CN106868227A - Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform - Google Patents
Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform Download PDFInfo
- Publication number
- CN106868227A CN106868227A CN201710299035.3A CN201710299035A CN106868227A CN 106868227 A CN106868227 A CN 106868227A CN 201710299035 A CN201710299035 A CN 201710299035A CN 106868227 A CN106868227 A CN 106868227A
- Authority
- CN
- China
- Prior art keywords
- yak
- rotavirus
- pipe
- constant temperature
- isolation type
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform and application, it is made up of reaction buffer, the lyophilized pipe of fluorescence quantitative PCR reaction solution, positive reference substance and the lyophilized pipe of negative controls.The lyophilized pipe of fluorescence quantitative PCR reaction solution is by Taq enzyme, reverse transcriptase, detection primer, probe and dNTPs is freeze-dried mixed forms.Kit detection time of the present invention short (only 42 minutes reaction time), high specificity, sensitivity is high, be easy to storage (4 DEG C preservation), constant temperature isolation type PCR instrument is coordinated to use, the field quick detection to yak rotavirus is especially suitable for, can be widely popularized in basic unit.
Description
Technical field
The invention belongs to biological technical field, specifically, it is related to a kind of yak based on constant temperature isolation type fluorescent PCR platform
Bovine rota detection kit and application.
Background technology
Nineteen sixty-eight Mebus et al. is found that bovine rota, and yak rotavirus (Yak RV) in 1986 in Zhang Min
Deng finding first and isolated in the diarrhoeal diseases research to yak, but do not studied (Zhang Min, Wang Qing further but
River, Liu Dengwei waits research [J] the Sichuan animal and veterinary of Hongyuan yak rotavirus, 1986,3:2-4.).Nineteen ninety, Li Ming
Auspicious grade finds the presence of yak rotavirus in the Yak calf diarrhoea in Lanzhou, and proves that yak rotavirus is to cause yak abdomen
(Li Mingrui, Li Chunling, Li Hao wait investigation [J] China beast of .1990. yak On The Pathogen of Calf Epidemic Diarrhea to the Important cause of disease for rushing down
Medical courses in general (2), 12-13.).It is mainly diarrhoea, normal secondary bacterial sense by the Yak calf clinical symptoms of yak rotavirus infection
Dye, causes death, and case fatality rate is high.Huge economic loss is brought to yak aquaculture.
Cause yak suffer from diarrhoea cause of disease it is more, including rotavirus, coronavirus, viral diarrhea virus, Escherichia coli,
Salmonella, Cryptosporidium and coccidia etc., symptom is similar, it is more difficult to distinguish, therefore the antidiastole of early stage is particularly important, cause of disease inspection
Survey can provide the positive evidence of infection, be most reliable diagnostic means, and PCR, quantitative fluorescent PCR are the most commonly used aetologies
Detection means, especially quantitative fluorescent PCR have the advantages that sensitivity high, high specificity, good stability, have become animal epidemic
The main flow means of diagnosis.But common fluorescence quantifying PCR method is due to needing large scale equipment, complex operation, reaction time long
Its application at the scene in terms of quick detection is constrained etc. factor.
Simultaneously as the particular surroundings of Qinghai-Tibet Platean result in yak rotavirus gene there occurs very big variation, it is special
It is not the VP6 genes frequently as target gene, the research such as 2016 weeks virtues shows the serious shadow of variation of the gene of yak rotavirus vp 6
(Zhou Fang, Yue Hua, Zhang Bin wait the gene sequencing of .2016. yaks rotavirus vp 6 and RT- to the recall rate of the existing PCR method of sound
The foundation of PCR detection method and application [J] journal of animal science and veterinary medicine, 47 (7), 1465-1473.).Therefore for yak colyliform disease
Poison is set up a kind of quick, sensitive, special, extremely urgent suitable for the detection method at scene.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of yak based on constant temperature isolation type fluorescent PCR platform
Bovine rota detection kit and application, the present invention are a kind of simple, quick, sensitive based on constant temperature isolation type fluorescent PCR
Degree strong detection method high and special, detection that can be with scene to yak rotavirus provides scientific basis, to ensureing that yak is supported
Grow industry sound development significant.
