensitivity evaluation
Take the virus of 10 kinds of common poultry diseases or DNA(Avian pneumo-encephalitis virus (NDV), Mareks disease virus (I type MDV), avian viral arthritis virus, infections chicken cloacal bursa virus, egg drop syndrome virus, avian infectious bronchitis virus, bird pox virus, Salmonellas, intestinal bacteria, the staphylococcus of bacterium) and avian leukosis J subgroup virus [HPRS-103 strain (Z46390)] DNA as template, take the primer of embodiment 1 as the pcr amplification primer, carry out pcr amplification, with the specificity of checking primer of the present invention.Only found that at avian leukosis J subgroup viral DNA to be that the band about 500bp appears in the swimming lane of template, and band does not all appear in other viral template, shows as feminine gender.Presentation of results, primer of the present invention have avian leukosis J subgroup virus-specific.
The susceptibility of having carried out different DNA concentration gradients detects, be the avian leukosis J subgroup viral DNA positive template doubling dilution 8 times of 40 μ g/ml with concentration, concentration is respectively 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.313 μ g/ml, 0.157 μ g/ml.Detect according to the described step of test kit of the present invention, display density is that the above sample standard deviation of 0.625 μ g/ml can detect the positive as a result, the results are shown in Figure 2.Therefore, the minimal detectable concentration of primer of the present invention is 0.625 μ g/ml, shows that the detection avian leukosis J subgroup virus PCR reaction of using primer foundation of the present invention has higher sensitivity.
1, test kit comprises: the primer pair of embodiment 1, and DNA extraction agent, PCR reaction enzymes and Buffer, positive control are avian leukosis J subgroup viral DNA, negative control is the stoning sour water.
2,100 chickens to be measured are divided into 10 groups, and 10 every group, the DNA of every group of 10 chickens is made a biased sample, working method is seen the operation steps of embodiment 2.
3, the method according to embodiment 3 detects (in case find to amplify purpose band 525bp 5 groups of hybrid dnas, namely show the individuality that infects the J subgroup avian leucosis virus is arranged in this DNA pond), there is the purpose band to be amplified out if find, should use respectively the primer of embodiment 1 to carry out pcr amplification the DNA of this this 10 chickens, find the individuality that infects J type avian leukosis virus, and weed out immediately positive chicken.If have no the band of purpose clip size (525bp), then show 10 individualities that this DNA pond is corresponding, all do not infect the J subgroup avian leucosis virus.The method can be used for large flux screening intensive culture field bird and whether infects the J subgroup avian leucosis virus.
Extract to infect immediately among the chicken group of avian leukosis virus J subgroup totally 133 parts of blood samples, difference DNA extraction and serum, using test kit of the present invention and commercialization ELISA detection kit [available from gene U.S. company, test kit full name: avian leukosis virus J subgroup (ALV-J) enzyme immunoassay (ELISA)] detects respectively.The result shows that the specificity of the method is 95.6%(43/45), susceptibility is 94.3%(83/88), both coincidence rates are 94.7%(126/133), see the following form 1.Test kit of the present invention has advantages of detection time short (shortening half), the ELISA method on average for each person every day an instrument can detect about 400, this test kit then can detect nearly thousand.Cost low (saving 29 times), reliable results, the advantages such as convenient operation, making large-scale farming already carry out the rapid detection avian leukosis becomes possibility.
Test kit of the present invention and commercialization ELISA test kit are to the clinical sample detected result relatively
Claims (6)
1. an avian leukosis J subgroup virus PCR detects and uses primer, it is characterized in that, can the env gene of avian leukosis J subgroup virus be increased, and generates avian leukosis virus specific amplification fragment.
2. primer as claimed in claim 1 is characterized in that, the nucleotide sequence of described primer is shown in SEQ ID No.1 and SEQ ID No.2.
3. the diagnostic reagent that contains claim 1 or 2 described primers.
4. the detection kit that contains claim 1 or 2 described primers.
5. claim 1 or 2 described primers detect the diagnostic reagent of avian leukosis J subgroup virus or the application in the test kit in preparation.
6. detection kit as claimed in claim 4 is characterized in that, detecting step is:
1) extracts respectively 10 ~ 50 fowl blood, be mixed to get hybrid dna behind the individual DNA of extracting;
2) the hybrid dna application rights with step 1) requires 2 described primers to carry out pcr amplification, judges according to amplification purpose band; If the fragment of 525bp appears in the sample amplification band, then sample is avian leukosis J subgroup virus infection positive; If amplified band does not have the 525bp fragment, then negative.
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| CN105779649A (en) * | 2016-03-29 | 2016-07-20 | 扬州大学 | Immune PCR reagent kit for detecting avian leukemia virus |
| CN108085416A (en) * | 2017-12-21 | 2018-05-29 | 湖北省农业科学院畜牧兽医研究所 | A kind of fowl neoplastic disease triple fluorescent PCR detection kit and its detection method |
| CN110408727A (en) * | 2019-08-16 | 2019-11-05 | 华南农业大学 | A kind of CPA primer sets, CPA nucleic acid test strip kit and its application detecting J subgroup avian leucosis virus |
| CN113930546A (en) * | 2021-10-25 | 2022-01-14 | 华南农业大学 | RT-RAA fluorescence detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779649A (en) * | 2016-03-29 | 2016-07-20 | 扬州大学 | Immune PCR reagent kit for detecting avian leukemia virus |
| CN108085416A (en) * | 2017-12-21 | 2018-05-29 | 湖北省农业科学院畜牧兽医研究所 | A kind of fowl neoplastic disease triple fluorescent PCR detection kit and its detection method |
| CN110408727A (en) * | 2019-08-16 | 2019-11-05 | 华南农业大学 | A kind of CPA primer sets, CPA nucleic acid test strip kit and its application detecting J subgroup avian leucosis virus |
| CN113930546A (en) * | 2021-10-25 | 2022-01-14 | 华南农业大学 | RT-RAA fluorescence detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene |
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Application publication date: 20130227 |