CN108384895A - Porcine rotavirus digital pcr absolute quantitation detection kit - Google Patents
Porcine rotavirus digital pcr absolute quantitation detection kit Download PDFInfo
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- CN108384895A CN108384895A CN201810429940.0A CN201810429940A CN108384895A CN 108384895 A CN108384895 A CN 108384895A CN 201810429940 A CN201810429940 A CN 201810429940A CN 108384895 A CN108384895 A CN 108384895A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The present invention provides a kind of porcine rotavirus droplet type digital pcr absolute quantitation detection kits, the kit include 1 pair of specific primer pair and 1 with specific primer to the use of specific probe, nucleotide sequence is respectively as shown in SEQ ID No.1 3.The kit of the present invention using digitlization PCR instrument can quickly, it is special, delicately detect porcine rotavirus, detection sensitivity is copied down to 1.Kit application method of the present invention is simple, of low cost, is not necessarily to standard curve, can quickly, directly, quantitatively detect porcine rotavirus, be highly suitable for that disease surveillance, scene be emergent and the detection of clinical samples, be suitable for large-scale promotion and application.
Description
Technical field
The present invention relates to kit detection technique fields, absolutely fixed more particularly to a kind of porcine rotavirus digital pcr
Quantity measuring method and detection kit.
Background technology
Porcine rotavirus belongs to Reoviridae rotavirus, is to cause a variety of new born animals and young animal diarrhea
One of important enteropathogenic, and 1~5 age in days piglet of infection or weanling pig encountered pathogenic.With apocleisis, vomiting, diarrhea
It is characterized with dehydration, weight loss, the death of piglet, Adult Pig is caused to be lost flesh, the price of deed reduces, and increases labour cost and expenses for medicine
Spending etc..The aggravation if mixing infection with transmissible gastroenteritis and Escherichia coli, the death rate increase.Therefore clinically
Need a kind of detection method for capableing of quick diagnosis porcine rotavirus.
Traditional porcine rotavirus detection method includes mainly separation and identification, the immunoperoxidase single layer reality of virus
(IPMA), indirect immunofluorescence experiment (IFA) and Immunofluorescent antibody is tested to test (indirect ELISA) etc..It is the most frequently used at present
Nucleic acid detection method has reverse transcription-polymerase chain reaction to test (RT-PCR) and fluorescence RT-PCR (Realtime RT-PCR) etc.
Experiment.Traditional porcine rotavirus detection method, which exists, needs to use high cost instruments equipment, longer the time required to experiment to wait not
Foot.Although method has the stronger, high sensitivity of specificity, reproducible to RT-PCR and Realtime RT-PCR as compared with the past, and
The features such as high degree of automation, but the above method can only realize the detection of quantitative and semi-quantitative, can not to viral nucleic acid into
Row accurate quantification, there are still certain limitations in sensitivity and Sensitivity Specificity.
The concept for digitizing PCR (Digital PCR, dPCR) was just used simultaneously by Bert Vogelstein early in 1999
Pertinent literature is delivered, original intention is to be able to a large amount of normal from clinical sample (such as urine, lymph, blood plasma, excrement)
Micro mutant cell is detected in body cell, but since the consumptive material that can be used for dilute sample at that time is 384 orifice plates, also
The core concept of digital pcr cannot very well be embodied --- " infinite dilution " (terminal dilution).Bio-Rad is public
A sample can be divided into the droplet of 20,000 nanoliter level by the droplet technology of the QX200 system cores of department, substantially will
A test of traditional quantitative PCR becomes 20,000 test, substantially increase nucleic acid sequence detection sensitivity and precisely
Degree is deduced to the perfection of " infinite dilution " this concept, and Method And Principle can be described as droplet type digitlization PCR (Droplet
Digital PCR, ddPCR).QX200ddPCR systems include two instruments：Drop generator and droplet analyzer, and its it is related
Consumptive material.Each sample is divided into 20,000 uniform nanoliter level droplet by drop generator, wherein each droplet or without waiting for
Nucleic acid target molecule is examined, or containing there are one extremely several nucleic acid target molecules to be checked.Each droplet is as an independent PCR reaction
Device.Subsequent droplet is transferred in 96 hole PCR plates, carries out terminal PCR amplification.Using droplet analyzer (droplet reader) by
A to be detected to each droplet, it is 1 to have the droplet interpretation of fluorescence signal, and the not droplet interpretation of fluorescence signal is 0, final root
The concentration or copy number for providing target molecule to be checked can be calculated according to Poisson distribution principle and the ratio of positive droplet, analysis software.
