CN102230035B - Myxomatosis polymerase chain reaction (PCR) detection kit and use thereof - Google Patents
Myxomatosis polymerase chain reaction (PCR) detection kit and use thereof Download PDFInfo
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- CN102230035B CN102230035B CN 201110188307 CN201110188307A CN102230035B CN 102230035 B CN102230035 B CN 102230035B CN 201110188307 CN201110188307 CN 201110188307 CN 201110188307 A CN201110188307 A CN 201110188307A CN 102230035 B CN102230035 B CN 102230035B
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Abstract
The invention discloses a myxomatosis polymerase chain reaction (PCR) detection kit, which is used for diagnosing and detecting myxomatosis. The kit is developed by designing specific primers according to the sequence of the M150 gene of the myxomatosis, constructing a PCR detection method and optimizing PCR reaction conditions. The invention also provides a method for detecting the myxomatosis by using the kit. The PCR detection kit disclosed by the invention can quickly and accurately detect the myxomatosis.
Description
Technical field
The present invention relates to infectious myxoma virus diagnosis and detection technical field, in particular, relate to a kind of infectious myxoma virus PCR detection kit and detection method.The method is sensitive, special, rapid detection.
Background technology
Infectious myxoma virus (Myxomavirus) belongs to the member that Poxviridae (Poxviridae), rabbitpox virus belong to (Leporipox virus).Virus particle is brick shape, and size is about 280～230nm * 75nm, can be caused a kind of height contact, the lethality transmissible disease of rabbit by infectious myxoma virus.This disease around the subcutaneous particularly facial area of whole body and the natural hole swelling of subcutaneous generation myxoma as principal character.This disease found at uruguayan Meng Deweiya early than 1898, subsequently soon should disease namely propagates into the countries such as Brazil, Argentina, Colombia and Panama in South America.1930, this disease was imported California State, USA into through Mexico, and it is popular to be region in each state of Western United States at present.The harm that Australia causes for eliminating hare was introduced myxoma virus in 1950 artificially.This disease was imported Europe in 1952, had spread all over the states such as French, Belgian, German and Dutch in 18 months, and had crossed the English Channel and pass to the Britain, and while Scandinavia and north African country be Epidemics also.Up to the present, this disease occured in existing at least 56 countries and regions.China not yet finds this disease at present, but experimental study proved in recent years, and Chinese Rabbit is 100% to the M ﹠ M of infectious myxoma virus, and OIE and China classify it as respectively epidemic disease or the two class animal infection epidemic diseases of statutory report.
Rabbit is unique susceptible animal of this disease, and people and other animals are susceptible not; The equal susceptible of the rabbit at various ages, but as if young rabbit more have resistibility than adult rabbits.This disease is seasonal and occurs, and be onset peak season summer and autumn, and per 8～10 years are once popular, Major Epidemic in Oceania, America, European all states, there is not yet report in China.This disease mainly is mechanical transmission, namely comprises that through HAEMATOPHAGOUS ARTHROPODS the mouthpart of various mosquitoes, flea, buffalo gnat fly, tick, rabbit flea and mite etc. carries virus and propagates.Rabbit contact or contact with contaminated feed, drinking-water and utensil etc. and can cause infection, but contact transmission is not main circulation way.Mosquito is the main communication media of this disease, but slightly variant at the different areas communication media.
According to Clinical symptoms, pathological change, can make tentative diagnosis in conjunction with epidemiology.Make a definite diagnosis and to carry out laboratory inspection.Get pathological tissues and make section or plate coating checking stellate cell and inclusion body, or get fresh pathological material of disease inoculation rabbit, chicken embryo, rabbit kidney primary cell or RK13 passage cell isolated viral, carry out the evaluation of virus with immunofluorescent test, neutralization test and agar diffusion test.
