CN103045762B - Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit - Google Patents

Abstract

The invention provides a kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV). The kit comprises (a) fluorescent PCR (polymerase chain reaction) liquid reactant, (b) a fluorescent probe, (c) hot start Tag enzyme, (d) a standard positive template and (e) negative control, wherein the fluorescent PCR liquid reactant contains forward primers and reverse primers; the sequence of the forward primers is shown in SEQ ID NO:2; the sequence of the reverse primers is shown in SEQ ID NO:3; the sequence of the fluorescent probe is shown in SEQ ID NO:4; fluorescent substance is designed at the 5' end of the sequence of the fluorescent probe; cancellation substance is designed at the 3' end of the sequence of the fluorescent probe; and the nucleotide insertion sequence of the standard masculine template is shown in SEQ ID NO:5. The invention further provides application of the kit to detecting proviral DNA of BLV in whole blood. The PCR detection kit provided by the invention can be used for quickly and accurately detecting BLV and is applicable to quick diagnosis of BLV, epidemiological investigation and risk assessment.

Description

A kind of test kit and application thereof that detects the front DNA of bovine leukemia virus

Technical field

The invention belongs to field of biological detection, be specifically related to a kind of test kit and application thereof that detects the front DNA of bovine leukemia virus.

Background technology

Enzootic bovine leukemia is the chronic neoplastic disease of one of the ox that caused by bovine leukemia virus (Bovine leukemiavirus, BLV).It is characterized by lymphoidocyte neoplasm, the high mortality after carrying out property emaciation and morbidity.At present almost each cowboying all over the world country of this disease, was found in Hefei ,Anhui in China first in 1976, then in succession occurred in each province and city.In recent years, this disease seroprevalence of China cows is soaring year by year, is increased to 30%～50% from 4% left and right.The pollution-free food raising dairy cattle animal doctor all clear and definite regulations of criterion (NY 5047-2001) and pollution-free food beef cattle feeding epidemic prevention criterion (NY 5126-2002) of preventing epidemic, once bovine leucosis occurs in cattle farm, must implement superseded control techniques in time.According to another bibliographical information, although this sick public hygienics meaning is not also finalized, from statistical significance, infecting BLV virus can increase people and suffer from the possibility of mammary cancer, needs further investigation and the investigation carried out this disease badly.

Although this disease is worldwide propagated, also do not have effectively to control or to treat this sick vaccine or method at present, thereby as early as possible diagnosis, in time eliminate sick ox just seem particularly important.Existing diagnostic method in group BLV infect generaI investigation normally based on serological test as agar diffusion test, enzyme linked immunosorbent assay etc., although serological test can be estimated the popularity of BLV in cows, but because the antibody of the passive antibody of BLV virus and initiatively infection generation cannot be distinguished, it is all the difficult problem during serology detects that the antibody test in the maternal antibody of calf and close to calving and postpartum there will be the factors such as false negative, and serological method need to just can detect after 3 ~ 16 weeks that in infection antibody and antibody recall rate are very low, can not get rid of so only rely on antibody test the possibility that BLV infects, also find the morning that is unfavorable for epidemic disease, early diagnosis, take as early as possible corresponding measure.Therefore for the screening of a large amount of sample BLV, detect, make a definite diagnosis, be necessary very much to develop high specificity, highly sensitive, operate relatively easy PCR detection reagent, determine the infection state of animal.

Various countries control the infection of this disease to have taked all comprehensive preventive health measuress, comprise superseded, isolation and correct management, but these measures all be unable to do without effective detection means.At present, the detection method of this disease agar diffusion test that China's current standards only has work out for 2002, the standard of not yet working out this disease fluorescence PCR method, also needleless emerges to the Taqman fluorescent PCR kit of this disease virus.Traditional B LV detects the general method that adopts sleeve type PCR amplification, object sequencing fragment and sequence alignment, this method is generally adopted in round pcr field always, but the shortcoming of himself is also obvious: waste time and energy, program is loaded down with trivial details, step complexity, cycle is long, and cost of labor is high, is unfavorable for the detection of great amount of samples; Be applied to clinical gene diagnosis and also have many limitation, being mainly manifested in can not accurate quantitative analysis and easily produce false positive etc. because of crossed contamination; And personnel require high, the result of order-checking need to have certain expertise personnel and carry out sequence alignment ability final decision result, and the judgement of fluorescent PCR result is simple, directly perceived.