Compared with traditional quantitative PCR, the accuracy of digital pcr and sensitivity are more preferably.Using droplet type digital pcr technology, people is studied
Member can detect rare mutation, accurate to measure copy number variation, and carry out absolute quantitation to gene expression.Cancer related mutation because
Concentration is relatively low, often escapes from detection.By the high sensitivity of QX200 systems, nowadays researcher can detect concentration down to 1/1,
000,000 target molecule.
Invention content
The object of the present invention is to provide one kind having high sensitivity, high specific, high accuracy, pinpoint accuracy, can be achieved
The digital pcr detection method of accurate quantification and digital PCR detection kit, are used for the quick accurate detection of porcine rotavirus.
First technical purpose of the present invention is to provide specific primer and probe combinations for detecting porcine rotavirus,
Including 1 pair of specific primer and 1 specific probe being used cooperatively with primer pair, nucleotide sequence is respectively：
F2：ACCATCTWCACRTRACCCTC；
R2：GGTCACATAACGCCCC；
P2：FAM-ATGAGCACAATAGTTAAAAGCTAACACTGTCAA-BHQ1
The present invention provides above-mentioned specific primers and probe combinations to prepare porcine rotavirus detection kit or inspection
Application in test agent.
The kit containing above-mentioned specific primer and probe combinations that the present invention provides a kind of.The kit is preferably
Droplet digital pcr absolute quantitation detection kit.
Another technical purpose of the present invention, which is to provide, a kind of having high sensitivity, high specific, high accuracy and accurately
The detection kit of degree, easy to operate detection porcine rotavirus, wherein including 1 pair of specific primer and 1 and primer pair
The specific probe being used cooperatively, nucleotide sequence are respectively：
F2：ACCATCTWCACRTRACCCTC；
R2：GGTCACATAACGCCCC；
P2：FAM-ATGAGCACAATAGTTAAAAGCTAACACTGTCAA-BHQ1
Preferably, the kit also includes virus total RNA extracts reagent and detection reagent；The detection reagent includes nothing
The distilled water of RNA enzyme, one-step method ddPCR sonde method premixed liquids, sonde method ddPCR droplets occur oil, control liquid, negative control and
Positive control.
Preferably, the virus total RNA extracts reagent contains TRIzoL, chloroform or isoamyl alcohol, isopropanol.The one-step method
The formula of its 900 μ L of ddPCR sonde methods premixed liquid is：The 2X One-step RT-ddPCR Supermix for of 500 μ L
Probes, upstream and downstream primer are respectively 90 μ L, and specific probe is 40 μ L, and the concentration of primer and probe is 10 μM, Yi Jiwu
180 μ L of RNase enzymes distilled water.
Preferably, the positive control is the extracting solution containing porcine rotavirus geneome RNA, and negative control is no RNA
Enzyme distilled water.
Its operation principle of the kit of the present invention is using droplet type digitlization PCR (ddPCR).
The working procedure of kit provided by the invention is：
(1) ddPCR reaction solutions are prepared；20 μ L of ddPCR reaction solutions, which are formulated, is：18 μ of one-step method ddPCR sonde methods premixed liquid
L, sample to be tested RNA templates are 2 μ L；
(2) droplet is prepared, droplet is then transferred to PCR plate, is expanded in PCR instrument；
(3) PCR plate for completing PCR amplification is put into droplet analyzer, detects droplet, analyze data, display detection knot
Fruit.
Preferably, in one embodiment of the invention, the method for preparing droplet is the QX200 using Bio-Rad companies
Drop generator in system, to specifications operation carry out droplet preparation.