In order to solve the above-mentioned technical problem, the invention discloses a kind of yak based on constant temperature isolation type fluorescent PCR platform
The primer and probe of rotavirus detection, the primer and probe include：Yak rotavirus sense primer and yak colyliform disease
Malicious anti-sense primer and yak rotavirus probe, wherein,
The nucleotide sequence of the yak rotavirus sense primer is as shown in SEQ ID NO.1；The yak colyliform disease
The nucleotide sequence of malicious anti-sense primer is as shown in SEQ ID NO.2；The nucleotide sequence such as SEQ of the yak rotavirus probe
Shown in ID NO.3；The 5' ends of the nucleotide sequence of yak rotavirus probe are connected with FAM marks, and 3' ends are connected with non-fluorescence
Quenching group and BHQ.
The invention also discloses a kind of above-mentioned primer and probe answering in terms of yak rotavirus detection kit is prepared
With.
The invention also discloses a kind of yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform,
Including reaction buffer, the lyophilized pipe of fluorescence quantitative PCR reaction solution, the lyophilized pipe of positive reference substance and the lyophilized pipe of negative controls；
The lyophilized pipe of the fluorescence quantitative PCR reaction solution is included on the μ L of Taq enzyme 1, the μ L of reverse transcriptase 1.25, yak rotavirus
Trip primer, each 3.5 μ L of the yak rotavirus anti-sense primer and μ L of yak rotavirus probe 0.25, dNTPs 4 μ L；
The nucleotide sequence of yak rotavirus sense primer is as shown in SEQ ID NO.1；Under the yak rotavirus
The nucleotide sequence of trip primer is as shown in SEQ ID NO.2；The nucleotide sequence of the yak rotavirus probe such as SEQ ID
Shown in NO.3；The 5' ends of the nucleotide sequence of yak rotavirus probe are connected with FAM marks, and 3' ends are connected with non-fluorescence and are quenched
Group and BHQ.
Further, the final concentration of 5U μ L of Taq enzyme-1, the final concentration of 20U μ L of reverse transcriptase-1, yak colyliform
The final concentration of viral sense primer and yak rotavirus anti-sense primer is 10 μm of ol μ L-1, yak rotavirus probe
Final concentration of 10 μm of ol μ L-1, the final concentration of 2.5mM of dNTPs.
Further, the lyophilized pipe of the fluorescence quantitative PCR reaction solution is prepared by the following method and obtains：By Taq enzyme, reversion
Record enzyme, yak rotavirus sense primer, yak rotavirus anti-sense primer and yak rotavirus probe, dNTPs mixing add
- 50 DEG C are down in the PCR pipe for entering 0.5ml, 24h is then freezed under 10pa air pressure, form stopped pipe after dry powder-shaped, fluorescence is obtained and determines
The lyophilized pipe of amount PCR reaction solutions, the lyophilized pipe of the fluorescence quantitative PCR reaction solution is provided with 50 pipes.
Further, reaction buffer is made up of following components：500mM KCl、pH 8.3、100mM Tris-HCl、
15mM MgCl2, balance of ddH2O, with upper volume total amount as 3ml.
Further, the lyophilized pipe of positive reference substance is prepared by the following method and obtains：Yak rotavirus RNA piece will be contained
The μ L of RNA sample 100 of section load in PCR pipe, are down to -50 DEG C, and 24h is then freezed under 10pa air pressure, are closed after forming dry powder-shaped
Pipe is prepared from；
The lyophilized pipe of negative controls is prepared by the following method and obtains：By the sample 100 without yak rotavirus RNA fragment
μ L load in PCR pipe, are down to -50 DEG C, and 24h is then freezed under 10pa air pressure, and stopped pipe is prepared from after forming dry powder-shaped.