Along with the introducing to external each all rabbit and rabbit products material etc., this disease is very large to the potential threat of China's rabbit keeping, if import into, the harm that causes and financial loss can't be estimated.Especially in recent years, the appearance of atypical myxoma virus, often similar to the clinical symptom that fibroma virus produces, detection technique is had higher requirement, so in the urgent need to address have a fast and accurately gene diagnosis technology.The diagnostic method of report has all time-consuming, efforts such as pathological section, viral separation, immunofluorescence, neutralization test at present, it is unrealistic to be used for inspection and quarantining for import/export, and because the poison operation of living, require high to the laboratory Biosafety, adopt PCR method to detect the nucleic acid of the infectious myxoma virus in the sample, highly sensitive, only needed several hours both can obtain the result fast, be particularly suitable for inspection and quarantining for import/export inspection routine needs.The present invention is exactly test kit and the detection method that can detect fast, with sensitivity infectious myxoma virus of developing on the basis of conventional PCR method.
Summary of the invention
The technical problem that (one) will solve
Purpose of the present invention aims to provide a kind of PCR detection kit of infectious myxoma virus, is used for detection and the Molecule Epidemiology Investigation of infectious myxoma virus.
(2) technical scheme
The M150R gene of infectious myxoma virus is one section conservative special sequence, by the comparison to the infectious myxoma virus M150R gene order of GenBank issue, designed, designed a pair of special primer, developed the PCR detection kit and a kind of method of using infectious myxoma virus in this test kit rapid detection sample is provided, to realize the quickly and accurately detection to infectious myxoma virus.
The PCR detection kit of wherein said infectious myxoma virus comprises:
(1) dna cleavage liquid
The Nacl of 100mM, the Tris-HCl of the 10mM of pH 8.0, the EDTA of the 25mM of pH 8.0, volume ratio content is the mixing solutions of the Proteinase K of 1% SDS and 4 μ g/ μ L by weight.
(2) PCR reaction solution
Each 50～400 μ M of final concentration 4 in dNTPs, final concentration is primer M150R-1 and the M150R-2 of 0.1-0.5 μ M, the Mg of 1.5～5.0 μ M
2+
(3) positive control
The positive plasmid pTM150R of infectious myxomatosis M150R gene.
(4) the Taq enzyme contains the 1.0U adding by each PCR reaction, does not contain the Taq enzyme in the reaction solution;
In addition, can also contain 3mol/L NaAC, phenol, phenol in the test kit of the present invention: chloroform: primary isoamyl alcohol, chloroform: primary isoamyl alcohol, Taq enzyme 5U/ μ L, agarose, ethidium bromide and tetrabromophenol sulfonphthalein point sample damping fluid.Can also contain the PCR reaction tubes.
The optimizing process of the optimization reaction conditions of test kit of the present invention:
(1) primer concentration optimization
Primer concentration is chosen in 0.1～0.5 μ M carries out pcr amplification test, the result shows that the primer of these several concentration can both amplify the purpose band, and when every primer amount with 20pmol/ μ L, i.e. best results during 0.2 μ M.
(2) MgCL
2
Concentration optimization
Mg is set
2+Concentration range is 1.5～5.0mM, and the result shows the Mg in this concentration range
2+Can both amplify the purpose band, and work as Mg
2+When concentration was 2.0mM, expanding effect was best, and not only without non-specific, and amplified band is also very limpid in sight.
(3) optimization of dNTPs concentration
4 kinds of isocyatic dNTPs take final concentration as 50～400 μ mol/L carry out pcr amplification, and the dNTPs in this concentration range all can amplify the purpose band as a result, and are the best when total dNTPs concentration is 200 μ mol/L.
(4) optimization of Taq archaeal dna polymerase concentration
Taq archaeal dna polymerase take final concentration as 0.01～0.1U/ μ L carries out pcr amplification, and the dNTPs in this concentration range all can amplify the purpose band as a result, and is the best when Taq enzyme final concentration is 0.05U/ μ L.
