CN1405328A - Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use - Google Patents
Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use Download PDFInfo
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Abstract
The invention discloses a fluorescence quantitative PCR agent box for quick-quantitative detecting swine-fever virus and swine-fever rabbit weak-virus bacterin, the method and the application. The agent box overcomes the shortage in traditional and modern method and can effectively make quantitative detection and quality supervisory control.
Description
Technical field
The present invention relates to the PCR kit for fluorescence quantitative that a kind of fast quantification detects Pestivirus suis and hog cholera lapinised virus seedling, real monitoring also relates to the purposes in detecting Pestivirus suis and hog cholera lapinised virus seedling when being applicable to the detection by quantitative of Pestivirus suis and hog cholera lapinised virus seedling and vaccine quality.
Background technology
Swine fever is a kind of height contagious disease of pig, often causes serious economy loss to pig industry.At present, slaughter and the vaccine inoculation of the early stage specific diagnosis of eqpidemic disease, epidemic-infected animal are the important means of effective prevention and control swine fever.
Traditional swine fever detection method mainly comprises: the separation of virus and biological test, electron microscopy, immunofluorescence technique, complement fixation test (CFT), nucleic acid hybridization, polyacrylamide gel electrophoresis etc., these methods have played vital role in virus diagnostic and import and export inspection and quarantine, but ubiquity significantly not enough, long such as the cycle, complex operation, specificity and susceptibility are poor, and is lack of standardization, be not suitable for mass detection, bring great difficulty for the detection of swine fever.
China hog cholera lapinised virus vaccine (Hog Cholera Lapinized Virus, HCLV) it is fast to have an immune response of inducing with it, boar, sucking pig are not had remaining virulence, and immune swine can be resisted the characteristics such as attack of swine fever virulent strain, becomes the attenuated vaccine the most safely and effectively of generally acknowledging in the world.For many years, at home and abroad be widely used, the control of swine fever is played an important role, and (Fu Liezhen is etc. the weak malicious NS2-3 Gene Sequence Analysis [J] of Chinese swine fever rabbitization for Wang Jiafu, Zhang Chuyu.The virus journal, 2000,16:150-154.).
The height of vaccine valence is the whether qualified key index of vaccine.The proliferative ability of HCLV in cell a little less than, do not produce cytopathogenic effect, its mensuration of tiring is continued to use rabbit body-shaping thermal response method (The Ministry of Agriculture of the People's Republic of China, MOA, People's Republic of China's veterinary biologics rules [S] .2000 version .) always.But the outstanding deficiency that this method exists is: can not be accurately quantitative; Survey malicious result and be subject to rabbit body individual difference and weather condition influence; Grow, waste time and energy experimental period.Therefore, measuring vaccine valence how fast, accurately is one of main difficult problem that faces in the swine Fever Vaccine production.It is reported, adopt micro-cell cultures ELISA (Du Nianxing, the reviews and prospects of swine fever [J]. Chinese livestock and poultry transmissible disease, 1998,20:317-319) with reverse indirect hemagglutination experiment (Li Baochen, Guo Xingfu, Han Zheng is rich, Deng. utilization reverse indirect hemagglutination method is imitated seizure test [J] to swine fever cell toxicant stoste. Heilungkiang herding beast, and 1999,5:46-46.) measure HCLV to tire, can be corresponding shortening detection time, reduce production costs, but still can not be accurately quantitative to malicious valency, and do not have widespread usage in production practice at present yet.