Preferably, the amplification program of PCR is 50 DEG C of reverse transcription 10min in step (2)；95 DEG C of pre-degeneration 10min；94 DEG C of changes
Property 30sec, 57~60 DEG C annealing 60sec, totally 40 cycle；Reaction was completed by 98 DEG C of 10min, often walks and is all arranged 2.5 DEG C/sec's
Cooling rate.
Application of the kit provided by the invention in schweineseuche disease detects and prevents also belongs to protection scope of the present invention.
The determination method of kit testing result of the present invention is：(1) positive control：20 ± 2 copies；(2) negative control：
<1 copy；Sample to be tested result judgement：(1) positive：Detection result of specimen >=1 copies.(2) negative：Detection result of specimen<
1 copy.
The present invention further discloses the working concentrations of the primer and probe of most suitable droplet type digital pcr.And PCR reactions
Most suitable warming and cooling rate in the process enables the method for the present invention and BIO-RAD companies to improve the amplification efficiency of digital pcr
QX200 systems be combined.
Kit of the present invention have rapidly and efficiently, easy to operate, high specific, high sensitivity, can direct quantitative, without mark
Directrix curve, it is easy to operate the advantages that, can epidemic situation break out in advance before convenient within early stage determine epidemic disease, prevention is carried out, from source
Control epidemic situation.It is in particular in：
(1) high specific：The present invention devises specific primer and special spy according to porcine rotavirus NSP3 gene orders
Needle, specificity is high, and highly stable, ensure that being smoothed out for reaction, further ensures that the spy of porcine rotavirus detection
It is anisotropic；
(2) highly sensitive：Target molecule of the detectable concentration down to 1/1,000,000；It can be in direct quantitative sample to be tested
Copy number；
(3) low template：Since the sensitivity of detection is very high, low-abundance target gene can be detected, it is possible to drop
Low template consumption dilutes template concentrations, and for some preciousnesses, rare sample can have more researchs.
(4) can direct quantitative, be not necessarily to standard curve：Compared with traditional porcine rotavirus detection method, which can
Direct quantitative is not necessarily to standard curve, easy to operate, accurate, effectively prevents the large-scale outbreak of schweineseuche disease.
Description of the drawings
Fig. 1 primer screenings are as a result, the primer combination matched from left to right is followed successively by：F1R1、 F1R2、F2R1、F2R2.
Fig. 2 probe the selection results, the from left to right pairing of primer and probe are followed successively by： F2R2+P1、F2R2+P2.
Fig. 3 primers, probe concentration optimization as a result, be followed successively by from left to right according in table 2 1-3 select concentration.
As a result, wherein Fig. 4 A are the testing result of other viruses, Fig. 4 B take turns the specific detection of Fig. 4 different virus for pig
The testing result of shape virus.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The design and screening of 1 primer of embodiment and probe
1, primer of the present invention, probe design：It has been delivered according to Genbank according to the porcine rotavirus that Genbank has been delivered
Porcine rotavirus genome sequence KJ450847.1, select porcine rotavirus NSP3 for target gene, carry out suitable for ddPCR
Specific primer and probe are designed as target gene, carry out the design of the specific primer sum suitable for ddPCR.
This gives the process for screening best primer, selection Software for Design go out it is wherein several to alternative primer into
Row screening, alternative primer is as follows, is shown in Table 1.
Table 1：For the specificity amplification primer and probe of porcine rotavirus NSP3 genes
2, the screening of primer
(1) it is four pairs that primer is matched at random：F1R1、F1R2、F2R1、F2R2；
(2) and then with dye method quantitative fluorescent PCR, PCR system formula are done：2Xone-step SYBR Green
10 μ L of Supermix, sense primer, downstream primer are respectively 1 μ L, RNase Free dH23 μ L of O, 5 μ L of positive template.PCR
Amplification program be 50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 60 DEG C of annealing 60sec, totally 40
A cycle；65 DEG C -95 DEG C every five seconds for example increase 0.5 DEG C of solubility curve, and reaction was completed.