Summary of the invention

The object of the present invention is to provide a kind of test kit that detects DNA before bovine leukemia virus in whole blood, test kit high specificity, the susceptibility of described this detection bovine leukemia virus are high, cost is low.

The invention provides a kind of test kit that detects the front DNA of bovine leukemia virus, described test kit contains: a) Fluorescence PCR liquid, b) fluorescent probe, c) warm start Taq enzyme, d) standard positive template, e) negative control, in described Fluorescence PCR liquid, contain upstream and downstream primer, wherein, upstream primer sequence, as shown in SEQ ID NO:2, is C1:5 '-AACCCCACCTTCCCATGAC-3 '; Downstream primer sequence, as shown in SEQ ID NO:3, is C2:5 '-TGGTAGGAGGTTAATCTGATTGTGAGT-3 '; Its effect be can specific amplification BLV before the pol gene fragment of DNA, 5 ' end of described fluorescent probe is designed with fluorescent substance, 3 ' end is designed with cancellation material, sequence is as shown in SEQ ID NO:4, for fluorescent substance-CAGGCCCTTTCTCGAGCCCTCTG-cancellation material, the Nucleotide insertion sequence of described standard positive template is as shown in SEQ ID NO:5.

Described in when detection, the final concentration of upstream and downstream primer wall scroll is 0.05-1 μ M, is preferably 0.24 μ M.

In described Fluorescence PCR liquid, also contain Mg ²⁺, when detection described in Mg ²⁺final concentration be 1.5-5.0mM, be preferably 2mM.

In described Fluorescence PCR liquid, also contain four kinds of dNTPs, comprise dATP, dCTP, dGTP, dTTP, when detection described in the final concentration of various dNTPs be respectively 0.1-1mM, be preferably 0.25mM.

Described in when detection, the final concentration of fluorescent probe is 0.05-1 μ M, is preferably 0.2 μ M.

Described in when detection, the final concentration of warm start Taq enzyme is 0.01-5U/uL, is preferably 0.5U/ μ L.

The fluorescent substance of the sequence 5 ' end of described fluorescent probe is the one in FAM, HEX or VIC, and the cancellation material of 3 ' end is TAMRA.

Preferably, the sequence of described fluorescent probe is:

FAM-CAGGCCCTTTCTCGAGCCCTCTG-TAMRA。

The present invention also provides a kind of preferred test kit composition, and this test kit contains: by the each dNTPs for 0.3mM, and 2.4mM Mg ²⁺, be respectively the described Fluorescence PCR liquid of the upstream and downstream primer composition of 0.288 μ M, 2.5mL/ pipe × 2; 6 μ M fluorescent probes, 200 μ L/ pipes; 7.5U/ μ L warm start Taq enzyme, 200 μ L/ pipes; Standard positive template, 250 μ L/ pipes; Negative control, 250 μ L/ pipes.

Described standard positive template be by DNA before the primer pair bovine leukemia virus of described upstream and downstream carry out pcr amplification acquisition nucleotide fragments insert the recombinant plasmid that pBS-T vector construction forms.

The invention still further relates to a kind of test kit application in DNA before bovine leukemia virus in detection whole blood that detects the front DNA of bovine leukemia virus.Wherein, upstream and downstream primer is combined with BLV pol gene specific, and probe discharges FAM fluorescent signal in pcr amplification; In the time that fluorescent PCR instrument gathers fluorescence by corresponding passage, just can specific observations arrive amplification curve, thereby reach the object that detects BLV virus.