The quantitative fluorescent PCR that development in recent years is got up (Fluorogenetic Quantitative PCR, FQ-PCR) technology is highly sensitive with it, speed is fast, advantages such as high specificity are at gene expression dose analysis (Nellemann C, Vinggaard AM, Dalgaard M, et al.Quantification of Antiandrogen Effect Determinedby LightCycler Technology[J] .Toxicology, 2001,163:29-38), qualitative (the McGoldrick A of pathogenic agent, Lowings JP, Ibata G, et al.A Novel Approach to the Detection ofClassical Swine Fever Virus by RT-PCR with a Fluorogenic Probe (TaqMan) [J] .J.Virol.Methods, 1998,72:125-135.; Bhudevi B, Weinstock D.Fluorogenic RT-PCRassay (TaqMan) for Detection and Classification of Bovine Viral Diarrhea Virus[J] Vet.Microbiol., 2001,83:1-10.) and detection by quantitative (Desire N, Dehee A, Schneider V, et al.Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqManReal-Time PCR Assay[J] .J.Clin.Microbiol, 2001,39:1303-1310.; Martell M, Gomez J, Esteban JI, et al.High-Throughput Real-Time Reverse Transcription-PCRQuantitation of Hepatitis C Virus RNA[J] .J.Clin.Microbiol, 1999,37:327-332.; Keams AM, Turner AJL, Eltringham GJA, et al.Rapid Detection and Quantificationof CMV DNA in Urine using Lightcycler-based Real-time PCR[J] .J.Clin.Virol, 2002,24:131-134; Florence KP, Glaucia PB, Mireille S, et al.Quantitation of HCVRNA using real-time PCR and fluorimetry[J] .J.Virol.Methods, 2001,95:111-119.) etc. the aspect be used widely, and become the quantitative main method of current viral nucleic acid with, domestic at present also existing test kit listing about third liver, hepatitis B, mycoplasma, AIDS, tuberculosis detection by quantitative.
Summary of the invention
The object of the present invention is to provide a kind of fast quantification to detect the PCR kit for fluorescence quantitative of Pestivirus suis and hog cholera lapinised virus vaccine, this PCR test kit not only can use all types fluorescent quantitation instrument that exists at present on the market, the more important thing is and can carry out detection by quantitative to Pestivirus suis and hog cholera lapinised virus vaccine apace.
Another object of the present invention provides a kind of PCR kit for fluorescence quantitative in the application of Pestivirus suis and hog cholera lapinised virus vaccine being carried out in the detection by quantitative.
To achieve these goals, the present invention will use following technical measures: a kind of fast quantification detects the PCR kit for fluorescence quantitative of Pestivirus suis and hog cholera lapinised virus vaccine, this test kit comprises a) RNA lysate, b) reversed transcriptive enzyme, c) RNA enzyme inhibitors, d) Taq archaeal dna polymerase, e) standard positive template, f) reverse transcription reaction liquid and g) the fluorescent quantitation reaction solution, it is characterized in that: f) to contain forward primer PF be the nucleotide sequence shown in 5 '-TAGCCATGCCCATAGTAGG-3 ' (SEQ ID NO:1) to reverse transcription reaction liquid, g) the fluorescent quantitation reaction solution contains primer and fluorescent probe, primer is forward primer and reverse primer, be respectively the nucleotide sequence shown in 5 '-TAGCCATGCCCATAGTAGG-3 ' (SEQ ID NO:1) and the 5 '-ATCAGGTCGTACTCCCATCAC-3 ' (SEQ ID NO:2), fluorescent probe is the nucleotide sequence shown in 5 '-TACAGGACAGTCGTCAGTAGTTCGACGTGA-3 ' (SEQID NO:3), the fluorescence report group of fluorescent probe 5 ' end mark is FAM, the fluorescent receptor group of 3 ' end mark is ROX, e) standard positive template pGS contains the pGEM-T carrier that 400 nucleotide fragments of hog cholera lapinised virus strain 5 ' end constitute, and this carrier can be bred in bacillus coli DH 5 alpha.
In a preferred version of the present invention, reverse transcription reaction liquid is by forward primer (SEQ ID NO:1), RT5 * buffer solution, dNTPs solution and aseptic double-distilled water are formed, in a concrete scheme of the present invention, reverse transcription reaction liquid is 10 μ mol/L forward primers (SEQ ID NO:1), 3 μ l, RT5 * buffer 10 μ l, 10mmol/LdNTPs 5 μ l, reversed transcriptive enzyme 1 μ l, RNA enzyme inhibitors 1 μ l, sterilization distilled water 10 μ l.
In a preferred version of the present invention, the fluorescent quantitation reaction solution is by forward primer (SEQ ID NO:1) and reverse primer (SEQ ID NO:2), fluorescent probe FP, PCR 10 * buffer solution, MgCl
2Solution, dNTPs solution, Taq archaeal dna polymerase and aseptic double-distilled water are formed; In a concrete scheme of the present invention, the fluorescent quantitation reaction solution is by PCR 10 * buffer 2 μ l, 10 μ mol/L forward primers (SEQ ID NO:1) and reverse each 0.5 μ l of primer (SEQ ID NO:2), l μ mol/L fluorescent probe 1 μ l, 25mmol/L MgCl
23.6 μ l, 10mmol/L dNTPs 0.4 μ l, 3U/ μ l Taq archaeal dna polymerase 0.5 μ l, aseptic double-distilled water 9.5 μ l form.Wherein fluorescent probe is the nucleotide sequence shown in the SEQ ID NO:3, and the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent receptor group of 3 ' end mark is ROX.