Further, the kit is stored at 4 DEG C.
The invention also discloses a kind of yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform
Application method, comprises the following steps：
1) RNA templates are prepared：The μ L of fecal sample supernatant 200 are taken, with reference to the auspicious deep victory limited public affairs of (Xiamen) biotechnology of gold
The PetNAD kits of department extract specification and extract tested sample supernatant total serum IgE, prepare RNA templates；
2) preparation of Fluorescence PCR system：Being prepared in for Fluorescence PCR system is operated on ice；Take 100 μ L reactions slow
Fliud flushing mixes in being added to the lyophilized pipe of positive reference substance, prepares positive reference substance, then takes 50 μ L reaction buffers and 5 μ L
Positive reference substance is added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepares positive control reaction system；Take 100 μ L reactions
Buffer solution is added in the lyophilized pipe of negative controls, prepares negative controls, then takes 50 μ L reaction buffers and 5 μ L are negative
Reference substance is added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepares negative control reaction system；Take 50 μ L reaction buffers
In the 5 μ L detection sample rna lyophilized pipes of addition fluorescence quantitative PCR reaction solution, detection sample rna reaction system is prepared；So
Take from each reaction system respectively afterwards during 50 μ L mixtures move into constant temperature isolation type Fluorescence PCR pipe；Note keeping away during sample-adding
Light is operated；The instantaneous high speed centrifugation 5s of quantitative fluorescent PCR reaction tube that will be prepared, it is to avoid bubble occur；
3) augmentation detection：PCR reaction tubes are positioned in constant temperature isolation type fluorescent PCR instrument, operation key is pressed, that is, started anti-
Should；
4) testing result judges："+" represents positive, and "-" represents negative.
Compared with prior art, the present invention can be obtained including following technique effect：
1) the invention provides a kind of fluorescence PCR detection reagent kit of yak rotavirus, its advantage is high specificity, spirit
Sensitivity is high, detection time is short, it is pollution-free, without electrophoresis, can quick detection at the scene.
2) present invention can quickly and accurately detect the yak rotavirus RNA in tested sample, while can also detect
The rotavirus RNA of common ox, for the Molecule Epidemiology Investigation of bovine rota.
3) fluorescence quantitative PCR reaction solution of the invention includes reverse transcriptase and PCR amplification enzymes, make nucleic acid reverse transcription and
Amplification carried out in same pipe, simplify operation, save reagent simultaneously stopped pipe operate reduce RNA degraded and pollution can
Can property.
4) fluorescence quantitative PCR reaction solution, positive and negative controls are prepared using lyophilized mode, reduce kit
Preservation condition, can 4 DEG C of preservation one-year ages, be readily transported, convenient storage, while improve the stability of reagent.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is the structure chart of POCKIT Micro instruments of the present invention；
Fig. 2 is positive sample sensitivity technique result of the present invention, wherein, 1：9.66×104copies/μL；2：9.66×
103copies/μL；3：9.66×102copies/μL；4：9.66×101copies/μL。
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
Embodiment 1 is based on the yak rotavirus detection kit of constant temperature isolation type fluorescent PCR platform
A kind of yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform, including following components：
(1) reaction buffer：3ml, by 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl2And ddH2O
(surplus) is constituted.
(2) the lyophilized pipe of fluorescence quantitative PCR reaction solution (50 pipe)：
Fluorescence quantitative PCR reaction solution includes that the μ L/ of Taq enzyme 1 manage (5U μ L-1), the μ L/ of reverse transcriptase 1.25 pipe (20U μ L-1), detection manage (10 μm of ol μ L with each 3.5 μ L/ of upstream and downstream primer-1) and the μ L/ of probe 0.25 pipes (10 μm of ol μ L-1)、dNTPs
4 μ L/ manage (2.5mM)；- 50 DEG C are down in the PCR pipe that thing mixed above is added 0.5ml, are then freezed under 10pa air pressure
24h, forms stopped pipe after dry powder-shaped, and the lyophilized pipe of fluorescence quantitative PCR reaction solution is obtained.Wherein, detection yak rotavirus is upper and lower
Trip primer and probe totally 3, the length for amplifying the gene of yak rotavirus vp 6 are 92bp.Upstream primer sequence:5’-
GCTAACCACTTGGTATCCG-3 ', SEQ ID NO:1；Downstream primer sequence:5 '-GCCATCTGAGTGATTACTCTG-3 ',
SEQ ID NO:2；Probe sequence：FAM-TACGTATTCGCTACACAGAGTAATCA-BHQ1, SEQ ID NO:3.
