CN102586479A - Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method - Google Patents
Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method Download PDFInfo
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Abstract
The invention relates to the technical field of avian reovirus detection, in particular to primers for loop-mediated isothermal amplification of the avian reoviruses, comprising three pairs of specific primers, and the sequences of the primers are shown in a sequence list. A detection kit of the avian reoviruses comprises loop-mediated isothermal amplification reaction liquid which contains the three pairs of specific primers. A detection method of the avian reoviruses is characterized in that the detection kit is used for carrying out the loop-mediated isothermal amplification reaction on an RNA sample, a positive control group and a negative control group are set, and the conditions of the loop-mediated isothermal amplification reaction are that the temperature is 59-65 DEG C, reaction time is 30-75 minutes. The detection kit of the avian reoviruses has high specificity and sensitivity, and by using the detection kit, 100 copies of RNA can be detected, the operation is simple, and observation is convenient.
Description
Technical field
The present invention relates to Avianreovirus detection technique field, the particularly primer of ring mediated isothermal amplification Avianreovirus also relates to a kind of detection kit of Avianreovirus, also relates to the detection method of said Avianreovirus.
Background technology
(Avian reoviruses ARV) belongs to the Reoviridae Orthoreovirus to Avianreovirus, multiple birds such as main infected chicken, turkey, duck, goose.Avianreovirus mainly causes bird sacroiliitis, tenosynovitis, also can cause growth retardation, pericarditis, myocarditis, hepatitis, osteoporosis and acute and chronic respiratory tract disease etc. simultaneously.In addition, the polyinfection of Avianreovirus and other immunosuppressive disease is ubiquity in the chicken crowd, causes chicken poor growth, price of deed rate low, influences immune effect, causes enormous economic loss to aviculture.
At present; The Avianreovirus diagnostic method has a lot, mainly contains immune agar diffusion (AGP) test, enzyme linked immunological absorption (ELISA) test, immunoblotting (IBT), polymerase chain reaction (PCR) and real time fluorescent quantitative (real-time PCR) etc.But aforesaid method exist the test period length, complex operation, examined materials limitations, to the high different shortcomings of laboratory apparatus and testing staff's technical requirements, therefore be not suitable for laboratory applications in basic unit.
Dong Jiawen, Sun Minhua etc. mention 4 primers that designed 6 constant gene segment Cs of special corresponding target sequence according to the Avianreovirus gene order in " Chinese animal doctor's journal " July the 31st in 2011 in volume the 7th interim article of delivering " foundation of Avianreovirus RT-LAMP method for quick ", and the reaction conditions of this method is optimized.But disclosed detection method sensitivity is low in this article, and lower limit of detectability is 4ELD50, and uses SYBR Green I to be optical dye, very easily causes crossed contamination.
Summary of the invention
In order to solve the low and easy pollution problems of the sensitivity that exists in the above LAMP method detection, the invention provides the primer of the high ring mediated isothermal amplification Avianreovirus of a kind of detection sensitivity.
The present invention also provides a kind of detection kit that can effectively avoid the crossed contamination Avianreovirus.
Another object of the present invention has provided the detection method of Avianreovirus.
The present invention realizes in the following manner:
A kind of ring mediated isothermal amplification (loop-mediated isothermal amplification, the LAMP) primer of Avianreovirus, comprise altogether outside primer to, inboard primer to annular primer to three pairs of Auele Specific Primers, sequence is following:
A pair of primer is can be right with p10 gene (sequence 1 in the sequence table) bonded outside primer in the Avianreovirus S1133 strain (GeneBank Accession Number AF330703):
Upstream primer F3:5 '-ATGCTGCGTATGCCTCCC-3 ' sees sequence 2 in the sequence table,
Downstream primer B3:5 '-CCAGCTCACGATGGAAGAC-3 ' sees sequence 3 in the sequence table,
A pair of primer be can with Avianreovirus S1133 strain (the inboard primer of p10 gene bonded is right among the GeneBank Accession Number AF3307030:
Upstream primer FIP:
5 '-ACGTAGCTTGCAAATCACCACCTTTTGTGTAACGGTGCGACTGCT-3 ' sees sequence 4 in the sequence table,
Downstream primer BIP:
5 '-CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3 ' sees sequence 5 in the sequence table,
A pair of primer is can be right with p10 gene bonded annular primer in the Avianreovirus S1133 strain (9GeneBank Accession Number AF3307030):
Upstream primer LF:5 '-GCCTGACAATGAACGTTACC-3 ' sees sequence 6 in the sequence table,
Downstream primer LB:5 '-AGCGGCGGGTGGTGGTTTC-3 ' sees sequence 7 in the sequence table,
Outside primer is 1:8:4 to, inboard primer to, the right mol ratio of annular primer.