The concrete grammar that this test kit uses comprises the following steps:

(1) the front DNA extraction of sample to be checked;

(2) fluorescent PCR detects: according to amplification number of objects, get Fluorescence PCR liquid, probe, warm start Taq enzyme and be mixed in a centrifuge tube, and concussion, packing, lid upper tube cap is for subsequent use; The DNA of negative control, positive control, each sample is added in corresponding point tubulature, centrifugal after each pipe mark, take out and put in fluorescent PCR instrument;

(3) result is judged: reaction solution is measured Ct value, and to be greater than 40 sample negative; The positive sample of sample of Ct value≤35; The sample suggestion repetition measurement of measuring 35 < Ct value≤40, repetition measurement result Ct value≤35 are positive, and repetition measurement result Ct value >'s 35 is negative.

Wherein, Ct value (Threshould cycle) refers to that generation can be detected the required minimal circulation number of fluorescent signal, is the corresponding cycle index of flex point that fluorescent signal is started to enter exponential growth phase by background in PCR working cycle.There is linear relationship in the logarithm of the initial copy number of this Ct value and template, initial copy number is more, and Ct value is less.

This test kit utilizes bovine leukemia virus to have can be incorporated into the characteristic that forms front DNA in host cell, save the step of viral RNA reverse transcription, there is convenient, quick, reliable advantage, be conducive to investigate for this disease epidemiology infection conditions background.

The present invention compares with prior art, reagent preparation is simple, method is simple and efficient, high specificity, susceptibility are high, result judgement easily.Can be for multiple fields such as the diagnosis of bovine leukemia virus, epidemiology and molecular biology researches, adopt single stage method can reach the rapid detection object of bovine leukemia virus, there is important using value for this sick differential diagnosis, epidemiology survey and risk assessment thereof.

Brief description of the drawings

Fig. 1 is that the positive pathological material of disease of the check samples such as bovine viral diarrhea virus, infectious bovine rhinotrachetis virus, Brucella abortus, sheep placenta nephrocyte (FLK) and BLV carries out real-time fluorescence PCR detected result figure, can confirm the specificity of test kit.

Fig. 2 is fluorescent PCR canonical plotting, by positive plasmid according to 10 times of gradient dilutions, from 10-1 ~ 10-6 doubly do identical dilution group totally 3 groups carry out fluorescent PCR, can confirm the susceptibility of test kit.

Fig. 3 is fluorescent PCR figure, and wherein, the test kit that is respectively 1d ~ 6d 37 DEG C of storage times detects BLV positive sample, can confirm the stability of test kit.

Embodiment

The following example is intended to further describe for example invention, instead of restriction invention by any way, any change that those of ordinary skill in the art made for the present invention easily realize or change do not deviating under the prerequisite of the spirit and principles in the present invention, within all will fall into the interest field that awaits the reply of the present invention.

The design of embodiment 1 primer and probe is with synthetic

The genome total length of BLV accession number on GenBank is FJ914764.1, and what the present invention is directed to is the pol gene of BLV, this full length gene 2559bp, and encoding sequence is positioned at 2320 ~ 4878bp of the full gene of BLV, and sequence is as shown in SEQ ID NO:1.

Design a pair of Auele Specific Primer according to BLV pol gene order as follows:

Upstream primer C1:5 '-AACCCCACCTTCCCATGAC-3 ';

Downstream primer C2:5 '-TGGTAGGAGGTTAATCTGATTGTGAGT-3 ';

Its effect be can specific amplification BLV before the pol gene fragment of DNA.

According to BLV pol gene order design fluorescent probe, its sequence is as follows:

FAM-CAGGCCCTTTCTCGAGCCCTCTG-TAMRA

Wherein, the fluorescent substance of probe sequence 5 ' end is except selecting FAM(hydroxyl fluorescein), also can be selected from HEX (chlordene-6 methyl fluorescein) or VIC(ABI house journal) in one, can realize equally technique effect of the present invention.

Wherein, one end of fluorescent probe connects fluorescent substance, and one end connects cancellation material, and its DNA chain can be combined with specific DNA object fragment; This carries out on the one hand the synthetic of DNA fragmentation under the effect of TaqDNA polysaccharase to Auele Specific Primer, when to tat probe binding site, it brings into play again restriction enzyme digestion effect simultaneously, and the DNA chain that connects fluorescent substance and cancellation material in probe is cut off, and discharges fluorescent substance; In the time that fluorescent substance is free in reaction system, be subject to exciting and just can send fluorescence, and collected by instrument, form amplification curve, thereby reach the object that detects BLV virus.