(3) the lyophilized pipe of positive reference substance (1 pipe)：The μ L of RNA sample 100 containing yak rotavirus RNA fragment are loaded
In PCR pipe, -50 DEG C are down to, 24h is then freezed under 10pa air pressure, stopped pipe is prepared from after forming dry powder-shaped；Wherein, yak
The sequence of rotavirus RNA such as SEQ ID NO:Shown in 4.
(4) the lyophilized pipe of negative controls (1 pipe)：The μ L of sample 100 without yak rotavirus RNA fragment are loaded into PCR pipe
It is interior, -50 DEG C are down to, 24h is then freezed under 10pa air pressure, stopped pipe is prepared from after forming dry powder-shaped.
This kit is stored in 4 DEG C, and transport needs bag on the rocks.
Embodiment 2 is based on the application method of the yak rotavirus detection kit of constant temperature isolation type fluorescent PCR platform
The method is comprised the following steps：
1) RNA templates：The μ L of fecal sample supernatant 200 are taken, with reference to auspicious deep victory (Xiamen) bio tech ltd of gold
PetNAD kits extract specification and extract tested sample supernatant total serum IgE, prepare RNA templates；
2) preparation of Fluorescence PCR system：Being prepared in for Fluorescence PCR system is operated on ice；
Take during 100 μ L reaction buffers are added to the lyophilized pipe of positive reference substance and mix, prepare positive reference substance, then
Take in 50 μ L reaction buffers and the lyophilized pipe of 5 μ L positive reference substances addition fluorescence quantitative PCR reaction solution, prepare positive control
Reaction system；Take in the 100 μ L reaction buffers lyophilized pipe of addition negative controls, prepare negative controls, then take 50 μ
L reaction buffers and 5 μ L negative controls are added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepare negative control reaction
System；Take in 50 μ L reaction buffers and the 5 μ L detection sample rna lyophilized pipes of addition fluorescence quantitative PCR reaction solution, prepare inspection
Survey sample rna reaction system；Then taking from each reaction system 50 μ L mixtures respectively, to move into constant temperature isolation type fluorescent PCR anti-
Ying Guanzhong.Notice that lucifuge is operated during sample-adding.The instantaneous high speed centrifugation 5s of quantitative fluorescent PCR reaction tube that will be prepared, it is to avoid occur
Bubble.
3) augmentation detection：PCR reaction tubes are positioned in constant temperature isolation type fluorescent PCR instrument (POCKIT Micro) (such as
Shown in Fig. 1), press operation key, that is, start reaction；
4) testing result judges："+" represents positive, and "-" represents negative.
The standard items of embodiment 3 prepare the measure with sensitivity
The μ L of fecal sample supernatant 200 are taken, with reference to the PetNAD reagents of auspicious deep victory (Xiamen) bio tech ltd of gold
Box extracts specification and extracts tested sample supernatant total serum IgE, referring next to the reverse transcription examination of precious bioengineering (Dalian) Co., Ltd
Agent box specification is by total serum IgE reverse transcription into cDNA.