Eight zones of primer identification are to mate all bird reovirus p10 gene conservative districts in recent years; So primer can be discerned all bird reovirus p10 genes; Described outside primer to, inboard primer to annular primer to doing as a whole use, should avoid the formation of primer dimer during use as far as possible.
A kind of detection kit of avian infectious bronchitis virus comprises loop-mediated isothermal amplification liquid, contains above-mentioned three pairs of Auele Specific Primers in the loop-mediated isothermal amplification liquid.
Described detection kit; Contain outside primer in per 24 μ L loop-mediated isothermal amplification liquid to upstream primer and each 5pmol of downstream primer; Inboard primer is to upstream primer and each 40pmol of downstream primer, and annular primer is to upstream primer and each 20pmol of downstream primer.
Described detection kit also contains Tris-HCl 20mM, KCl 10mM, (NH in per 24 μ L loop-mediated isothermal amplification liquid
4)
2SO
410mM, MgSO
44-16mM, Tween20 0.1%, fluorexon 0.05 mM, MnCl
20.6mM, AMV ThermoScript II 0.2U, trimethyl-glycine 0.8M, deoxynucleotide dNTPs 1.4mM, Bst polysaccharase 8U.
Described detection kit also contains Tris-HCl 20mM, KCl 10mM, (NH in per 24 μ L loop-mediated isothermal amplification liquid
4)
2SO
410mM, MgSO
48mM, Tween20 0.1%, fluorexon 0.05 mM, MnCl
20.6mM, AMV ThermoScript II 0.2U, trimethyl-glycine 0.8M, deoxynucleotide dNTPs 1.4mM, Bst polysaccharase 8U.
Described detection kit also comprises fluorescent color-developing agent in the described detection kit.
Described detection kit, fluorescent color-developing agent are fluorexon and MnCl
2
A kind of detection method of avian infectious bronchitis virus; Use described detection kit that sample RNA is carried out loop-mediated isothermal amplification; Positive controls and negative control group are set simultaneously, and said loop-mediated isothermal amplification condition is: 59-65 ℃, 30-75 minute.
Described detection method, the sample that will finish loop-mediated isothermal amplification reacted 2 minutes under 80 ℃ of conditions.
Use test kit provided by the invention and detect Avianreovirus, can whether contain Avianreovirus through directly inspecting judgement sample.Directly inspection method is:
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.If the reaction tubes of sample to be checked is housed the bright green visible fluorescence is arranged, explain that then the Avianreovirus detected result is positive in the sample to be checked.Still do not have fluorescence liquid if the reaction tubes of sample to be checked is housed, explain that then the Avianreovirus detected result is negative in the sample to be checked for pale brown look.
It is following that Avianreovirus detection kit provided by the invention detects principle:
8 particular combination zones of described primer sets and described Avianreovirus AF330703 sequence p10 gene combine, and see table 1.
Table 1 primer sets and described Avianreovirus AF330703 sequence p10 gene calmodulin binding domain CaM
Above-mentioned primer sets can be accomplished the amplification to template ribonucleic acid under the effect of AMV ThermoScript II and Bst archaeal dna polymerase.Amplification procedure is divided into three phases, and is specific as follows:
(1) the reverse transcription stage
Under the effect of AMV ThermoScript II, sample rna is inverted records into cDNA.
(2) circulation initial period
The inboard primer of one end combines prior to template and starts DNA to synthesize.The outside primer of mutually same end combines to start DNA subsequently with template synthetic, and the generation strand displacement discharges the dna single chain that contains inboard primer sequence.This single stranded DNA successively combines with the inboard primer and the outside primer of the other end as template, starts synthetic merging of DNA strand displacement to take place, and forms an initial stem circular DNA at last.