(3) interpretation of result selects the primer pair F2R2 of not non-specific amplification.F1R1, F1R2, F2R1, F2R2 are copied
Number is followed successively by：1200copies/μL、1070copies/μL、 10710copies/μL、12410copies/μL.
3, the screening of probe
(1) primed probe is combined as：1, F2R2+P1,2, F2R2+P2
(2) and then on ddPCR platforms, ddPCR system formulations：2X One-step RT-ddPCR Supermix 10μ
L, sense primer, downstream primer are respectively 1 μ L, 0.5 μ L, RNase Free dH of probe23.5 μ L of O, 4 μ L of positive template, always
20 μ L of volume (concentration of primer and probe is 10 μM).Then droplet is generated.
(3) PCR instrument expands on, is 50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C denaturation 30sec, 60 DEG C
Anneal 60sec, totally 40 cycles；Reaction was completed by 98 DEG C of 10min.Upper droplet digital pcr detector detection.
(4) analysis result：F2R2+P2 primer combination of probe is selected according to experimental result.F2R2+P1, F2R2+P2 are copied
Number is followed successively by：10000copies/μL、15920copies/μL.
Embodiment 2 detects the optimization of the PCR method annealing temperature of porcine rotavirus on ddPCR platforms.
1, being combined as primed probe is selected：F2R2+P2.
2 and then on ddPCR platforms, ddPCR system formulations：10 μ L of 2X One-step RT-ddPCR Supermix,
Sense primer, downstream primer are respectively 1 μ L, 0.5 μ L, RNase Free dH of probe23.5 μ L of O, 4 μ L of positive template, total volume
20 μ L (concentration of primer and probe is 10 μM).8 multiple holes are done, droplet is then generated.
3, upper PCR instrument amplification, is 50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C denaturation 30sec, 50~60
DEG C (instrument is distributed 8 temperature gradients automatically) anneals 60sec, totally 40 cycles；Reaction was completed by 98 DEG C of 10min.Upper droplet number
PCR detectors detect.
4, analysis result：57~60 DEG C of annealing temperature is selected according to experimental result.
The Optimal Experimental of 3 primer of embodiment, concentration and probe concentration
Design primer concentration and probe concentration is 10 μM, is combined as F2R2P2, and system configurations method is shown in Table 2.
Table 2：The optimization of digital pcr primer and probe
Then droplet is generated on ddPCR platforms, be transferred in 96 orifice plates, seal aluminium film.
The amplification program of upper PCR instrument amplification, PCR is 50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation
30sec, 60 DEG C of annealing 60sec, totally 40 recycle；Reaction was completed by 98 DEG C of 10 min.Upper droplet digital pcr detector detection.
Analysis result：F2R2+P2 primed probe formulas are selected according to experimental result, it is respectively 1.8 μ L, probe to select primer
For 0.8 μ L.It is followed successively by 12410 copies/ μ L, 17010copies/ μ L, 14180copies/ μ L by 1,2,3 sequential copy numbers.
The Optimal Experimental of 4 PCR amplification warming and cooling rate of embodiment
1 and then on ddPCR platforms, primer combination of probe F2R2+P2 (a concentration of 10 μM), with ddPCR system formulations：
10 μ L of 2X One-step RT-ddPCR Supermix for probes, sense primer, downstream primer is respectively 1.8 μ L, is visited
Needle is 0.8 μ L, RNase Free dH23.6 μ L of O, 2 μ L of positive template, 20 μ L of total volume.Every instrument does 4 multiple holes.Then
Droplet is generated, is transferred in PCR tubules.
2, it is expanded on two same PCR instrument platforms, is 50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94℃
30sec, 58 DEG C of annealing 60sec are denaturalized, totally 40 cycles；Reaction was completed by 98 DEG C of 10min, and a setting warming and cooling rate is set as
5 DEG C/sec, setting warming and cooling rate is set as 2.5 DEG C/sec on another platform, after amplification, the intratubular amplification productions of PCR
Object is transferred in 96 orifice plate of same, seals aluminium film, upper droplet digital pcr detector detection.