In a preferred version of the present invention, standard positive template is to contain the pGEM-T carrier that 400 nucleotide fragments of hog cholera lapinised virus strain 5 ' end constitute, and this carrier can be bred in bacillus coli DH 5 alpha.Storing concentration is 5 * 10
10Copy/μ l, serial dilution before using.Testing used standard substance is the plasmid pGS that contains the purpose amplified fragments, and alkaline lysis method of extracting is used in this plasmid transformation escherichia coli DH5 α propagation back, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 5 * 10
10Copy/μ l ,-20 ℃ of preservations.
In the invention provides the PCR kit for fluorescence quantitative that detects Pestivirus suis and hog cholera lapinised virus vaccine, there are two ends to be marked with the specificity fluorescent probe of fluorophor, when probe is complete, two groups distance on space structure is close mutually, the fluorescence that 5 ' end reporter group produces is because FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, so there is not the variation of fluorescent signal in the system.In PCR annealing and extension process, probe combines with template specificity, extension along with primer, the Taq archaeal dna polymerase utilizes its 5 '-3 ' circumscribed activity that probe cutting is discharged reporter group, destroyed the FRET between two groups like this, the photofluorometer that the fluorescence that reporter group discharged can be built in the detection by quantitative instrument detects, and the accumulation volume of the increase of fluorescence volume and PCR product is proportionlity.To HCLV quantitatively can comparing draws by the circulation thresholding (Ct, Threshold Cycle) with standard substance.The Ct value is in the PCR process, and the accumulation of fluorescence volume surpasses the circulation number of turns of substrate fluorescence volume, and Ct value and initial modulus are the certain proportion relation, and the Ct value is more little, and the starting template number is many more, and on the contrary, the Ct value is big more, and the starting template number is few more.Utilize the Ct value of positive gradient standard form to make typical curve, can accurately measure the initial copy number of this sample again according to the Ct value of testing sample.
In the invention provides the PCR kit for fluorescence quantitative that detects Pestivirus suis and hog cholera lapinised virus vaccine, at the singularity in the hog cholera lapinised virus vaccine detection, different target fragments is carried out reaction system, optimization as primer and concentration and probe concentration, Mg2+ concentration, annealing temperature etc., and FQ-PCR technology and detection by quantitative system (comprised the LigthCycler system, Roche; ABI Prism system, PE Applied Bioasystems; ) combine, use it for the detection by quantitative of Pestivirus suis and hog cholera lapinised virus vaccine.Pass through prioritization scheme, experiment repeatedly, and compare with traditional rabbit body-shaping thermal response method, detection by quantitative HCV and HCLV method have been set up, and develop the test kit of Pestivirus suis and hog cholera lapinised virus seedling detection by quantitative, whether qualified the sensitivity of this test kit can detect 200Copies in each reaction system, can satisfy the quick specific detection of Pestivirus suis fully and detect hog cholera lapinised virus vaccine requirement.
In another aspect of the present invention, the method for using test kit of the present invention that Pestivirus suis and hog cholera lapinised virus vaccine are detected also is provided, this method comprises the following steps:
A) use e) standard positive template prepares the positive criteria product, and quantitative with ultraviolet spectrophotometer;
B) use a) that the RNA lysate extracts RNA from sample to be measured, add b then) reversed transcriptive enzyme, c) RNA enzyme inhibitors and f) reverse transcription reaction liquid reverse transcription becomes cDNA;
C) get B respectively) A of the cDNA in the step and the serial dilution of same amount) positive criteria product in the step join and contain d) Taq archaeal dna polymerase and g) carry out the PCR detection with the fluorescent quantitation detector in the PCR reaction system of fluorescent quantitation reaction solution;
D) the initial copy number to testing sample carries out quantitatively by the circulation thresholding that compares testing sample and standard substance.
The detection Pestivirus suis that provides in the present invention and the PCR kit for fluorescence quantitative of hog cholera lapinised virus vaccine can carry out the time real monitoring of detection by quantitative and vaccine quality to Pestivirus suis and hog cholera lapinised virus vaccine, and the rabbit body-shaping thermal response method of alternative Pestivirus suis traditional detection method of always continuing to use and hog cholera lapinised virus vaccine detection.