(3) the reaction cycle stage
Inboard primer combines with initial stem circular DNA, and it is synthetic to start strand displacement DNA, can produce the stem circular DNA of another an initial stem circular DNA and a new double length.It is synthetic that the template that can be used as these stem circular DNAs continues to combine to start strand displacement DNA with inboard primer, and each synthesizing all can make the stem length of stem circular DNA be doubled and redoubled.
After getting into the cycle stage, can produce the different stem circular DNA of many molecular sizes, these stem circular DNAs can also combine with annular primer as template, start the synthetic and generation strand displacement of more DNA.Through one hour isothermal duplication, goal gene can add up 10 at last
9Copy.
Beneficial effect of the present invention: use Avianreovirus detection kit provided by the invention and detect, specificity and susceptibility are high, can detect the RNA of 100 copies.Compare with conventional P CR detection method, use Avianreovirus detection kit provided by the invention and detect, do not need expensive instrument, only need the ortho-water bath to get final product, and detected result can be through visual inspection fluorescence, simple to operate, it is convenient to observe.The detection of carrying Avianreovirus in Clinical Veterinary Medicine that this test kit can be applicable to carry out at the basic unit scene and the food.
Description of drawings
Fig. 1 is the gel electrophoresis spectrogram of embodiment 1 amplification,
Wherein: M:Marker III, 1:0mM Mg
2+, 2:4mM Mg
2+, 3:8mM Mg
2+, 4:12mM Mg
2+, 5:16mM Mg
2+ Embodiment
Below in conjunction with specific embodiment, further illustrate related content of the present invention.Not marked concrete TP in the following instance, usually according to the condition described in the molecular cloning laboratory manual, or the condition that provides according to manufacturer.
Detection method provided by the invention comprises the steps:
(1) extraction of RNA
Test kit provided by the invention can be used for detecting the Avianreovirus in various internal organs, the secretory product, like nasopharyngeal secretions, lungs, kidney, ight soil etc.The total RNA of different sources extracts can be with reference to corresponding data, or uses corresponding RNA to extract test kit.
(2) isothermal duplication of ring mediation
1. in the reaction tubes of the LAMP reaction solution that 24 μ L are housed, add 1 μ L template ribonucleic acid;
2. on water-bath or metal constent temperature heater 59-65 ℃ placed 30-75 minute, take out.
After 2. step was accomplished, before the taking-up, water transfer bath temperature to 80 ℃ reaction was 2 minutes again, and purpose is can termination reaction, so that the prolonged preservation reaction result.
(3) result judges
Can whether contain Avianreovirus through directly inspecting judgement sample.
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.Still do not have fluorescence liquid if the reaction tubes of detected sample is housed, explain that then the Avianreovirus detected result is negative in the sample to be checked for pale brown look; If the sample pipe of detected sample is housed the bright green visible fluorescence is arranged, then the Avianreovirus detected result is positive in the interpret sample.
The preparation of embodiment 1, Avianreovirus detection kit
One, primer is synthetic
3 pairs of primers below the synthetic, outside primer is right:
Upstream primer F3:5 '-ATGCTGCGTATGCCTCCC-3 ',
Downstream primer B3:5 '-CCAGCTCACGATGGAAGAC-3 ',
Inboard primer is right:
Upstream primer FIP:
5’-?ACGTAGCTTGCAAATCACCACCTTTTGTGTAACGGTGCGACTGCT-3’,
Downstream primer BIP:
5’-?CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3’,
Annular primer is right:
Upstream primer LF:5 '-GCCTGACAATGAACGTTACC-3 ',
Downstream primer LB:5 '-AGCGGCGGGTGGTGGTTTC-3 ',
Two, preparation LAMP reaction solution
Per 24 μ L LAMP reaction solutions contain following component: 20mM Tris-HCl, 10mM KCl, 10mM (NH
4)
2SO
4, 4-16mM MgSO
4, 0.1% Tween20,0.05 mM fluorexon, 0.6mM MnCl
2, 0.2U AMV ThermoScript II, 0.8M trimethyl-glycine, 1.4mM deoxynucleotide dNTPs, 8U Bst polysaccharase, the outside, 5pmol upper reaches primer (F3), the outside, 5pmol downstream primer (B3), the inboard primer (FIP) in the 40pmol upper reaches, 40pmol downstream interior side primer (BIP), 20pmol upper reaches annular primer, 20pmol downstream annular primer.