3, analysis result：It is found according to analysis of experimental results, the slow droplet number finally detected of warming and cooling rate is than lifting
Warm fireballing more, amplification efficiency is high, and what the distance between the slow negative droplet of warming and cooling rate and positive particle divided opens very much, very
Easy to distinguish, what the distance between the fast negative droplet of warming and cooling rate and positive particle divided does not open, it is not easy to distinguish, therefore select
2.5 DEG C/sec warming and cooling rates.
5 viral RNA of embodiment extracts
It takes 100 μ L tissue samples grinding supernatant to set in 1.5mL eppendorf pipes, adds 500 μ L solution As, mixing, room
Warm acutely concussion 15s, is stored at room temperature 5min.Add 120 μ L solution Bs, carefully covers cap, room temperature acutely shakes 15s, is placed at room temperature for
5min.12,000rpm, 4 DEG C, centrifuge 15min, it is seen that be divided into three layers, upper strata aqueous phase contains RNA.Water phase is shifted to one new
Eppendorf is managed, and equivalent solution C (about 500 μ L) is added, mixing is placed at room temperature for 15min.12,000rpm, 4 DEG C, centrifugation
10min has glue sample RNA precipitate eppendorf tube edges and bottom are visible after centrifugation.Wash RNA：Supernatant is abandoned, 1000 μ L 75% are added
Ethyl alcohol (adding absolute ethyl alcohol to configure with solution D using preceding, -20 DEG C of precoolings) rinsing precipitation, 12000rpm, 4 DEG C, centrifugation
5min abandons supernatant.The fully dry RNA precipitate of room temperature.Add 15 μ L RNase Free dH2O, you can be used for PCR amplification.Can with-
20 DEG C save backup.
Embodiment 6 detects the foundation of the ddPCR methods of porcine rotavirus
1, the assembling of droplet digital pcr absolute quantitation detection kit
Following reagent, labelling (mark title, lot number, date of manufacture, term of validity etc.) are packed with suitable external packing box.
Solution A (RNase Free dH2O) 1,1mL；Solution B (one-step method ddPCR sonde methods premixed liquid) one, 900 μ
L；Solution C (oil occurs for sonde method ddPCR droplets) one, 7mL；Solution D is ddPCR sonde method control liquids, 3.5mL；Solution E
(positive control) one, 40 μ L；Solution F (negative control) one, 40 μ L；The use of geneome RNA is positive template, uses
Freezen protective after Tris-EDTA buffer solutions (0.01M pH8.0) dilution.Qualified positive control preparation will be examined to determine by 400 μ L
Amount packing.Use RNase Free dH2O is negative control.
The formula of above-mentioned its 900 μ L of one-step method ddPCR sonde methods premixed liquid is：The 2X One-step RT- of 500 μ L
DdPCR Supermix for probes, upstream and downstream primer are respectively 90 μ L, and specific probe is 40 μ L, primer and probe
Concentration is 10 μM.RNase Free dH2O 180μL。
In use, the RNA templates of 2 μ L are added in the ddPCR sonde method premixed liquids of 18 μ L, the ddPCR for obtaining 20 μ L is anti-
Liquid is answered, droplet is used to prepare.
2, the foundation of ddPCR methods
(1) ddPCR reaction solutions, 20 μ L of total volume are prepared.Following reactants are added into PCR amplification pipe：
| Single reaction system formulation | One-step method ddPCR sonde method premixed liquids | 18μL |
| Template | RNA | 2μL |
Blank control：With RNase Free dH2O replaces template, is expanded under similarity condition.
(2) droplet is taken to be placed in Kato fixed, 20 μ L reaction solutions, which are added to droplet, to be occurred to block the 8 of an intermediate row
It in a hole, is supplied with 20 μ 1 × solution Ds of L when less than 8 samples, it is proposed that (cannot using 8 channel, the 20 μ L volley of rifle fires and 20 μ L pipette tips
With 200uL pipette tips), one side bottom of pipette tips access hole when sample-adding is in about 15 ° of angles with side wall, slowly gets liquid, get one
Slowly promote pipette tips position after point and get remaining liquid again, rifle not pressed to more than the first file location in order to avoid introducing bubble.