The present invention compared with prior art has the following advantages and effect:
1, quantitatively accurately;
2, detection speed is fast, and only 1 hour, add the extraction of nucleic acid and the preparation of cDNA, only need 3-4 hour altogether;
3, step is simple;
4, can carry out high-throughout sample detection simultaneously;
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); D.L. Spector etc., Science Press, 200l, cell experiment guide; Lv Hongsheng, Science Press, 1982, or connect the condition of advising according to manufacturer.
The PCR kit for fluorescence quantitative of embodiment 1 swine fever attenuated vaccine is formed and preparation
A). reagent is formed:
It is GIBCOL company product that TRIZOL RNA extracts test kit.DNTPs (10mM), Taq archaeal dna polymerase (3U/ μ l), RNA enzyme inhibitors (40U/ μ l), MgCl
2(25mM) all available from magnificent company.MMLV reversed transcriptive enzyme (200U/ μ l) is available from Promega company.
B) .MMLV 5 * Buffer forms:
50mM?Tris-HCl(PH8.3?25℃)、3mM?MgCl2、75mM?KCl、
10mM?DDT；
C). fluorescent PCR 10 * Buffer forms:
500mM?KCl、100mM?Tris-HCl(PH9.0?25℃)、
1.0％Triton?X-100；
D). reverse transcription reaction liquid: forward primer PF (SEQ ID NO:1) 3 μ l (10 μ mol/L), RT5 * buffer 10 μ l, dNTPs 5 μ l (10mmol/L), aseptic double-distilled water 10 μ l.
E). fluorescent quantitation reaction solution: PCR 10 * buffer 2 μ l, each 0.5 μ l (10 μ mol/L) of forward primer PF (SEQ ID NO:1) and reverse primer PR (SEQ ID NO:2), fluorescent probe 1 μ l (1 μ mol/L), MgCl
23.6 μ l (25mmol/L), dNTPs 0.4 μ l (10mmol/L), Taq archaeal dna polymerase 0.5 μ l (3U/ μ l), aseptic double-distilled water 9.5 μ l.Fluorescent probe FP sequence is SEQ ID NO:3, and the fluorescence report group of its 5 ' end mark is FAM, and the fluorescent receptor group of 3 ' end mark is ROX.Working concentration is 1 μ mol/L.
F). provide reagent for oneself: 75% ethanol of the sterilization distilled water that chloroform, Virahol, DEPC are handled, the sterilization distilled water preparation handled with DEPC.
The PCR kit for fluorescence quantitative of embodiment 2 usefulness hog cholera lapinised virus seedlings detects the hog cholera lapinised virus seedling
A). getting three kinds, dissimilar (rabbit all produces the typing thermal response; A rabbit has typing heat, and one does not produce typing heat; Two rabbits do not produce the typing thermal response) each 100 μ l of the vaccine liquid through the checking of rabbit body-shaping thermal response duplicate detection (producing) by Wuhan Zhongbo Biochemical Co., Ltd, add RNA lysate 1ml mixing respectively, room temperature left standstill 10 minutes, add the 0.2ml chloroform, room temperature left standstill 3 minutes, in centrifugal 15 minutes of 4 ℃ of 12000g/min, supernatant liquor is transferred to another centrifuge tube, adds the 0.5ml Virahol, precipitation at room temperature 10 minutes, centrifugal 10 minutes of 4 ℃ of 12000g/min, decant supernatant, add 1ml 75% ethanol, abandon supernatant in centrifugal 5 minutes of 4 ℃ of 7500g/min, the RNA precipitation is at room temperature dried naturally, adds the 20ul that DEPC handled.
B). get A) the step RNA that carries 20 μ l, hatch 5min for 85 ℃, put 5min on ice rapidly, add reverse transcription reaction liquid 28 μ l then, RNA enzyme inhibitors 1 μ l, MMLV reversed transcriptive enzyme 1 μ l.42 ℃ of 60min, 95 ℃ of 5min deactivation MMLV.
C). with the dilution of positive criteria moulding plate series is 5 * 10
5Copy number/μ l, 5 * 10
4Copy number/μ l, 5 * 10
3Copy number/μ l.