Three, the assembling of test kit
This test kit is made up of following material: the LAMP reaction solution of step 2 preparation, Avianreovirus RNA (positive control) (ARV/S1133 strain), sterilization distilled water (negative control), reaction tubes.
Mg in the reaction
2+Concentration is the important factor that influences amplification efficiency.Mg
2+Concentration is low more, and atopic is strong more, but amplification efficiency reduces; Mg
2+Concentration is high more, and the efficient of reaction increases, but specificity weakens.In order to confirm MgSO
4The best add concentration, prepare MgSO respectively
4Concentration is respectively the LAMP reaction solution of 4mM, 8 mM, 12 mM and 16 mM, on water-bath or metal constent temperature heater, places 60 minutes for 60 ℃, takes out, and carries out the gel electrophoresis test, and the result sees Fig. 1, and embodiment result shows, MgSO in the LAMP reaction system
4The most suitable during for 8mM, so test kit MgSO
4The preferred 8mM of concentration.
MgSO in the LAMP reaction solution that uses in following examples and sensitivity, the specific detection
4Concentration is 8mM.
Get two chickens, 1 is diagnosed as the sick chicken that Avianreovirus infects, 1 healthy chicken.Respectively 2 chickens are sampled with the cloaca cotton swab.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Collect the cloaca cotton swab respectively, in centrifuge tube, wash cotton swab repeatedly with the phosphate buffered saline buffer of 0.2mol/L pH7.4, take out cotton swab after, the centrifugal 1min of 500Orpm gets supernatant, obtains the detection liquid of 2 chickens.
The operation of extracting RNA is existing technology, only enumerates wherein a kind of concrete operations, as follows, but is not restricted to this kind operation at present:
In centrifuge tube, add 250 μ L and detect liquid and 750 μ L TRIzol Reagent, thermal agitation, ice bath 5min; Add chloroform 200 μ L, ice bath 5min; 12000rpm, centrifugal 10min under 4 ℃ of conditions, the water intaking phase is in another centrifuge tube; Add Virahol 500 μ L (20 ℃ of precoolings), hatch 10min under-20 ℃; 12000rpm, centrifugal 10min under 4 ℃ of conditions abandons supernatant; In deposition, adding 1mL 75% ethanol (preparation of 0.1%DEPC water) washs; 12000rpm, centrifugal 5min under 4 ℃ of conditions behind the repeated washing, abandons supernatant again; To precipitate room temperature and dry 10min, and with resolution of precipitate, and under 55-60 ℃, hatch 10min with 0.1%DEPC water, prepared RNA should detect or be stored in-80 ℃ immediately and is equipped with inspection.
Two, ring mediated isothermal amplification
In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains the disease chicken, as test set 1; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains healthy chicken, as test set 2; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add 1 μ L positive control, as positive controls; At 3 LAMP reaction solutions that 24 μ L are housed
Reaction tubes in respectively add 1 μ L sterilization distilled water, as negative control group.
Above-mentioned reaction tubes simultaneously on water-bath 59 ℃ placed 75 minutes, take out.
Three, inspect
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.Three reaction tubess of healthy chicken test set are pale brown look, and three reaction tubess of sick chicken test set all have the bright green visible fluorescence.
Get two chickens, 1 is diagnosed as the sick chicken that Avianreovirus infects, 1 healthy chicken.Respectively 2 chickens are sampled with the cloaca cotton swab.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Two, ring mediated isothermal amplification
In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains the disease chicken, as test set 1; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains healthy chicken, as test set 2; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add 1 μ L positive control, as positive controls; At 3 LAMP reaction solutions that 24 μ L are housed
Reaction tubes in respectively add 1 μ L sterilization distilled water, as negative control group.
