CN102586479B - 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 anRNA 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 described Avianreovirus.
Background technology
Avianreovirus (Avian reoviruses, ARV) belongs to Reoviridae Orthoreovirus, the multiple birds such as main infection 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 group, causes chicken poor growth, price of deed rate low, affects immune effect, causes huge financial loss to aviculture.
At present, the Avianreovirus diagnostic method has a lot, mainly contains immune agar diffusion (AGP) test, Enzyme-linked Immunosorbent Assay (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 using at laboratories.
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 " 2011 the 31st volume the 7th interim article of delivering in July " 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 limit of identification is 4ELD50, and uses SYBR Green I to be fluorescence dye, very easily causes crossed contamination.
Summary of the invention
The sensitivity that exists in the above LAMP method detection is hanged down and the problem of easy pollution in order to solve, and 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 is achieved in the following ways:
The primer of a kind of ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) Avianreovirus comprises outside primer pair, inboard primer pair and annular primer altogether to three pairs of Auele Specific Primers, and sequence is as follows:
Pair of primers be can with the outside primer pair of p10 gene (sequence 1 in the sequence table) combination 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,
Pair of primers be can with Avianreovirus S1133 strain (the inboard primer pair that the p10 gene is combined 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,
Pair of primers be can with the annular primer that be combined of p10 gene in the Avianreovirus S1133 strain (9GeneBank Accession Number AF3307030) pair:
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 pair, inboard primer pair, the right mol ratio of annular primer are 1:8:4.
Eight zones of primer identification are to mate the in recent years conserved regions of all bird reovirus p10 genes, so primer can be identified all bird reovirus p10 genes, described outside primer pair, inboard primer pair and annular primer should be avoided the formation of primer dimer to doing as a whole use as far as possible during use.
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 pair upstream primer and each 5pmol of downstream primer in per 24 μ L loop-mediated isothermal amplification liquid, inboard primer pair upstream primer and each 40pmol of downstream primer, 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 described loop-mediated isothermal amplification condition is: 59-65 ℃, 30-75 minute.
Described detection method, the sample that will carry out 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 by 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 still is that brown color is without fluorescence liquid.If the reaction tubes of sample to be checked is housed the bright green visible fluorescence is arranged, illustrate that then the Avianreovirus detected result is positive in the sample to be checked.If the reaction tubes that sample to be checked is housed still for brown color without fluorescence liquid, illustrate that then the Avianreovirus detected result is negative in the sample to be checked.
It is as follows that Avianreovirus detection kit provided by the invention detects principle:
8 particular combination zone combinations of described primer sets and described Avianreovirus AF330703 sequence p10 gene see Table 1.
Table 1 primer sets and described Avianreovirus AF330703 sequence p10 gene calmodulin binding domain CaM
Above-mentioned primer sets can be finished 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 prior to template in conjunction with and to start DNA synthetic.The outside primer of mutually same end is combined with template subsequently that to start DNA synthetic, and strand displacement occurs discharges the dna single chain that contains inboard primer sequence.This single stranded DNA successively is combined with inboard primer and the outside primer of the other end as template, starts synthetic merging of DNA strand displacement to occur, and forms at last an initial stem circular DNA.
(3) the reaction cycle stage
Inboard primer is combined with initial stem circular DNA, starts strand displacement DNA synthetic, can produce the stem circular DNA of another 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 be combined with inboard primer to start strand displacement DNA, and synthesize at every turn the stem length of stem circular DNA is doubled and redoubled.
After entering the cycle stage, can produce the different stem circular DNA of many molecular sizes, these stem circular DNAs can also be combined with annular primer as template, start more DNA synthetic and strand displacement occurs.Finally by the isothermal duplication of spending one hour, goal gene can add up 10
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 the normal PCR 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 by visual inspection fluorescence, simple to operate, it is convenient to observe.This test kit can be applicable to the detection of carrying Avianreovirus in the Clinical Veterinary Medicine of carrying 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 test method in the following example, 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, such as 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 finished, 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 by 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 still is that brown color is without fluorescence liquid.If the reaction tubes that detected sample is housed still for brown color without fluorescence liquid, illustrate that then the Avianreovirus detected result is negative in the sample to be checked; 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
The following 3 pairs of primers of synthetic, outside primer pair:
Upstream primer F3:5 '-ATGCTGCGTATGCCTCCC-3 ',
Downstream primer B3:5 '-CCAGCTCACGATGGAAGAC-3 ',
Inboard primer pair:
Upstream primer FIP:
5’- ACGTAGCTTGCAAATCACCACCTTTTGTGTAACGGTGCGACTGCT-3’,
Downstream primer BIP:
5’- CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3’,
Annular primer pair:
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 upstream primer (F3), the outside, 5pmol downstream primer (B3), the inboard primer (FIP) in 40pmol upstream, 40pmol downstream interior side primer (BIP), 20pmol upstream annular primer, 20pmol downstream annular primer.