The preparation of embodiment 2 standard positive templates

1, get the blood sample of infected cattle leukosis virus, it is carried out to DNA extraction.These concrete steps can be referring to the AxyPrep of AXYGEN company body fluid viral DNA/RNA small volume of reagent box specification sheets.Detailed step is as follows:

A) these 200 μ L of sampling, add DNA cleavage liquid Buffer V-L, and vortex concussion mixes, and leaves standstill 5min.

B) add 75 μ L Buffer V-N, vortex vibration mixes, the centrifugal 5min of 12000 × g.

C) supernatant is transferred in new 2ml centrifuge tube, added 300 μ L Virahols (1% glacial acetic acid), turned upside down 6 ~ 8 times, mixes.

D) be placed in 2ml centrifuge tube by preparing pipe, get mixed solution immigration in step 3 and prepare in pipe, the centrifugal 1min of 6000 × g.

E) abandon filtrate, put and get back in 2ml centrifuge tube preparing pipe, add 500 μ L Buffer W1A, room temperature leaves standstill 1min.The centrifugal 1min of 12000 × g.

F) abandon filtrate, put and get back in 2ml centrifuge tube preparing pipe, add 800 μ L Buffer W2, the centrifugal 1min of 12000 × g.

G) put and get back in 2ml centrifuge tube, the centrifugal 1min of 12000 × g preparing pipe.

H) be placed in clean 1.5ml centrifuge tube by preparing pipe, add 40 ~ 60 μ LBuffer TE preparing periosteum central authorities, room temperature leaves standstill 1min, and room temperature leaves standstill 1min.The centrifugal 1min wash-out of 12000 × g nucleic acid ,-20 DEG C save backup.

2, regular-PCR amplification

PCR reaction system is as follows: upstream and downstream primer C1, C2(10 μ molL ^-1) each 1 μ L, 10 × Buffer, 2 μ L, 2.5mmolL ^-1dNTP 2 μ L, rTaq archaeal dna polymerase 5U, it is nucleic acid-templated that 2 μ L extract, and sterilizing distilled water is supplied system to 25 μ L, and PCR cycling condition is: 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 40 circulations, 72 DEG C of 10min.After finishing, PCR carries out electrophoresis on 1% agarose gel, imaging under ultraviolet lamp after EB dyeing.

3, the structure of positive plasmid

Under ultraviolet lamp, the object band of above-mentioned PCR is taken out, the step that reclaims test kit according to the DNA of TAKARA company glue reclaims, fetching receipts product is connected with pBS-T carrier, ligation system is: the PCR product 7 μ L of recovery, 10 × connection Buffer, 1 μ L, pBS-T carrier 1 μ L, T4DNA ligase enzyme 1 μ L, 4 DEG C of connections are spent the night, then transform DH5 α competence bacteria, under IPTG, X-gal induction, cultivate 12-14h for 37 DEG C, after picking hickie colony inoculation LB liquid nutrient medium overnight incubation, extract plasmid.Getting recombinant plasmid send Invitrogen company to carry out sequencing, and compare together with the nucleotide sequence of the BLV pol gene of having delivered, be defined as after this gene masculine plasmid, plasmid is carried out to A260/A280 light absorption value with ultraviolet spectrophotometer and measure concentration,-20 DEG C save backup, are standard positive template.

The optimization of 3 test kit Fluorescence PCR conditions of embodiment

1, the optimization of primer concentration:

It is 50-1000nM that wall scroll primer final concentration scope is set, and carries out fluorescent PCR test with the primer of different concns gradient, and result shows the primer of this concentration range S type curve that can increase, and in the time that every primer final concentration is 0.24 μ M best results.

2, Mg ²⁺the optimization of concentration:

Mg is set ²⁺final concentration scope is 1.5-5.0mM, and result shows the Mg in this concentration range ²⁺all can amplify S type curve, and work as Mg ²⁺when final concentration is 2mM, expanding effect is best.