Above-mentioned reaction tubes simultaneously on water-bath 65 ℃ placed 60 minutes, take out.
Three, inspect
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.Three reaction tubess of healthy chicken test set are pale brown look, and three reaction tubess of sick chicken test set all have the bright green visible fluorescence.
Get two chickens, 1 is diagnosed as the sick chicken that Avianreovirus infects, 1 healthy chicken.Respectively 2 chickens are sampled with the cloaca cotton swab.Adopt the detection kit of embodiment 1 preparation that sample is detected.
Two, extract RNA
Two, ring mediated isothermal amplification
In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains the disease chicken, as test set 1; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains healthy chicken, as test set 2; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add 1 μ L positive control, as positive controls; At 3 LAMP reaction solutions that 24 μ L are housed
Reaction tubes in respectively add 1 μ L sterilization distilled water, as negative control group.
Above-mentioned reaction tubes simultaneously on water-bath 63 ℃ placed 45 minutes, take out.
Three, inspect
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.Three reaction tubess of healthy chicken test set are pale brown look, and three reaction tubess of sick chicken test set all have the bright green visible fluorescence.
Get two chickens, 1 is diagnosed as the sick chicken that Avianreovirus infects, 1 healthy chicken.Respectively 2 chickens are sampled with the cloaca cotton swab.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Two, ring mediated isothermal amplification
In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains the disease chicken, as test set 1; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add the RNA sample that 1 μ L step 1 obtains healthy chicken, as test set 2; In the reaction tubes of 3 LAMP reaction solutions that 24 μ L are housed, respectively add 1 μ L positive control, as positive controls; At 3 LAMP reaction solutions that 24 μ L are housed
Reaction tubes in respectively add 1 μ L sterilization distilled water, as negative control group.
Above-mentioned reaction tubes simultaneously on water-bath 65 ℃ placed 30 minutes, take out.
Three, inspect
The reaction tubes that positive control is housed has the bright green visible fluorescence, and the reaction tubes that negative control is housed does not still have fluorescence liquid for pale brown look.Three reaction tubess of healthy chicken test set are pale brown look, and three reaction tubess of sick chicken test set all have the bright green visible fluorescence.
Detection sensitivity and specificity evaluation
One, sensitivity
Preparation 10
10Copy/μ L, 10
9Copy/μ L, 10
8Copy/μ L, 10
7Copy/μ L, 10
6Copy/μ L, 10
5Copy/μ L, 10
4Copy/μ L, 10
3Copy/μ L, 10
2Copy/μ L, 10
1Copy/μ L contains the plasmid sample of Avianreovirus p10 gene.Adopt the detection kit of embodiment 1 preparation that the plasmid sample is detected.
1, the preparation of plasmid
Extract the RNA of Avianreovirus S1133 strain, reverse transcription obtains DNA, uses the p10 gene fragment that the amplification of RT-PCR method obtains this strain.Again with the plasmid that promptly obtains containing the p10 gene after the T carrier is connected, the p10 gene of a corresponding copy of plasmid molecule.Change plasmid over to competent cell, under appropriate condition, cultivate competent cell, plasmid can duplicate along with the breeding of competent cell.From competent cell, extract purifying p10 gene plasmid at last.
Copy number concentration measured and calculated to the plasmid that obtains can through spectrophotometer method.With distilled water plasmid is diluted to needed concentration, promptly 10
10Copy/μ L, 10
9Copy/μ L, 10
8Copy/μ L, 10
7Copy/μ L, 10
6Copy/μ L, 10
5Copy/μ L, 10
4Copy/μ L, 10
3Copy/μ L, 10
2Copy/μ L, 10
1Copy/μ L.Add each concentration plasmid 1 μ L during detection.(can be with reference to " molecular cloning test guide " third edition, needed reagent and instrument are that the market can buy.)