D). get each 18 μ l of fluorescence quantitative PCR reaction solution respectively, get B) step gained cDNA and C) go on foot each the 2 μ l of positive criteria template that dilute, add different PCR reaction tubess respectively, the parallel PCR that is detects on the fluorescent quantitation detector.Cycling condition is: 95 ℃ of pre-sex change 2min; 95 ℃ of 15s, 65 ℃ of 40s, 40 circulations of increasing.Carry out when the program of fluoroscopic examination is arranged on each round-robin second EOS, the detection wavelength is 530nm.
After the loop ends, the utilization instrument carries software, reads sample copy number to be checked.The result is:
Standard positive template 5 * 10
5Copy number/μ l, 5 * 10
4Copy number/μ l, 5 * 10
3The Ct value of copy number/μ l is respectively 21.20,24.77 and 28.62;
The Ct value that makes two rabbits all produce typing thermal response vaccine sample is 26.96, and the every ml vaccine sample copy number in back that converts is 7.13 * 10
6Copy number/ml; A rabbit has typing heat, and a Ct value that does not produce the hot vaccine sample of typing is 28.63, and the every ml vaccine sample copy number in back that converts is 3.35 * 10
6Copy number/ml; The Ct value that two rabbits do not produce the vaccine sample of typing thermal response is 31.86, and converting afterwards, every ml vaccine sample copy number is 4.70 * 10
5Copy number/ml.
The PCR kit for fluorescence quantitative of embodiment 3 hog cholera lapinised virus seedlings and traditional rabbit body-shaping thermal response method detect the comparison of hog cholera lapinised virus seedling
1. get the vaccine liquid (being produced by Wuhan Zhongbo Biochemical Co., Ltd) of 15 parts of different batches results, being 5 parts through the detection of rabbit body-shaping thermal response method is that two rabbits all produce the typing thermal response; 5 parts is that a rabbit has typing heat, and one does not produce typing heat; 5 parts is that two rabbits do not produce the typing thermal response.Be numbered 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 respectively.1-5 is that two rabbits all produce typing thermal response vaccine liquid; 6-10 is that a rabbit has typing heat, and one does not produce the hot vaccine liquid of typing; 12-15 is that two rabbits do not produce typing thermal response vaccine liquid.Through detect result such as following table with this test kit:
Vaccine sample rabbit body-shaping thermal response quantitative fluorescent PCR
Method FQ-PCR
Virus quantity
(copies/ml)
1 ++ 4.63×10
6
2 ++ 1.42×10
7
3 ++ 5.63×10
6
4 ++ 6.28×10
6
5 ++ 1.36×10
7
6 +- 2.19×10
6
7 +- 1.47×10
6
8 +- 6.50×10
6
9 +- 1.99×10
6
10 +- 3.44×10
6
11 -- 5.52×10
5
12 -- 4.34×10
6
13 -- 9.25×10
4
14 -- 1.22×10
4
15 -- 8.21×10
5
++: two rabbits all produce typing thermal response vaccine sample
Rabbit of+-: has typing heat, and one does not produce the hot vaccine sample of typing
--: two rabbits do not produce the typing thermal response
No. 8 sample rabbit body-shaping thermal response method detect for+-, and fluorescence quantitative PCR detection is 6.50 * 10
6Virus particle/ml, the virus particle amount has reached the amount that makes the rabbit body produce the typing thermal response; No. 12 sample rabbit body-shaping thermal response methods detect and are--, and fluorescence quantitative PCR detection 4.34 * 10
6Virus particle/ml, the virus particle amount has reached and has made the rabbit body produce a generation typing thermal response, an amount that does not produce the typing thermal response; We detect with two kinds of methods again to this two duplicate samples, and the result is that No. 8 sample rabbit body-shaping thermal response methods detections are ++, fluorescence quantitative PCR detection is 6.94 * 10
6Virus particle/ml; No. 12 sample rabbit body-shaping thermal response methods detect for+-, and fluorescence quantitative PCR detection 3.95 * 10
6Virus particle/ml.