3, the optimization of dNTPs concentration:

Four kinds of dNTPs equivalents mix, and it is 0.1-1mM that every kind of dNTPs final concentration scope is set, and result shows that the dNTPs in this concentration range all can amplify curve, and every kind of dNTPs final concentration is with 0.258mM, is the best when total dNTPs final concentration is 1.032mM.

4, the optimization of concentration and probe concentration:

It is 0.05-1 μ M that probe final concentration scope is set, and carries out fluorescent PCR test with the probe of different concns gradient, and result shows the primer of this concentration range S type curve that can increase, and when probe final concentration best results during with 0.2 μ M.

5, the optimization of warm start Taq enzyme concn:

It is 0.01-5U/ μ L that warm start Taq enzyme final concentration scope is set, carry out fluorescent PCR test with the warm start Taq enzyme of different concns gradient, result shows the warm start Taq enzyme of this concentration range S type curve that can increase, and in the time that warm start Taq enzyme concn is 0.5U/ μ L best results.

The test kit of DNA composition before embodiment 4 detection bovine leukemia virus according to a preferred embodiment of the present invention

Through after above-mentioned condition optimizing, before detection bovine leukemia virus according to a preferred embodiment of the present invention, the test kit of DNA is composed as follows: a) Fluorescence PCR liquid 2 is managed (2.5mL) and comprised dNTPs, Mg ²⁺and primer, dATP, dGTP, dCTP and dTTP final concentration in reaction solution are respectively 0.3mM, Mg ²⁺final concentration is 2.4mM, and primer C1, C2 final concentration are respectively 0.288 μ M; B) probe 1 is managed (200 μ L), and concentration is 6 μ M; Warm start Taq enzyme 1 is managed (200 μ L), and concentration is 7.5U/ μ L; Standard positive template 1 is managed (250 μ L/ pipe), and negative control 1 is managed (250 μ L/ pipe).Wherein, dNTPs, Mg ²⁺, warm start Taq enzyme is purchased from TAKARA company, primer and probe are synthesized by invitrogen company.

This test kit adopts 30 μ L reaction systems, and reaction solution consists of: Fluorescence PCR liquid is 25 μ L, enzyme 2 μ L, probe 1 μ L and template 2 μ L.

The using method of the test kit of DNA before embodiment 5 detection bovine leukemia virus according to a preferred embodiment of the present invention

1, sample process:

Extract voluntarily DNA, concrete steps can be referring to the AxyPrep of AXYGEN company body fluid viral DNA/RNA small volume of reagent box specification sheets, and positive control is without extraction, directly application of sample.

2, the detection of fluorescent PCR

A) according to detecting sample number n(n=sample number+2 to be checked) get PCR reaction solution, warm start Taq enzyme and probe and be mixed in a centrifuge tube, on vortex vibrator, vibrate, by every pipe packing, cover upper tube cap for subsequent use.

B) now negative controls is added in a point of tubulature, the DNA that gets each sample adds in corresponding reaction tubes; Finally take out positive control and add in another reaction tubes, centrifugal after each reaction tubes mark, take out and put in ABI7500 fluorescent PCR instrument.

C) Fluorescence PCR condition: 95 DEG C of denaturation 30sec; Circulate 40 times by following parameter: 95 DEG C of sex change 5sec, 60 DEG C of collection fluorescence 34sec.

3, quality control standard

A) the Ct value of negative control should equal none.

B) the Ct value of positive control answers≤25, otherwise it is invalid that this experiment is considered as.(Ct value should be as the criterion with the cycle number of S type knee point)

C) should meet above-mentioned 2 conditions simultaneously, otherwise invalidate the test.

4, result is judged

Reaction solution is measured Ct value, and to be all greater than 40 sample negative; The positive sample of sample of Ct value≤35; The sample suggestion repetition measurement of measuring 35 < Ct≤40, repetition measurement result Ct < 35 is positive, repetition measurement result Ct >=35 negative.