(5) response procedures determines
To the cycle number 10,20 of PCR reaction, 30 and 50 ℃～60 ℃ of annealing temperatures carry out pcr amplification, cycle number expanding effect when 30 annealing temperatures are 55 ℃ is best as a result.
PCR test kit of the present invention detects the method for infectious myxoma virus, and step is as follows:
1, the pre-treatment of sample: get the sample of detection, the abundant washing sample of physiological saline that adds 600 μ L sterilization obtains containing virulent suspension;
2, sample DNA extracts: behind above-mentioned sample multigelation, then add dna cleavage liquid, 30min is hatched in 37 ℃ of water-baths; Hatch and take out adding isopyknic balance phenol (pH 8.0), the mixing that turns upside down, the centrifugal 5min of 12000rpm gets supernatant, if protein content is high, can adds balance phenol again and repeat extracting once; Add isopyknic phenol in the supernatant: chloroform: primary isoamyl alcohol, chloroform: primary isoamyl alcohol respectively extracting is once got supernatant; The 3mol/L NaAC (pH 5.2) that in supernatant, adds 1/10 volume, the cold dehydrated alcohol of 2.5 times of volumes is placed 2h or is spent the night for-20 ℃; After the taking-up, the centrifugal 15min of 12000rpm abandons supernatant, and 75% ethanol is washed once; Naturally after drying, extract is dissolved in 10 μ L ultrapure waters, it is for subsequent use to make pcr template.
3, pcr amplification
(1) get the PCR reaction solution according to amplification number n (n=sample number+2), Taq enzyme mixing is in a centrifuge tube, and by every pipe packing, the lid upper tube cap is for subsequent use.
(2) first negative controls is added in the tubulature in minute, the DNA that gets each sample adds in the corresponding reaction tubes, takes out at last positive control and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR instrument.
(3) pcr amplification program: behind 95 ℃ of denaturation 5 min, 95 ℃ of sex change 1 min, 55 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min, should set up the positive and negative control simultaneously;
4, pcr amplification product analysis
Get the sample after 5-10 μ L increases, adopt in the sepharose of 1.2-1.5%, in the ethidium bromide solution of 0.5-1 μ g/mL, dye after electrophoresis 30-40 minute under the 120-150V, under ultraviolet lamp, observe, if there are 1000 bpDNA bands in positive hole negative hole without situation under, sample well has the 1000bpDNA band, contains infectious myxoma virus in the interpret sample, otherwise does not then have.
(3) beneficial effect
Positively effect of the present invention is: test kit preparation rationally, simple, the PCR reaction condition optimization of preparation, high specificity, susceptibility be high, and the result judges objective and accurate, and infectious myxoma virus nucleic acid is had diagnostic effect.
Cardinal principle of the present invention is: with the dna cleavage liquid in the test kit viral DNA in the sample is carried out extracting.The PCR Reagent Tube contains polymerase chain reaction reagent, by add infectious myxoma virus specific DNA fragment primer and
TaqThe compositions such as archaeal dna polymerase carry out specific amplification to the DNA that extracts in the sample.Owing to designed primer is that infectious myxoma virus institute is peculiar, therefore, if contain infectious myxoma virus in the sample, so behind its DNA process amplification and electrophoresis and the ethidium bromide staining, UV-light detects just can detect the band of certain molecular weight, if there is not infectious myxoma virus the band of same molecular weight also just can not occur in the sample.
The present invention has following advantage:
The present invention is at design infectious myxoma virus special primer and successfully set up on the basis of PCR detection method of infectious myxoma virus, further be developed into the PCR detection kit of Simple fast sensitivity, compare with other infectious myxoma virus detection technique, the outstanding feature of the inventive method is: 1) detect fast, can learn detected result in 3-4 hour, and other method such as viral isolation technique need at least 24 hours or one more than week; Be particularly suitable for inspection and quarantining for import/export inspection routine needs; 2) sensitivity that detects is higher, detects the DNA concentration that lower limit is low to moderate 0.3pg; 3) the present invention has guaranteed the accuracy and the specificity that detect according to the peculiar one section conserved sequence design special primer of infectious myxoma virus and the comparison of increasing.