2. get the vaccine liquid of 15 parts of different batches results, measure the virion subnumber with fluorescent quantificationally PCR detecting kit earlier, and then detect through rabbit body-shaping thermal response method.Numbering is respectively 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15.Result such as following table:
Vaccine sample rabbit body-shaping thermal response quantitative fluorescent PCR
Method FQ-PCR
Virus quantity
(copies/ml)
1 ++ 5.17×10
6
2 +- 7.42×10
6
3 -- 7.53×10
5
4 ++ 6.55×10
6
5 ++ 3.27×10
7
6 ++ 2.55×10
7
7 +- 1.48×10
6
8 +- 3.17×10
6
9 +- 6.36×10
6
10 ++ 1.17×10
7
11 -- 5.33×10
5
12 -- 4.70×10
6
13 ++ 9.55×10
6
14 +- 1.98×10
4
15 ++ 8.33×10
6
++: two rabbits all produce typing thermal response vaccine sample
Rabbit of+-: has typing heat, and one does not produce the hot vaccine sample of typing
--: two rabbits do not produce the typing thermal response
No. 2 sample rabbit body-shaping thermal response method detect for+-, and fluorescence quantitative PCR detection is 7.42 * 10
6Virus particle/ml, the virus particle amount has reached the amount that makes two rabbit bodies produce the typing thermal response; No. 9 sample rabbit body-shaping thermal response methods detect for+-, fluorescence quantitative PCR detection is 6.36 * 10
6Virus particle/ml, the amount of virus can make two rabbit bodies produce the typing thermal response; No. 12 sample rabbit body-shaping thermal response methods detect and are--, and fluorescence quantitative PCR detection 4.70 * 10
6Virus particle/ml, the virus particle amount has reached and has made a rabbit produce the typing thermal response, and one does not produce the typing thermal response; No. 14 sample rabbit body-shaping thermal response methods detect for+-, and fluorescence quantitative PCR detection 1.98 * 10
4Virus particle/ml, the virus particle quantity not sufficient is so that any rabbit body produces the typing thermal response;
We detect with two kinds of methods again to above-mentioned 4 samples, and the result is that No. 2, No. 9 sample rabbit body-shaping thermal response methods detections are ++, fluorescence quantitative PCR detection is respectively 6.78 * 10
6Virus particle/ml and 7.11 * 10
6Virus particle/ml; No. 12 sample rabbit body-shaping thermal response methods detect for+-, fluorescence quantitative PCR detection is 3.55 * 10
6Virus particle/ml; No. 14 sample rabbit body-shaping thermal response methods detect still be+-, fluorescence quantitative PCR detection is 5.32 * 10
4Virus particle/ml.
Can illustrate that from above-mentioned experiment contrast the fluorescence quantifying PCR method quantitative repeatability is good, quantitatively accurately, and rabbit body-shaping thermal response method can cause surveying malicious inaccurate because of rabbit individual reaction difference.And fluorescent quantificationally PCR detecting kit only needed just can finish in 4 hours to the detection of vaccine, and traditional rabbit body-shaping thermal response method needs just can finish about 1 week approximately, therefore, uses this test kit to shorten detection time greatly.
The implementation method that detects Pestivirus suis with PCR kit for fluorescence quantitative is identical with the detection hog cholera lapinised virus vaccine.Do not carry out rabbit body-shaping thermal response.
The operation of this test kit only needs 1 people can finish whole quantitative work process, once can detect 32-384 (by the model decision of detection by quantitative instrument) sample, has so also reduced waste of manpower resource.
SEQUENCE?LISTING
＜110〉Wuhan University
＜120〉a kind of fast quantification detects the PCR kit for fluorescence quantitative and the application of Pestivirus suis and hog cholera lapinised virus vaccine
＜130〉a kind of fast quantification detects the PCR kit for fluorescence quantitative and the application of Pestivirus suis and hog cholera lapinised virus vaccine
<160>4
<170>PatentIn?version?3.1
<210>1
<211>19
<212>DNA
＜213〉synthetic
<400>1
tagccatgcc?catagtagg 19
<210>2
<211>21
<212>DNA
＜213〉synthetic
<400>2
atcaggtcgt?actcccatca?c 21
<210>3
<211>30
<212>DNA
＜213〉synthetic
<400>3