The specific test of the test kit of DNA before embodiment 6 detection bovine leukemia virus of the present invention

Get bovine viral diarrhea virus (planting malicious AV69), infectious bovine rhinotrachetis virus (planting malicious AV20), Brucella abortus, FLK cell as 4 check samples (these four kinds of check samples are all purchased from China Veterinery Drug Inspection Office), extract respectively DNA, respectively get 2 μ L and carry out the specific PCR amplification of test kit as template, establish negative control group simultaneously.

Pcr amplification condition: 95 DEG C of denaturation 30Sec, circulate 40 times by following parameter: 95 DEG C of sex change 5sec, 60 DEG C of collection fluorescence 34sec.

Fluorescent PCR result shows that only the front DNA cloning of BLV goes out curve, and the DNA of the bovine viral diarrhea virus of sample, infectious bovine rhinotrachetis virus, Brucella abortus, FLK cell all occurs without this amplification curve in contrast, as shown in Figure 1.

The sensitivity test of the test kit of DNA before embodiment 7 detection bovine leukemia virus of the present invention

DNA positive plasmid intestinal bacteria before BLV after counting are carried out to 10 times of gradient dilutions, positive plasmid intestinal bacteria (3.2 × 10 ⁶individual/mL) press 1:10 ¹, 1:10 ², 1:10 ³, 1:10 ⁴, 1:10 ⁵, 1:10 ⁶carry out 3 groups of identical dilutions, extracting DNA.From each gradient DNA, respectively getting 2 μ L template fluorescent PCR kits detects.Amplification condition, with embodiment 5, is established negative control simultaneously.The Fluorescence PCR of the plasmid DNA by 6 concentration gradients, makes typical curve, and curve R value has reached more than 0.99, as shown in Figure 2.

The replica test of the test kit of DNA before embodiment 8 detection bovine leukemia virus of the present invention

Using identical dilution standard plasmid as template, and become the template (1:10 of three different gradients with 10 times of gradient dilutions ¹, 1:10 ², 1:10 ³), repeat 10 secondary responses, the results are shown in following table.

Table 1

Wherein, CV=s/x, the demonstration of repetition measurement result, under all dilution gradients, Ct value variation coefficient CV is all in 0.67 ~ 1.06% left and right, show that this detection system is better to the repeatability of positive plasmid detection, represent that the suitable formation of this detection system has the reagent of good reproducibility.

The stability test of the test kit of DNA before embodiment 9 detection bovine leukemia virus of the present invention

Detection reagent is put into the test that accelerates the failure of 37 DEG C of thermostat containers by Fig. 3, monitors continuously the variation that reaction reagent detected performance in 6 days, thereby judge the result figure of the stability of reaction system.Detect post analysis result, its Ct value is respectively 20.33,20.67,20.95,21.42,21.85,22.01, and the variation coefficient is 3.14%, and from test-results, inspection system has satisfactory stability.

The contrast of 10 test kit fluorescent PCR methods of embodiment and sleeve type PCR method

1, the pre-treatment of sample

(1) aseptic root of the tail gathers totally 94 parts, District of Shanghai ox EDTA anticoagulation sample, puts into 4 DEG C of refrigerators to be checked after numbering.

(2) extraction of sample DNA (with reference to the AxyPrep of AXYGEN company body fluid viral DNA/RNA small volume of reagent box specification sheets).

A) these 200 μ L of sampling, add DNA cleavage liquid Buffer V-L, and vortex concussion mixes, and leaves standstill 5min.

B) add 75 μ L Buffer V-N, vortex vibration mixes, the centrifugal 5min of 12000 × g.

C) supernatant is transferred in new 2ml centrifuge tube, added 300 μ L Virahols (1% glacial acetic acid), turned upside down 6 ~ 8 times, mixes.

D) be placed in 2ml centrifuge tube by preparing pipe, get mixed solution immigration in step 3 and prepare in pipe, the centrifugal 1min of 6000 × g.

E) abandon filtrate, put and get back in 2ml centrifuge tube preparing pipe, add 500 μ L Buffer W1A, room temperature leaves standstill 1min.The centrifugal 1min of 12000 × g.