Description of drawings
Fig. 1. PCR test kit specific test, wherein 1, rabbit fibroma virus; 2, tularemia cause of disease; 3, rabbit hemorrhagic disease virus; 4, infectious myxoma virus 5, negative control; 6, Marker DL2000;
Fig. 2. the sensitivity test of PCR test kit, wherein 1, Marker DL2000; 2,3,4,5,6 are respectively 0.1pg, 0.3pg.0.5pg, 0.7pg, 0.9pg;
Fig. 3. the stability test of PCR test kit; Wherein 1 ~ 6 preserved respectively 30,60,90,120,150,180 days; 7, negative control; 8, Marker DL2000;
Fig. 4. the test of PCR test kit preservation condition; Wherein 1, Marker DL2000; 2, negative control; 3～6 preserved respectively 1 month, 3 months, 6 months, 9 months;
Fig. 5. PCR test kit test sample; 1. standard molecular weight DL-2000 wherein; 2. infectious myxoma virus 3.4.5, test sample.
Embodiment
The following example is intended to further describe for example the present invention, rather than limit by any way the present invention, under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that those of ordinary skills that the present invention is done realize easily all will fall within the claim scope that awaits the reply of the present invention.
Infectious myxoma virus M150R gene masculine clone's preparation
The infectious myxoma virus M150R gene order of delivering according to GenBank, precious biological by Dalian Takara() company is synthetic and be cloned in the pMD-T carrier, turns out to be infectious myxoma virus M150R gene by order-checking.Concrete gene order:
atggcttttg acccgctaca cgagtactgt ctggaggagg aacctataca cgtagatacg ttgaaggcgt acctcgccag gtataacccc aacgccgtct actacagaat atccacgttc agacgctatc tacaacgtaa agacgttaga caagatatcg tggaactgtt tatacaacac ggagccgccg taaacggcac cccagaggac aacggaacgc ctctctatac cctgttcgat aacagacgtg ccgtctataa gaacatcgtc gacgtggcca gatgcctgtt gtcgaacggt gcggatatca actgtaaagg tagaggagaa caccccttct gttgtttgct aaagaacccc aaaattaatc acaaggactt tatacaatat ctcattaaaa agtacccggt ggatatttac gtactagacg gcttaacgtt aaacgcgatc cagtgctacg tgcgatctgg aaacgtaaac tttgacgttc tccacttctt catagataac gacgtaccgt acaatagagt aggaccgctt aacacggata tattaatatg gtatatacag tataacgtgc atagattaga cgctaagatt atccaattgc ttatatcgaa gggagtgacg atcacgcagc ataaatatag ggagcattat ttgtatcacg cggccaaggc ctgtataaaa caacaacata gagtcgaatt tgacaaggcg acgttcgatt ttatcctttc ccacatggac gcaaattaca taaacattgt acacaagcag acgttattac agtgcgccat tcaacgggga tacgttcagg cgttcgatta cttactgagc aaaggtgcgg acattcacgt gtgcgacaga ttagccgaaa actgtctaca gatggcgatg ttaaaaggaa acaagtacat cctcaacaag gtcctgtatc gtaccagatt ggacgaatac gaaaaggcgt tcgaacgaat cgatttagat atcatgtacg acggcaaatg cgtctacagc
The composition of embodiment 1 test kit
Test kit contains dna cleavage liquid 15ml, wherein contains NaCl 100mM, and Tris-HCl(pH 8.0) 10mM, EDTA(pH 8.0) and 25mM, SDS 1%(w/v) and Proteinase K 4 μ g/ μ L.PCR reaction tubes (5 pipe) (25 μ L/ reaction * 20), dATP wherein, dTTP, dGTP and dTCP final concentration are 200 μ M, the final concentration of primer M150R-1 and M150R-2 is 0.2 μ M, Mg
2+Final concentration is 2.0mM.Other reagent: 3mol/L NaAC 2mL, phenol, phenol: chloroform: primary isoamyl alcohol, chloroform: each 10mL of primary isoamyl alcohol, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, Taq (5U/ μ L) 10 μ L; The positive plasmid 20 μ L of 1 infectious myxoma virus M150R gene.