2, respectively the plasmid sample of above-mentioned each concentration is detected, it is following to detect step:
(1) ring mediated isothermal amplification
Get step 1 and obtain 10
10Copy/μ L, 10
9Copy/μ L, 10
8Copy/μ L, 10
7Copy/μ L, 10
6Copy/μ L, 10
5Copy/μ L, 10
4Copy/μ L, 10
3Copy/μ L, 10
2Copy/μ L, 10
1Each 1 μ L of the plasmid sample of copy/μ L adds to respectively in 3 sample pipes, and every pipe adds the LAMP reaction solution of 24 μ L again, respectively as test set 1,2,3,4,5,6,7,8,9,10.Sterilization distilled water with equal volume replaces the plasmid sample to be equipped with negative control with legal system, and other gets 3 positive controls, adds the LAMP reaction solution of 24 μ L.
Above-mentioned reaction tubes simultaneously on water-bath 63 ℃ placed 60 minutes, water transfer bath temperature to 80 ℃ reaction 2min takes out.
(2) directly inspect
The result shows that three reaction tubess of positive controls all have the bright green visible fluorescence, and three reaction tubess of negative control group are pale brown look is not had fluorescence liquid.Three reaction tubess of test set 1 all have the bright green visible fluorescence; Three reaction tubess of test set 2 all have the bright green visible fluorescence, and three reaction tubess of test set 3 all have the bright green visible fluorescence, and three reaction tubess of test set 4 all have the bright green visible fluorescence; Three reaction tubess of test set 5 all have the bright green visible fluorescence; Three reaction tubess of test set 6 all have the bright green visible fluorescence, and three reaction tubess of test set 7 all have the bright green visible fluorescence, and three reaction tubess of test set 8 all have the bright green visible fluorescence; Three reaction tubess of test set 9 all have the bright green visible fluorescence, and three reaction tubess of test set 10 are pale brown look is not had fluorescence liquid.
It is thus clear that the detection kit of Avianreovirus of the present invention can detect 10
2The RNA of copy/μ L, detection sensitivity is very high, can detect accurately Avianreovirus.
Two, specificity
Extract Avianreovirus RNA sample, and with NDV La Sota strain (
Newcastle Disease Virus, NDV), the H9N2 subtype avian influenza virus (
Avian Influenza Virus, AIV), avian infectious bronchitis virus (
Infectious Bronchitis Virus, IBV), ILTV (
Infectious laryngotracheitis Virus, ILTV), egg-decreasing syndrome virus (
Egg Drop Syndrome Virus, EDS), escherichia coli (
E.coli) be the contrast cause of disease, adopt the test kit of embodiment 6 preparations that avian infectious bronchitis virus RNA sample and contrast cause of disease nucleic acid are carried out specific detection.
1, the extraction of nucleic acid
(1) extracts RNA
Extract the RNA of Avianreovirus, NDV, H9N2 subtype avian influenza virus, avian infectious bronchitis virus, step is with the step 1 of embodiment 5.
(2) extract DNA
Extract the DNA of ILTV, egg-decreasing syndrome virus and escherichia coli.Get 3 kinds of cause of disease liquid cultures and carry out the extraction of nucleic acid DNA, step is following:
Get 500 μ L nutrient solutions and add the SDS of 25 μ L10% and the Proteinase K of 10 μ L 20mg/mL, concussion evenly; Water-bath is 2 hours under 56 ℃ of conditions; Add the saturated phenol of 200 μ L Tris, the centrifugal 10min of 12000rpm behind the mixing; Get the upper strata water, add 100 saturated phenol of μ L Tris and 100 μ L chloroforms, mixing, the centrifugal 10min of 12000rpm; The upper water phase transition in new centrifuge tube, is added 200 μ L chloroforms, vibration, the centrifugal 10min of 12000rpm; Shift the new centrifuge tube of upper strata water to, add isopyknic Virahol ,-20 ℃ of centrifugal 10min of 12000rpm after freezing 10 minutes; Abandon supernatant, add 500 μ L, 70% ethanol, the centrifugal 2min of 12000rpm; Abandon supernatant, room temperature adds 50 μ L water dissolution DNA after placing 30min.