F) abandon filtrate, put and get back in 2ml centrifuge tube preparing pipe, add 800 μ L Buffer W2, the centrifugal 1min of 12000 × g.

G) put and get back in 2ml centrifuge tube, the centrifugal 1min of 12000 × g preparing pipe.

H) be placed in clean 1.5ml centrifuge tube by preparing pipe, add 40 ~ 60 μ LBuffer TE preparing periosteum central authorities, room temperature leaves standstill 1min, and room temperature leaves standstill 1min.The centrifugal 1min eluted dna of 12000 × g ,-20 DEG C save backup.

2, sleeve type PCR method detects

" terrestrial animal diagnostic test and vaccine handbook " (the Manualof Diagnostic Tests and Vaccines for Terrestrial Animals showing with reference to OIE, 2008) the sleeve type PCR primer sequence synthetic primer of recommending in 2.3.4 endemicity bovine leucosis, primer sequence is

1A:5’-CTTTGTGTGCCAAGTCTCCCAGATACA-3’

6A:5’-CCAACATATAGCACAGTCTGGGAAGGC-3’

3B:5’-CTGTAAATGGCTATCCTAAGATCTACTGGC-3’

5B:5’-GACAGAGGGAACCCAGTCACTGTTCAACTG-3’

The gp51(env that this primer target sequence is BLV) gene, sequence is taken from GenBank, and registration number is K02120.

(1) sleeve type PCR amplification

PCR reaction system is as follows:

A) upstream and downstream primer 1A, 6A(10 μ molL ^-1) each 1.5 μ L, 10 × Buffer, 5 μ L, dNTP(10mM) 4*1 μ L, bovine serum albumin (1mg/ml) 5 μ L, MgCL ₂(25mM) 5 μ L, rTaqDNA polysaccharase 2U, the viral nucleic acid that adds 5 μ L to extract, adding the sterilizing distilled water 21 μ L systems of supplying is 50 μ L.PCR cycling condition is: 94 DEG C of 45s, 60 DEG C of 60s, 72 DEG C of 90s, 5 circulations; 94 DEG C of 45s, 55 DEG C of 60s, 72 DEG C of 90s, 30 circulations; Last 72 DEG C of 420s.

Second step: upstream and downstream primer 3B, 5B(10 μ molL ^-1) each 1.5 μ L, taking the first step PCR product as template, the same the first step of all the other each components, system is 50 μ L.PCR cycling condition is: 94 DEG C of 45s, 58 DEG C of 60s, 72 DEG C of 90s, 5 circulations; 94 DEG C of 45s, 53 DEG C of 60s, 72 DEG C of 90s, 30 circulations; Last 72 DEG C of 420s.

After finishing, PCR carries out electrophoresis on 1% agarose gel, after EB dyeing, and imaging under ultraviolet lamp, product size is 341bp.

B) clone of gp51 gene and qualification

Under ultraviolet lamp, the object band of PCR is taken out, the step that reclaims test kit according to DNA glue reclaims, fetch receipts product and be connected with pBS-T carrier, ligation system is: the PCR product 7 μ L of recovery, 10 × connection Buffer, 1 μ L, pBS-T carrier 1 μ L, T4DNA ligase enzyme 1 μ L, 4 DEG C of connections are spent the night, and then transform DH5 α competence bacteria, under IPTG, X-gal induction, cultivate 12～16h for 37 DEG C, after picking hickie colony inoculation LB liquid nutrient medium overnight incubation, extract plasmid.

(3) mensuration of nucleotide sequence

Get above-mentioned positive recombinant plasmid and send Invitrogen company to carry out sequencing, and compare together with the nucleotide sequence of the BLV gp51 gene of having delivered.

3, fluorescence PCR method detects

(1) according to detecting sample number n(n=sample number+2 to be checked) get PCR reaction solution, warm start Taq enzyme and probe and be mixed in a centrifuge tube, on vortex vibrator, vibrate, by every pipe packing, cover upper tube cap for subsequent use.