Tacaggacag tcgtcagtag ttcgacgtga 30＜210〉4＜211〉400＜212〉DNA＜213〉Classical Swine Fever Virus Shimen Strain, fever virus lapinized Chinese Strain＜400〉4gtatacgagg ttagttcatt ctcgtataca cgattggaca aatcaaaatt ataatttggt 60tcagggcctc cctccagcga cggccgaact gggctagcca tgcccatagt aggactagca 120aaacggaggg actagccata gtggcgagct ccctgggtgg tctaagtcct gagtacagga 180cagtcgtcag tagttcgacg tgagcagaag cccacctcga gatgctacgt ggacgagggc 240atgcccaaga cacaccttaa ccctagcggg ggtcgctagg gtgaaatcac gccacgtgat 300gggagtacga cctgataggg cgccgcagag gcccactatt aggctagtat aaaaatctct 360gctgtacatg gcacatggag ttgaatcgct ttgaactttt 400
Claims (5)
1, a kind of fast quantification detects the PCR kit for fluorescence quantitative of Pestivirus suis and swine fever attenuated vaccine, this test kit comprises a) RNA lysate, b) reversed transcriptive enzyme, c) RNA enzyme inhibitors, d) Taq archaeal dna polymerase, e) standard positive template, f) reverse transcription reaction liquid and g) the fluorescent quantitation reaction solution, it is characterized in that: reverse transcription reaction liquid contains forward primer, its sequence is 5 '-TAGCCATGCCCATAGTAGG-3 ', the fluorescent quantitation reaction solution contains primer and fluorescent probe, primer is forward primer and reverse primer, its sequence is 5 '-ATCAGGTCGTACTCCCATCAC-3 ', the fluorescent probe sequence is 5 '-TACAGGACAGTCGTCAGTAGTTCGACGTGA-3 ', and the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent receptor group of 3 ' end mark is ROX, standard positive template pGS contains the pGEM-T carrier that 400 nucleotide fragments of hog cholera lapinised virus strain 5 ' end constitute, and this carrier can be bred in bacillus coli DH 5 alpha.
2, a kind of fast quantification according to claim 1 detects Pestivirus suis and swine fever attenuated vaccine PCR kit for fluorescence quantitative, it is characterized in that: reverse transcription damping fluid, dNTPs solution and aseptic double-distilled water.
3, a kind of fast quantification according to claim 1 detects Pestivirus suis and swine fever attenuated vaccine PCR kit for fluorescence quantitative, and it is characterized in that: the fluorescent quantitation reaction solution is forward primer and reverse primer, fluorescent probe, PCR damping fluid, MgCl
2Solution, dNTPs solution, Taq archaeal dna polymerase and aseptic double-distilled water.
4, a kind of Quantitative detection CSFV according to claim 1 and swine fever attenuated vaccine PCR kit for fluorescence quantitative, it is characterized in that: the nucleotides sequence of standard positive template pGS is classified as: gtatacgagg ttagttcatt ctcgtataca cgattggaca aatcaaaatt ataatttggt 60tcagggcctc cctccagcga cggccgaact gggctagcca tgcccatagt aggactagca 120aaacggaggg actagccata gtggcgagct ccctgggtgg tctaagtcct gagtacagga 180cagtcgtcag tagttcgacg tgagcagaag cccacctcga gatgctacgt ggacgagggc 240atgcccaaga cacaccttaa ccctagcggg ggtcgctagg gtgaaatcac gccacgtgat 300gggagtacga cctgataggg cgccgcagag gcccactatt aggctagtat aaaaatctct 360gctgtacatg gcacatggag ttgaatcgct ttgaactttt 400
5, the application of the described PCR kit for fluorescence quantitative of claim 1 in fast quantification detection Pestivirus suis and hog cholera lapinised virus vaccine.
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|---|---|---|---|
| CNB021392536A CN1164767C (en) | 2002-11-08 | 2002-11-08 | Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100350055C (en) * | 2003-06-26 | 2007-11-21 | 上海科华生物工程股份有限公司 | Reagent box for quantitatively inspecting number of copies of sars virus |
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2002
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| CN100350055C (en) * | 2003-06-26 | 2007-11-21 | 上海科华生物工程股份有限公司 | Reagent box for quantitatively inspecting number of copies of sars virus |
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| CN100451129C (en) * | 2006-08-11 | 2009-01-14 | 中国兽医药品监察所 | Pig pestivirus fluorescence quantitative RT-PCR diagnosis agent kit |
| CN101144775B (en) * | 2007-08-31 | 2010-05-26 | 浙江大学 | Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit |
| CN101348837B (en) * | 2008-07-18 | 2012-03-14 | 黄萍 | Probe, primer and reagent kit for realtime fluorescent quantitative RT-PCR detection of classical swine fever virus |
| CN101463396B (en) * | 2009-01-06 | 2011-06-29 | 天津出入境检验检疫局动植物与食品检测中心 | African hog cholera virus fluorescent quantitative PCR detecting reagent and preparation and use thereof |
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