The composition of embodiment 2 test kits
Test kit contains dna cleavage liquid 15ml, wherein contains NaCl 100mM, and Tris-HCl(pH 8.0) 10mM, EDTA(pH 8.0) and 25mM, SDS 1%(w/v) and Proteinase K 4 μ g/ μ L.PCR reaction tubes (5 pipe) (25 μ L/ reaction * 20), dATP wherein, dTTP, dGTP and dTCP final concentration are 50 μ M, the final concentration of primer M150R-1 and M150R-2 is 0.1 μ M, Mg
2+Final concentration is 1.5mM.Other reagent: 3mol/L NaAC 2mL, phenol, phenol: chloroform: primary isoamyl alcohol, chloroform: each 10mL of primary isoamyl alcohol, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, Taq (5U/ μ L) 10 μ L; The positive plasmid 20 μ L of 1 infectious myxoma virus M150R gene.
The composition of embodiment 3 test kits
Test kit contains dna cleavage liquid 15ml, wherein contains NaCl 100mM, and Tris-HCl(pH 8.0) 10mM, EDTA(pH 8.0) and 25mM, SDS 1%(w/v) and Proteinase K 4 μ g/ μ L.PCR reaction tubes (5 pipe) (25 μ L/ reaction * 20), dATP wherein, dTTP, dGTP and dTCP final concentration are 400 μ M, the final concentration of primer M150R-1 and M150R-2 is 0.5 μ M, Mg
2+Final concentration is 5.0mM.Other reagent: 3mol/L NaAC 2mL, phenol, phenol: chloroform: primary isoamyl alcohol, chloroform: each 10mL of primary isoamyl alcohol, agarose 10g, ethidium bromide (10 μ g/ μ L) 100 μ L and 6% tetrabromophenol sulfonphthalein point sample damping fluid, 100 μ L, Taq (5U/ μ L) 10 μ L; The positive plasmid 20 μ L of 1 infectious myxoma virus M150R gene.
Following examples, the test kit of employing be among the embodiment 1-3 any.
The specific test of embodiment 4 test kits
Each the 1 μ L of DNA that gets 3 check samples such as rabbit fibroma virus, tularemia cause of disease, rabbit hemorrhagic disease virus is the specific PCR amplification that template is carried out test kit, establishes simultaneously blank, yin and yang attribute contrast.The pcr amplification condition: behind 95 ℃ of denaturation 5 min, 95 ℃ of sex change 1 min, 55 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its specificity.
Electrophoresis result shows that the DNA cloning of infectious myxoma virus sample goes out the specific fragment of 1000bp, and the DNA of sample all occurs without this amplified band in contrast, sees Fig. 1.
The sensitivity test of embodiment 5 test kits
The gradient that pTM150R after quantitative is carried out respectively 10 times is diluted, and detects with the PCR test kit, and amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its susceptibility.Electrophoresis result shows, detects the DNA concentration that lower limit is low to moderate 0.3pg, all can amplify clear and legible band, sees Fig. 2.
The stability test of embodiment 6 test kits
Except Taq enzyme, PCR reaction solution, outside the positive template-20 ℃ storage, lysate and all the other reagent thereof are all preserved under 4 ℃ of conditions.When being 30,60,90,120,150,180 days, period of storage takes out, with the stability of known PCR positive plasmid detection kit.Amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its stability.The result shows at 30,60,90,120,150,180 days day parts and takes out respectively test kit, carries out pcr amplification with the infectious myxoma virus positive plasmid, all can amplify bright band, and specific band nothing but, and blank is all negative, sees Fig. 3.