(3) 70 μ L droplets are respectively added in card most 8 holes of bottom next row occur for droplet and generate oil, cannot equally have empty
Hole.
(4) rubber cushion is covered, notices that the aperture on both sides will hook jail.
(5) the above Kato is lightly steadily positioned over to droplet to generate in instrument, starts to generate droplet, pays attention to indicating on instrument
Lamp state is completed within general 2 minutes.
(6) droplet is created on droplet and occurs in one round of card the top, it is proposed that small using the 8 channel volley of rifle fires and 200 μ L pipette tips
The heart is slowly drawn, and adjustment volume aspirated is 40 μ L, Kato is laid flat, pipette tips touch hole to be put into 30~45° angle with hole wall
Bottom draws 40uL in about 5 seconds, then equally slowly squeezes into 96 orifice plate corresponding position holes (about 5 seconds), and pipette tips are close to hole wall access hole
Bottom pays attention to sealing up lid with grease proofing volatilization, discards used droplet every time and card and rubber cushion occurs.
(7) after the completion of transfer oil droplet enters in 96 orifice plates, sealer (the bright face of film is carried out to it with preheated PX1 heat-sealing instrument
Upward, dark face is downward), the operation program of recommendation is：180 DEG C, 10s, generally it is not necessarily to the secondary sealer of inverted orientation；
PCR reactions should be carried out after film in 30 minutes by sealing, or is put in 4 DEG C of refrigerators within 4 hours and is carried out PCR,
It can be completed in any one 96 hole PCR instruments, pay attention to warming and cooling rate≤2.5 DEG C/s.Recommendation response condition：
(8) 96 orifice plates for completing PCR before are put into plate holder and are assembled, pay attention to plate oblique angle orientation, assembled
Droplet is lightly steadily put into after good to read in instrument.
(9) QuantaSoft softwares are opened, it is proposed that primary system cleaning is done before experiment every time, if one week or more is not used
It is recommended that first do primary filling oil circuit does cleaning systems again.Sample message in 96 orifice plates is configured later, is mainly to provide reality
Test title, experiment type and detecting probe information etc., instrument can be run after the completion, after result can be analyzed automatically, manually
Result is preserved after verification.
4, interpretation of result and judgement
The determination method of kit testing result of the present invention is：(1) positive control：20 ± 2 copies；(2) negative control：
<1 copy；Sample to be tested result judgement：(1) positive：Detection result of specimen >=1 copies.(2) negative：Detection result of specimen<
1 copy.
7 specific assay of embodiment
Pig healthy cell culture is detected respectively with the kit of preparation, and pig circular ring virus, pseudorabies virus, pig is tiny
As a result virus, each 5 parts of transmissible gastro-enteritis virus are all negative；Detect tissue and the cell culture of porcine rotavirus infection
As a result each 5 parts of object is all positive.
The above testing result demonstrate the present invention kit specificity is good, high sensitivity, can direct quantitative, it is easy to detect
Fast, result is accurate and reliable.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>One hundred gull Venture Capital bio tech ltd of Beijing
<120>Porcine rotavirus digital pcr absolute quantitation detection kit
<130> P1810132
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213>Artificial sequence
<223> w is a,or t
r is a,or g
<400> 1
accatctwca crtraccctc
20
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence
<400> 2
ggtcacataa cgcccc 16
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
atgagcacaa tagttaaaag ctaacactgt caa
34
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
cagtggttga tgctcaagat gga 23
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
tcattgtaat catattgaat accca 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
acaactgcag cttcaaaaga agwgt 25
Claims (10)
1. a kind of specific primer and probe combinations for detecting porcine rotavirus, which is characterized in that special including following 1 pair
Property primer pair and 1 specific probe being used cooperatively respectively with primer pair, nucleotide sequence are respectively：
F2:ACCATCTWCACRTRACCCTC；
R2:GGTCACATAACGCCCC；
P2:FAM-ATGAGCACAATAGTTAAAAGCTAACACTGTCAA-BHQ1。
2. specific primer described in claim 1 and probe combinations are preparing porcine rotavirus detection kit or detection reagent
In application.