(2) now negative controls is added in a point of tubulature, the DNA that gets each sample adds in corresponding reaction tubes; Finally take out positive control and add in another reaction tubes, centrifugal after each reaction tubes mark, take out and put in fluorescent PCR instrument.

(3) Fluorescence PCR condition: 95 DEG C of denaturation 30sec; Circulate 40 times by following parameter: 95 DEG C of sex change 5sec, 60 DEG C of collection fluorescence 34sec.

4, detected result

In sum, the test kit of detection bovine leukemia virus of the present invention has overcome the deficiency of sleeve type PCR detection method.By sleeve type PCR method and this test kit fluorescent PCR method, 94 parts of District of Shanghais ox clinical sample is carried out to DNA detection before bovine leukemia virus, 92 parts of results are consistent simultaneously, and coincidence rate has reached 97.87%.Result shows that the positive rate of sleeve type PCR method and fluorescent PCR method is respectively 34.04% and 36.17%, fluorescent PCR is detected and 2 parts of doubtful positive amplified productions that sleeve type PCR fails to detect check order, result shows to be bovine leukemia virus, proves that fluorescence PCR method susceptibility is a little more than sleeve type PCR.

This test kit compared with prior art has better positive coincidence rate, fully demonstrated this test kit high specificity, susceptibility is high, cost is low, quick, personnel require the advantages such as low, result judgement is convenient, directly perceived, this test kit adopts single stage method can reach the testing goal of bovine leukemia virus, has important using value for this sick differential diagnosis, epidemiology survey and risk assessment thereof.

Claims (9)

1. one kind is detected the test kit of the front DNA of bovine leukemia virus, described test kit contains: a) Fluorescence PCR liquid, b) fluorescent probe, c) warm start Taq enzyme, d) standard positive template, e) negative control, it is characterized in that, in described Fluorescence PCR liquid, contain, downstream primer, wherein the sequence of upstream primer is as shown in SEQ ID NO:2, the sequence of downstream primer is as shown in SEQ ID NO:3, the sequence of described fluorescent probe is as shown in SEQ ID NO:4, sequence 5 ' the end of described fluorescent probe is designed with fluorescent substance, 3 ' end is designed with cancellation material, the Nucleotide insertion sequence of described standard positive template is as shown in SEQID NO:5, wherein, described test kit is taking ox EDTA anticoagulation sample as detecting sample.

2. test kit according to claim 1, is characterized in that, when detection described in the final concentration of upstream and downstream primer wall scroll be 0.05-1 μ M.

3. test kit according to claim 1, is characterized in that, in described Fluorescence PCR liquid, also contains Mg ²⁺, when detection described in Mg ²⁺final concentration be 1.5-5.0mM.

4. test kit according to claim 1, is characterized in that, also contains four kinds of dNTPs in described Fluorescence PCR liquid, when detection described in the final concentration of various dNTPs be 0.1-1mM.

5. test kit according to claim 1, is characterized in that, when detection described in the final concentration of fluorescent probe be 0.05-1 μ M.

6. test kit according to claim 1, is characterized in that, when detection described in the final concentration of warm start Taq enzyme be 0.01-5U/uL.

7. test kit according to claim 1, is characterized in that, the fluorescent substance of the sequence 5 ' end of described fluorescent probe is the one in FAM, HEX or VIC, and the cancellation material of 3 ' end is TAMRA.

8. test kit according to claim 1, is characterized in that, described standard positive template is that the nucleotide fragments that carries out pcr amplification acquisition by DNA before the primer pair bovine leukemia virus of described upstream and downstream inserts the recombinant plasmid that pBS-T vector construction forms.

9. test kit according to claim 1, is characterized in that, described test kit contains: by being respectively the dNTPs of 0.3mM, and 2.4mM Mg ²⁺, be respectively the described Fluorescence PCR liquid of the upstream and downstream primer composition of 0.288 μ M, 2.5mL/ pipe × 2; 6 μ M fluorescent probes, 200 μ L/ pipes; 7.5U/ μ L warm start Taq enzyme, 200 μ L/ pipes; Standard positive template, 250 μ L/ pipes; Negative control, 250 μ L/ pipes.