The preservation condition test of embodiment 7 test kits
To positive plasmid be detected at the test kit (the Taq enzyme is-20 ℃ of preservations all the time) that room temperature, 4 ℃ and-20 ℃ were preserved 1 month, 3 months, 6 months, 9 months.Amplification condition is the same, establishes simultaneously blank.Amplified production carries out electrophoretic analysis with 1.0% agarose, to determine its preservation condition.The result shows that this test kit (except the Taq enzyme) can preserve 6 months at least under room temperature, 4 ℃ and-20 ℃, all can amplify bright band, and specific band nothing but, and blank is all negative.Taq enzyme in the suggestion test kit, the PCR reaction solution, positive template-20 ℃ storage, lysate and all the other reagent thereof are all preserved under 4 ℃ of conditions, and the effect that detects like this can be better, sees Fig. 4.
Gathered certain plant's rabbit brush,throat and ight soil swab sample, and totally 121 parts of rabbit product samples, numbering, it is to be checked then to put into 4 ℃ of refrigerators.
1, the pre-treatment of sample: get the sample of detection, the abundant washing sample of physiological saline that adds 600 μ L sterilization obtains containing virulent suspension;
2, sample DNA extracts: behind above-mentioned sample multigelation, then add dna cleavage liquid, 30min is hatched in 37 ℃ of water-baths; Hatch and take out adding isopyknic balance phenol (pH 8.0), the mixing that turns upside down, the centrifugal 5min of 12000rpm gets supernatant, if protein content is high, can adds balance phenol again and repeat extracting once; Add isopyknic phenol in the supernatant: chloroform: primary isoamyl alcohol, chloroform: primary isoamyl alcohol respectively extracting is once got supernatant; The 3mol/L NaAC (pH 5.2) that in supernatant, adds 1/10 volume, the cold dehydrated alcohol of 2.5 times of volumes is placed 2h or is spent the night for-20 ℃; After the taking-up, the centrifugal 15min of 12000rpm abandons supernatant, and 75% ethanol is washed once; Naturally after drying, extract is dissolved in 10 μ L ultrapure waters, it is for subsequent use to make pcr template.
3, pcr amplification
(1) get the PCR reaction solution according to amplification number n (n=sample number+2), Taq enzyme mixing is in a centrifuge tube, and by every pipe packing, the lid upper tube cap is for subsequent use.
(2) first negative controls is added in the tubulature in minute, the DNA that gets each sample adds in the corresponding reaction tubes, takes out at last positive control and adds in another reaction tubes, and is centrifugal behind each reaction tubes mark, takes out and puts on the PCR instrument.
(3) pcr amplification program: behind 95 ℃ of denaturation 5 min, 95 ℃ of sex change 1 min, 55 ℃ of renaturation 1 min, 72 ℃ are extended 1 min, so carry out 30 circulations; Last 72 ℃ are extended 10 min, should set up the positive and negative control simultaneously;
4, pcr amplification product analysis
Get the sample after 5-10 μ L increases, adopt in the sepharose of 1.2-1.5%, in the ethidium bromide solution of 0.5-1 μ g/mL, dye after electrophoresis 30-40 minute under the 120-150V, under ultraviolet lamp, observe, the results are shown in Figure 5.
As seen from Figure 5: there is the 1000bpDNA band in positive hole, negative hole without, sample well has no the 1000bpDNA band, does not have infectious myxoma virus in the interpret sample.
Detected result: do not detect infectious myxoma virus in institute's sample thief.