Three, the assembling of test kit
This test kit is comprised 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 affects amplification efficiency.Mg
2+Concentration is lower, and atopic is stronger, but amplification efficiency reduces; Mg
2+Concentration is higher, and the efficient of reaction increases, but specificity weakens.In order to determine MgSO
4The best add concentration, prepare respectively MgSO
4Concentration is respectively the LAMP reaction solution of 4mM, 8 mM, 12 mM and 16 mM, places 60 minutes for 60 ℃ on water-bath or metal constent temperature heater, takes out, and carries out the gel electrophoresis test, the results are shown in Figure 1, embodiment result and 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.
Embodiment 2
Get two chickens, 1 is diagnosed as the sick chicken that Avianreovirus infects, 1 healthy chicken.Respectively 2 chickens are sampled with cloacal swabs.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Collect respectively cloacal swabs, in centrifuge tube, repeatedly wash cotton swab 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, now only enumerates wherein a kind of concrete operations, and is as follows, but is not restricted to this kind operation:
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 ℃ precoolings), hatch 10min under-20 ℃; 12000rpm, centrifugal 10min under 4 ℃ of conditions abandons supernatant; Adding 1mL 75% ethanol (preparation of 0.1%DEPC water) in the precipitation washs; 12000rpm, centrifugal 5min under 4 ℃ of conditions behind the repeated washing, abandons supernatant again; To precipitate room temperature and dry 10min, and use 0.1%DEPC water with resolution of precipitate, and hatch 10min under 55-60 ℃, prepared RNA should detect or be stored in-80 ℃ of standby inspections immediately.
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 still is that brown color is without fluorescence liquid.Three reaction tubess of healthy chicken test set are brown color, 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 cloacal swabs.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Step 1 with embodiment 2.
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 still is that brown color is without fluorescence liquid.Three reaction tubess of healthy chicken test set are brown color, 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 cloacal swabs.Adopt the detection kit of embodiment 1 preparation that sample is detected.
Two, extract RNA
Step 1 with embodiment 2.
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 still is that brown color is without fluorescence liquid.Three reaction tubess of healthy chicken test set are brown color, 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 cloacal swabs.Adopt the detection kit of embodiment 1 preparation that sample is detected.
One, extracts RNA
Step 1 with embodiment 2.
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 still is that brown color is without fluorescence liquid.Three reaction tubess of healthy chicken test set are brown color, and three reaction tubess of sick chicken test set all have the bright green visible fluorescence.
Detection sensitivity and Evaluation on specificity
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 namely 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, cultivate competent cell under suitable condition, plasmid can copy along with the breeding of competent cell.From competent cell, extract at last purifying p10 gene plasmid.
Copy number concentration be measured and be calculated to the plasmid that obtains can by spectrophotometer method.With distilled water plasmid is diluted to needed concentration, namely 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, detecting step is as follows:
(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 with the standby negative control of 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, three reaction tubess of positive controls all have the bright green visible fluorescence, and three reaction tubess of negative control group are brown color without 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, three reaction tubess of test set 3 all have the bright green visible fluorescence, 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, three reaction tubess of test set 7 all have the bright green visible fluorescence, 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 brown color without fluorescence liquid.