2, respectively above-mentioned cause of disease sample nucleic acid is detected, it is following to detect step:
(1) ring mediated isothermal amplification
Get RNA and ILTV, the egg-decreasing syndrome virus of H9N2 subtype avian influenza virus that step 1 obtains, NDV, Avianreovirus, avian infectious bronchitis virus, each 1 μ L of DNA of escherichia coli; Add to respectively in 3 sample pipes; Every pipe adds the LAMP reaction solution (detector tube of ILTV, egg-decreasing syndrome virus, escherichia coli dna then water replaces AMV wherein) of 24 μ L again, respectively as test set 1,2,3,4,5,6,7.Sterilization distilled water with equal volume replaces sample of nucleic acid to be equipped with negative control with legal system, and other gets 3 positive controls, adds the LAMP reaction solution of 24 μ L.
Above-mentioned sample pipe simultaneously on water-bath 65 ℃ placed 60 minutes, water transfer bath temperature to 80 ℃ reaction 2min takes out.
(2) directly inspect
The result shows that three reaction tubess of positive controls all have the bright green visible fluorescence, and three reaction tubess of negative control group are pale brown look is not had fluorescence liquid.Three reaction tubess of test set 1 are pale brown look is not had fluorescence liquid; Three reaction tubess of test set 2 are pale brown look is not had fluorescence liquid; Three reaction tubess of test set 3 all have the bright green visible fluorescence, and three reaction tubess of test set 4 are pale brown look is not had fluorescence liquid, and three reaction tubess of test set 5 are pale brown look is not had fluorescence liquid; Three reaction tubess of test set 6 are pale brown look is not had fluorescence liquid, and three reaction tubess of test set 7 are pale brown look is not had fluorescence liquid.
It is thus clear that, the detection kit of Avianreovirus of the present invention to NDV La Sota strain (
Newcastle Disease Virus, NDV), the H9N2 subtype avian influenza virus (
Avian Influenza Virus, AIV), avian infectious bronchitis virus (
Infectious Bronchitis Virus, IBV), ILTV (
Infectious laryngotracheitis Virus, ILTV), egg-decreasing syndrome virus (
Egg Drop Syndrome Virus, EDS), escherichia coli (
E.coli) all there is not amplification, have excellent specificity, can detect accurately Avianreovirus.
< 110>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul
< 120>detection kit of the primer of ring mediated isothermal amplification Avianreovirus, Avianreovirus and detection method
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Claims (9)
1. the primer of a ring mediated isothermal amplification Avianreovirus, it is characterized in that comprising altogether outside primer to, inboard primer to annular primer to three pairs of Auele Specific Primers, sequence is following:
Outside primer is right:
Upstream primer F3:5 '-ATGCTGCGTATGCCTCCC-3 ',
Downstream primer B3:5 '-CCAGCTCACGATGGAAGAC-3 ',
Inboard primer is right:
Upstream primer FIP:
5’-?ACGTAGCTTGCAAATCACCACCTTTTGTGTAACGGTGCGACTGCT-3’,
Downstream primer BIP:
5’-?CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3’,
Annular primer is right:
Upstream primer LF:5 '-GCCTGACAATGAACGTTACC-3 ',
Downstream primer LB:5 '-AGCGGCGGGTGGTGGTTTC-3 ',
Outside primer is 1:8:4 to, inboard primer to, the right mol ratio of annular primer.
2. the detection kit of an avian infectious bronchitis virus is characterized in that comprising loop-mediated isothermal amplification liquid, contains three pairs of Auele Specific Primers in the claim 1 in the loop-mediated isothermal amplification liquid.
3. detection kit according to claim 2; It is characterized in that containing in per 24 μ L loop-mediated isothermal amplification liquid outside primer to upstream primer and each 5pmol of downstream primer; Inboard primer is to upstream primer and each 40pmol of downstream primer, and annular primer is to upstream primer and each 20pmol of downstream primer.
4. according to claim 2 or 3 described detection kit, it is characterized in that also containing Tris-HCl 20mM, KCl 10mM, (NH in per 24 μ L loop-mediated isothermal amplification liquid
4)
2SO
410mM, MgSO
44-16mM, Tween20 0.1%, fluorexon 0.05 mM, MnCl
20.6mM, AMV ThermoScript II 0.2U, trimethyl-glycine 0.8M, deoxynucleotide dNTPs 1.4mM, Bst polysaccharase 8U.