3. a kind of kit containing specific primer described in claim 1 and probe combinations.
4. kit as claimed in claim 3 is droplet digital pcr absolute quantitation detection kit.
5. kit as claimed in claim 4, which is characterized in that also include virus total RNA extracts reagent and detection reagent；Institute
The distilled water that detection reagent includes no RNA enzyme, one-step method ddPCR sonde method premixed liquids are stated, oil occurs for sonde method ddPCR droplets,
Control liquid, negative control and positive control.
6. kit as claimed in claim 5, which is characterized in that the positive control is to contain porcine rotavirus genome
The extracting solution of RNA, negative control are no RNA enzyme distilled water.
7. kit as claimed in claim 5, which is characterized in that its 900 μ L's of one-step method ddPCR sonde methods premixed liquid
Formula is：The 2X One-step RT-ddPCR Supermix for probes of 500 μ L, upstream and downstream primer is respectively 90 μ L,
Specific probe is 40 μ L, and the concentration of primer and probe is 10 μM, and without 180 μ L of RNase enzymes distilled water.
8. the kit as described in claim 4-7 is any, which is characterized in that the working procedure of the kit is：
(1) ddPCR reaction solutions are prepared；20 μ l of ddPCR reaction solutions, which are formulated, is：One-step method ddPCR sonde methods premixed liquid 18 μ L, it is to be measured
Sample RNA templates are 2 μ L.
(2) droplet is prepared, droplet is then transferred to PCR plate, is expanded in PCR instrument；
(3) PCR plate for completing PCR amplification is put into droplet analyzer, detects droplet, analyze data, show testing result.
9. kit as claimed in claim 8, which is characterized in that the amplification program of PCR is 50 DEG C of reverse transcriptions in step (2)
10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 57~60 DEG C of annealing 60sec, totally 40 recycle；98 DEG C of 10min knots
Shu Fanying often walks the cooling rate that 2.5 DEG C/sec is arranged.
10. application of any kits of claim 3-9 in inspection for food hygiene quarantine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810429940.0A CN108384895A (en) | 2018-05-08 | 2018-05-08 | Porcine rotavirus digital pcr absolute quantitation detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810429940.0A CN108384895A (en) | 2018-05-08 | 2018-05-08 | Porcine rotavirus digital pcr absolute quantitation detection kit |
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| Publication Number | Publication Date |
|---|---|
| CN108384895A true CN108384895A (en) | 2018-08-10 |
Family
ID=63071222
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| CN201810429940.0A Pending CN108384895A (en) | 2018-05-08 | 2018-05-08 | Porcine rotavirus digital pcr absolute quantitation detection kit |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222300A (en) * | 2016-08-04 | 2016-12-14 | 北京出入境检验检疫局检验检疫技术中心 | The test kit of a kind of accurate quantification detection A rotavirus and detection method |
| US20170088828A1 (en) * | 2015-09-29 | 2017-03-30 | Agenovir Corporation | Compositions and methods for treatment of latent viral infections |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170088828A1 (en) * | 2015-09-29 | 2017-03-30 | Agenovir Corporation | Compositions and methods for treatment of latent viral infections |
| CN106222300A (en) * | 2016-08-04 | 2016-12-14 | 北京出入境检验检疫局检验检疫技术中心 | The test kit of a kind of accurate quantification detection A rotavirus and detection method |
Non-Patent Citations (3)
| Title |
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| S ORISTO, HJ LEE, L MAUNULA等: "Performance of pre-RT-qPCR treatments to discriminate infectious human rotaviruses and noroviruses from heat‐inactivated viruses: applications of PMA/PMAxx, benzonase and RNase", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 郭延青; 李丹地; 段招军: "A组轮状病毒实时定量PCR检测方法的研究进展", 《病毒学报》 * |
| 金征宇，张雪宁，赵阳，等: "《基因与纳米探针-医学分子成像理论与实践 上》", 30 November 2017, 天津科学技术出版社 * |
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