Performing PCR amplification is entered to the cDNA of above-mentioned preparation respectively with the primer of yak rotavirus, is with corresponding cause of disease cDNA
Positive control, is negative control without template.Reaction system is：The μ L of 2.0 μ L, 2 × Taq PCR Master Mix of cDNA 12.5,
Each μ L of 1.0 μ L, ddH2O 8.5 of upstream and downstream primer, cumulative volume totally 25 μ L.PCR amplification conditions are:95 DEG C of 5min, 95 DEG C of 30s, 52
DEG C 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C of 10min.PCR primer identifies through 3% agarose gel electrophoresis, uses glue reclaim reagent
Box reclaims purpose fragment, and is cloned into pMD19-T carriers (precious bioengineering Co., Ltd), and converts bacillus coli DH 5 alpha
Competent cell, is filtered out in positive colony LB fluid nutrient mediums of the access containing ampicillin, and 37 DEG C of culture 8h are carried with plasmid
Take kit and extract recombinant plasmid, send raw work biology Co., Ltd to be sequenced.Correct positive plasmid is sequenced as yak colyliform disease
Malicious positive criteria product, nucleic acid-protein detector determines its concentration, and nucleic acid concentration is calculated according to the following equation：Positive criteria product concentration
(copies/ μ L)=(6.02 × 1023copies/mol)×(29.75ng/μl)×10-3/(1.85*106G/mol)=9.66 ×
1010copies/μL.By 10 times of gradient dilutions (10 of positive criteria product-1~10-10), to 103、102、101With 100Four dilution factors
Sample is detected that each reaction tube is put into POCKIT Micro reacting holes, presses " operation key ", and reaction terminates after 42 minutes,
Positive findings is "+", and negative findings is "-", as a result as shown in Fig. 2 wherein 103、102、101Result is "+" 100Result is
"-", illustrates that the lowest detection of this kit is limited to 96.6copies/ μ L.
The specificity of embodiment 4 and stability test
With the kit of present invention development to 22 parts of yak rotavirus positive samples, 8 parts of negative samples and 10 kinds of phases
Close cause of disease yak coronavirus, yak viral diarrhea virus, yak astrovirus, yak enteritis virus, yak Escherichia coli,
Yak salmonella, yak Bacillus perfringens, yak campylobacter jejuni, yak Cryptosporidium, yak coccidia detected,
And yak rotavirus positive criteria product is set up as control, with the specificity of kits for evaluation, as a result show to 22 portions of yaks
The test positive of rotavirus positive sample of nucleic acid, 8 parts of negative samples cause of disease testing result related to 10 kinds is feminine gender, is demonstrate,proved
Bright the method specificity is good.
8 positive criteria products of dilution factor of yak rotavirus are detected 3 times respectively with the kit developed, to evaluate
The stability of the kit, as a result shows that 3 testing results are consistent, shows having good stability for kit.
The clinical practice of the kit of embodiment 5 and compare with common fluorescent quantifying PCR method
With kit of the present invention and Rashi etc. (Gautam, R., Esona, M.D., Mijatovicrustempasic, S.,
Tam,K.I.,Gentsch,J.R.,&Bowen,M.D.(2014).Real-time rt-pcr assays to
differentiate wild-type group a rotavirus strains fromand
vaccine strains in stool samples.Human Vaccines&Immunotherapeutics,10(3),
767.) fluorescence quantifying PCR method of report enters to the 30 parts of yak rotavirus positive samples extracted and 8 parts of negative sample nucleic acid
Row detection, compares two kinds of coincidence rates of PCR method.Result shown in 22 parts of yak rotavirus positive samples, reagent of the present invention
The positive sample number of box detection is 22, and coincidence rate is 100%；And the fluorescence quantifying PCR method of the report such as Rashi is to total serum IgE
The positive sample number of detection is 11, and coincidence rate is 50%.Two methods are feminine gender to 8 parts of negative sample detection results, accord with
Conjunction rate is 100%.By quantitative fluorescent PCR do not detect and kit detection is positive PCR primer sequencing, sequencing result is confirmed
It is the distinguished sequence of the gene of yak rotavirus vp 6.Prove detection effect of the kit for yak rotavirus better than existing
Some fluorescence quantifying PCR methods, at the same it is again more simple to operate than existing PCR method, portable, Site Detection diagnosis is more suitable for, be
The diagnosis and epidemiology survey of yak rotavirus provide a kind of strong easily instrument.