5. according to claim 2 or 3 described detection kit, be characterised in that in per 24 μ L loop-mediated isothermal amplification liquid and also contain Tris-HCl 20mM, KCl 10mM, (NH
4)
2SO
410mM, MgSO
48mM, Tween20 0.1%, fluorexon 0.05 mM, MnCl
20.6mM, AMV ThermoScript II 0.2U, trimethyl-glycine 0.8M, deoxynucleotide dNTPs 1.4mM, Bst polysaccharase 8U.
6. according to claim 2 or 3 described detection kit, be characterised in that also to comprise fluorescent color-developing agent in the described detection kit.
7. detection kit according to claim 6 is characterised in that fluorescent color-developing agent is fluorexon and MnCl
2
8. the detection method of an avian infectious bronchitis virus; It is characterized in that using claim 2 or 3 described detection kit that sample RNA is carried out loop-mediated isothermal amplification; Positive controls and negative control group are set simultaneously, and said loop-mediated isothermal amplification condition is: 59-65 ℃, 30-75 minute.
9. detection method according to claim 8 is characterized in that the sample that finishes loop-mediated isothermal amplification was reacted 2 minutes under 80 ℃ of conditions.
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| CN103184298A (en) * | 2012-08-09 | 2013-07-03 | 中国农业科学院上海兽医研究所 | Primer for detecting infectious bronchitis viruses, detection method and kit |
| CN106119423A (en) * | 2016-08-31 | 2016-11-16 | 中国农业大学 | The general PCR primer of detection Avianreovirus and detection kit thereof |
| CN107058615A (en) * | 2017-01-17 | 2017-08-18 | 佛山科学技术学院 | A kind of calcein visualization RT LAMP kits and its application |
| CN108411041A (en) * | 2018-05-22 | 2018-08-17 | 山东农业大学 | A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application |
| CN109022618A (en) * | 2018-08-22 | 2018-12-18 | 上海实验动物研究中心 | A kind of primer set and its application for quickly detecting 3 type of mouse reovirus |
| CN111926113A (en) * | 2020-07-14 | 2020-11-13 | 安阳工学院 | Primer group, probe, kit and method for rapidly detecting avian reovirus |
| CN115820638A (en) * | 2022-09-30 | 2023-03-21 | 扬州大学 | Exogenous artificial miRNA for inhibiting replication of waterfowl-derived avian reovirus and application thereof |
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| CN103184298A (en) * | 2012-08-09 | 2013-07-03 | 中国农业科学院上海兽医研究所 | Primer for detecting infectious bronchitis viruses, detection method and kit |
| CN106119423A (en) * | 2016-08-31 | 2016-11-16 | 中国农业大学 | The general PCR primer of detection Avianreovirus and detection kit thereof |
| CN107058615A (en) * | 2017-01-17 | 2017-08-18 | 佛山科学技术学院 | A kind of calcein visualization RT LAMP kits and its application |
| CN108411041A (en) * | 2018-05-22 | 2018-08-17 | 山东农业大学 | A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application |
| CN108411041B (en) * | 2018-05-22 | 2021-11-30 | 山东农业大学 | Fluorescent quantitative RT-PCR kit for detecting novel chicken reovirus and application thereof |
| CN109022618A (en) * | 2018-08-22 | 2018-12-18 | 上海实验动物研究中心 | A kind of primer set and its application for quickly detecting 3 type of mouse reovirus |
| CN109022618B (en) * | 2018-08-22 | 2019-08-13 | 上海实验动物研究中心 | A kind of primer set and its application for quickly detecting 3 type of mouse reovirus |
| CN111926113A (en) * | 2020-07-14 | 2020-11-13 | 安阳工学院 | Primer group, probe, kit and method for rapidly detecting avian reovirus |
| CN111926113B (en) * | 2020-07-14 | 2023-11-21 | 安阳工学院 | Primer group, probe, kit and method for rapidly detecting avian reovirus |
| CN115820638A (en) * | 2022-09-30 | 2023-03-21 | 扬州大学 | Exogenous artificial miRNA for inhibiting replication of waterfowl-derived avian reovirus and application thereof |
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