As seen, 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 Avian pneumo-encephalitis virus 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), infectious laryngotracheitis virus (
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, Avian pneumo-encephalitis virus, 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 infectious laryngotracheitis virus, 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 as follows:
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; 56 ℃ of Water Unders were bathed 2 hours; Add the saturated phenol of 200 μ L Tris, the centrifugal 10min of 12000rpm behind the mixing; Get the upper strata water, add the 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, detecting step is as follows:
(1) ring mediated isothermal amplification
Get each 1 μ L of DNA of the RNA of H9N2 subtype avian influenza virus that step 1 obtains, Avian pneumo-encephalitis virus, Avianreovirus, avian infectious bronchitis virus and infectious laryngotracheitis virus, egg-decreasing syndrome virus, escherichia coli, add to respectively in 3 sample pipes, every pipe adds the LAMP reaction solution (detector tube of infectious laryngotracheitis virus, egg-decreasing syndrome virus, escherichia coli dna is water replacement AMV wherein then) 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 with the standby negative control of 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, three reaction tubess of positive controls all have the bright green visible fluorescence, and three reaction tubess of negative control group are brown color without fluorescence liquid.Three reaction tubess of test set 1 are brown color without fluorescence liquid, three reaction tubess of test set 2 are brown color without fluorescence liquid, three reaction tubess of test set 3 all have the bright green visible fluorescence, three reaction tubess of test set 4 are brown color without fluorescence liquid, three reaction tubess of test set 5 are brown color without fluorescence liquid, three reaction tubess of test set 6 are brown color without fluorescence liquid, and three reaction tubess of test set 7 are brown color without fluorescence liquid.
As seen, the detection kit of Avianreovirus of the present invention to Avian pneumo-encephalitis virus 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), infectious laryngotracheitis virus (
Infectious laryngotracheitis Virus, ILTV), egg-decreasing syndrome virus (
Egg Drop Syndrome Virus, EDS), escherichia coli (
E.coli) all without amplification, having good 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
<160>7
<210>1
<211>297
<212>DNA
＜213〉Avianreovirus (Avian reoviruses, ARV)
<220>
<223>
<440>1
atgctgcgta tgcctcccgg ttcatgtaac ggtgcgactg ctgtatttgg taacgttcat 60
tgtcaggcag ctcagaacac ggcaggtggt gatttgcaag ctacgtcatc cataattgca 120
tattggcctt atctagcggc gggtggtggt ttcttattaa ttgttatcat tttcgctctt 180
ctatactgtt gtaaggctaa gatcaaggcg gacgctgcac gtagtgtctt ccatcgtgag 240
ctggtagcgt tgagttctgg taagcacaat gcaatggctc cgccatacga cgtttga 297
<210>2
<211>18
<212>DNA
＜213〉synthetic
<220>
<223>
<440>2
ATGCTGCGTA TGCCTCCC 18
<210>3
<211>19
<212>DNA
＜213〉synthetic
<220>
<223>
<440>3
CCAGCTCACG ATGGAAGAC 19
<210>4
<211>45
<212>DNA
＜213〉synthetic
<220>
<223>
<440>4
ACGTAGCTTG CAAATCACCA CCTTTTGTGT AACGGTGCGA CTGCT 45
<210>5
<211>47
<212>DNA
＜213〉synthetic
<220>
<223>
<440>5
CATAATTGCA TATTGGCCTT ATCTTTTTTA CGTGCAGCGT CCGCCTT 47
<210>6
<211>20
<212>DNA
＜213〉synthetic
<220>
<223>
<440>6
GCCTGACAAT GAACGTTACC 20
<210>7
<211>19
<212>DNA
＜213〉synthetic
<220>
<223>
<440>7
AGCGGCGGGT GGTGGTTTC 19
Claims (7)
1. the primer of a ring mediated isothermal amplification Avianreovirus is characterized in that comprising altogether outside primer pair, inboard primer pair and annular primer to three pairs of Auele Specific Primers, and sequence is as follows:
Outside primer pair:
Upstream primer F3:5 '-ATGCTGCGTATGCCTCCC-3 ',
Downstream primer B3:5 '-CCAGCTCACGATGGAAGAC-3 ',
Inboard primer pair:
Upstream primer FIP:
5’- ACGTAGCTTGCAAATCACCACCTTTTGTGTAACGGTGCGACTGCT-3’,
Downstream primer BIP:
5’- CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3’,
Annular primer pair:
Upstream primer LF:5 '-GCCTGACAATGAACGTTACC-3 ',
Downstream primer LB:5 '-AGCGGCGGGTGGTGGTTTC-3 ',
Outside primer pair, inboard primer pair, the right mol ratio of annular primer are 1:8:4.
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 pair upstream primer and each 5pmol of downstream primer, inboard primer pair upstream primer and each 40pmol of downstream primer, 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
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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 |
| 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 |
| 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 |
| CN111926113B (en) * | 2020-07-14 | 2023-11-21 | 安阳工学院 | Primer group, probe, kit and method for rapidly detecting avian reovirus |
| CN115820638B (en) * | 2022-09-30 | 2023-07-18 | 扬州大学 | Exogenous artificial miRNA for inhibiting replication of waterfowl-derived avian reovirus and application thereof |
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