Due to the variation situation of rotavirus on plateau, cause existing fluorescence quantifying PCR method recall rate reduction, this reagent
Box overcomes the problem, and can detect the rotavirus of yak can also detect the rotavirus of common ox.
This kit is used cooperatively with POCKIT Micro instruments, simple to operate, portable, it is adaptable to clinic detection, at present
Also without commercially available kit.
The present invention is freezed to reaction reagent, reduces kit preservation condition, is readily transported and clinical practice.
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>Southwest University for Nationalities
<120>Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform
<130> 2017
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
gctaaccact tggtatccg 19
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
gccatctgag tgattactct g 21
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence
<400> 3
tacgtattcg ctacacagag taatca 26
<210> 4
<211> 92
<212> DNA
<213>Yak rotavirus
<400> 4
gctaaccact tggtatccgt gtagcgaata cgtagacgca tccttactta cagagttcaa 60
acagcttggc gtagctacat acttaccaaa gt 92
Claims (10)
1. a kind of yak rotavirus based on constant temperature isolation type fluorescent PCR platform is detected primer and probe, it is characterised in that
The primer and probe include：Yak rotavirus sense primer and yak rotavirus anti-sense primer and yak rotavirus are visited
Pin, wherein,
The nucleotide sequence of the yak rotavirus sense primer is as shown in SEQ ID NO.1；Under the yak rotavirus
The nucleotide sequence of trip primer is as shown in SEQ ID NO.2；The nucleotide sequence of the yak rotavirus probe such as SEQ ID
Shown in NO.3；The 5' ends of the nucleotide sequence of yak rotavirus probe are connected with FAM marks, and 3' ends are connected with non-fluorescence and are quenched
Group and BHQ.
2. application of the primer and probe described in claim 1 in terms of yak rotavirus detection kit is prepared.
3. a kind of yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform, it is characterised in that including anti-
Answer the lyophilized pipe of buffer solution, fluorescence quantitative PCR reaction solution, the lyophilized pipe of positive reference substance and the lyophilized pipe of negative controls；
The lyophilized pipe of the fluorescence quantitative PCR reaction solution includes that the μ L of Taq enzyme 1, the μ L of reverse transcriptase 1.25, yak rotavirus upstream are drawn
Each 3.5 μ L of thing, the yak rotavirus anti-sense primer and μ L of yak rotavirus probe 0.25, dNTPs 4 μ L；
The nucleotide sequence of yak rotavirus sense primer is as shown in SEQ ID NO.1；Draw in the yak rotavirus downstream
The nucleotide sequence of thing is as shown in SEQ ID NO.2；The nucleotide sequence of the yak rotavirus probe such as SEQ ID NO.3
It is shown；The 5' ends of the nucleotide sequence of yak rotavirus probe are connected with FAM marks, and 3' ends are connected with non-fluorescence quenching group
And BHQ.
4. the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform according to claim 3, its
It is characterised by, the final concentration of 5U μ L of the Taq enzyme-1, the final concentration of 20U μ L of reverse transcriptase-1, yak rotavirus
The final concentration of sense primer and yak rotavirus anti-sense primer is 10 μm of ol μ L-1, the end of yak rotavirus probe is dense
It is 10 μm of ol μ L to spend-1, the final concentration of 2.5mM of dNTPs.
5. the yak rotavirus detection reagent based on constant temperature isolation type fluorescent PCR platform according to claim 3 or 4
Box, it is characterised in that the lyophilized pipe of the fluorescence quantitative PCR reaction solution is prepared by the following method and obtains：By claim 3 or 4
Described Taq enzyme, reverse transcriptase, yak rotavirus sense primer, yak rotavirus anti-sense primer and yak rotavirus
- 50 DEG C are down in probe, the PCR pipe of dNTPs mixing additions 0.5ml, 24h is then freezed under 10pa air pressure, form dry powder-shaped
Stopped pipe, is obtained the lyophilized pipe of fluorescence quantitative PCR reaction solution afterwards.