SEQUENCE LISTING
＜110 〉
Jiangsu Bureau of Emigration ﹠ Ingression Examination ﹠. Quarantine, PRC
＜120〉infectious myxoma virus PCR detection kit and application thereof
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 1
atggcttttg acccgctaca cgag 24
<210> 2
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 2
catttgccgt cgtacatgat atct 24
<210> 3
<211> 1020
<212> DNA
＜213〉artificial sequence
<400> 3
atggcttttg acccgctaca cgagtactgt ctggaggagg aacctataca cgtagatacg 60
ttgaaggcgt acctcgccag gtataacccc aacgccgtct actacagaat atccacgttc 120
agacgctatc tacaacgtaa agacgttaga caagatatcg tggaactgtt tatacaacac 180
ggagccgccg taaacggcac cccagaggac aacggaacgc ctctctatac cctgttcgat 240
aacagacgtg ccgtctataa gaacatcgtc gacgtggcca gatgcctgtt gtcgaacggt 300
gcggatatca actgtaaagg tagaggagaa caccccttct gttgtttgct aaagaacccc 360
aaaattaatc acaaggactt tatacaatat ctcattaaaa agtacccggt ggatatttac 420
gtactagacg gcttaacgtt aaacgcgatc cagtgctacg tgcgatctgg aaacgtaaac 480
tttgacgttc tccacttctt catagataac gacgtaccgt acaatagagt aggaccgctt 540
aacacggata tattaatatg gtatatacag tataacgtgc atagattaga cgctaagatt 600
atccaattgc ttatatcgaa gggagtgacg atcacgcagc ataaatatag ggagcattat 660
ttgtatcacg cggccaaggc ctgtataaaa caacaacata gagtcgaatt tgacaaggcg 720
acgttcgatt ttatcctttc ccacatggac gcaaattaca taaacattgt acacaagcag 780
acgttattac agtgcgccat tcaacgggga tacgttcagg cgttcgatta cttactgagc 840
aaaggtgcgg acattcacgt gtgcgacaga ttagccgaaa actgtctaca gatggcgatg 900
ttaaaaggaa acaagtacat cctcaacaag gtcctgtatc gtaccagatt ggacgaatac 960
gaaaaggcgt tcgaacgaat cgatttagat atcatgtacg acggcaaatg cgtctacagc 1020
Claims (6)
1. infectious myxoma virus PCR detection kit is characterized in that it contains:
(1), dna cleavage liquid;
(2), the PCR reaction solution, it contains the Auele Specific Primer of each 0.1-0.5 μ M of final concentration:
M150R-1: 5'- atggcttttg acccgctacacgag-3'
M150R-2: 5'- catttgccgt cgtacatgatatct-3'
(3), the positive plasmid pTM150R of infectious myxomatosis M150R gene
(4) Taq enzyme.
2. a kind of infectious myxoma virus PCR detection kit according to claim 1 is characterized in that dna cleavage liquid wherein is NaCl, Tris-HCl, EDTA, the mixing solutions of SDS and Proteinase K.
3. a kind of infectious myxoma virus PCR detection kit according to claim 1, it is characterized in that, dna cleavage liquid wherein is: the NaCl of 100mM, the Tris-HCl of the 10mM of pH 8.0, the EDTA of the 25mM of pH 8.0, volume ratio content is the mixing solutions of the Proteinase K of 1% SDS and 4 μ g/ μ L by weight.
4. a kind of infectious myxoma virus PCR detection kit according to claim 1 is characterized in that, PCR reaction solution wherein also contains the dATP of each 50～400 μ M of final concentration, dTTP, dGTP and dTCP, the Mg of 1.5～5.0mM
2+
5. a kind of infectious myxoma virus PCR detection kit according to claim 4 is characterized in that wherein dATP in the PCR reaction solution, and dTTP, dGTP and dTCP final concentration are 200 μ M, and the final concentration of primer M150R-1 and M150R-2 is 0.2 μ M, Mg
2+Final concentration is 2.0mM.
6. described a kind of infectious myxoma virus PCR detection kit according to claim 1 is characterized in that described Taq enzyme is 1U.
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