6. the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform according to claim 5, its
It is characterised by, the lyophilized pipe of the fluorescence quantitative PCR reaction solution is provided with 50 pipes.
7. the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform according to claim 3, its
It is characterised by, the reaction buffer is made up of following components：500mM KCl、pH 8.3、100mM Tris-HCl、15mM
MgCl2, balance of ddH2O, with upper volume total amount as 3ml.
8. the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform according to claim 3, its
It is characterised by, the lyophilized pipe of positive reference substance is prepared by the following method and obtains：By the RNA containing yak rotavirus RNA fragment
The μ L of sample 100 load PCR pipe in, be down to -50 DEG C, then under 10pa air pressure freeze 24h, formed dry powder-shaped after stopped pipe prepare and
Into；
The lyophilized pipe of negative controls is prepared by the following method and obtains：By the μ L of sample 100 dresses without yak rotavirus RNA fragment
Enter in PCR pipe, be down to -50 DEG C, 24h is then freezed under 10pa air pressure, stopped pipe is prepared from after forming dry powder-shaped.
9. the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform according to claim 3, its
It is characterised by, the kit is stored at 4 DEG C.
10. a kind of application method of the yak rotavirus detection kit based on constant temperature isolation type fluorescent PCR platform, its feature
It is to comprise the following steps：
1) RNA templates are prepared：The μ L of fecal sample supernatant 200 are taken, with reference to auspicious deep victory (Xiamen) bio tech ltd of gold
PetNAD kits extract specification and extract tested sample supernatant total serum IgE, prepare RNA templates；
2) preparation of Fluorescence PCR system：Being prepared in for Fluorescence PCR system is operated on ice；Take 100 μ L reaction buffers
Mixing in the lyophilized pipe of positive reference substance is added to, positive reference substance is prepared, 50 μ L reaction buffers is then taken and 5 μ L is positive
Reference substance is added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepares positive control reaction system；Take 100 μ L reaction bufferings
Liquid is added in the lyophilized pipe of negative controls, prepares negative controls, then takes 50 μ L reaction buffers and 5 μ L negative controls
Product are added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepare negative control reaction system；Take 50 μ L reaction buffers and 5 μ
L detection sample rnas are added in the lyophilized pipe of fluorescence quantitative PCR reaction solution, prepare detection sample rna reaction system；Then divide
Do not taken from each reaction system 50 μ L mixtures move into constant temperature isolation type Fluorescence PCR pipe in；Notice that lucifuge is grasped during sample-adding
Make；The instantaneous high speed centrifugation 5s of quantitative fluorescent PCR reaction tube that will be prepared, it is to avoid bubble occur；
3) augmentation detection：PCR reaction tubes are positioned in constant temperature isolation type fluorescent PCR instrument, operation key is pressed, that is, start reaction；
4) testing result judges："+" represents positive, and "-" represents negative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710299035.3A CN106868227A (en) | 2017-04-27 | 2017-04-27 | Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710299035.3A CN106868227A (en) | 2017-04-27 | 2017-04-27 | Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106868227A true CN106868227A (en) | 2017-06-20 |
Family
ID=59161445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710299035.3A Pending CN106868227A (en) | 2017-04-27 | 2017-04-27 | Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106868227A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988434A (en) * | 2017-12-22 | 2018-05-04 | 西南民族大学 | Infectious bovine rhinotrachetis virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108085417A (en) * | 2017-12-22 | 2018-05-29 | 西南民族大学 | A kind of yak coronavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108707696A (en) * | 2018-06-07 | 2018-10-26 | 西南民族大学 | A kind of gene C type duck hepatitis A virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108866156A (en) * | 2018-07-19 | 2018-11-23 | 北京世纪元亨动物防疫技术有限公司 | The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system |
| CN111719020A (en) * | 2020-06-19 | 2020-09-29 | 内蒙古农业大学 | Kit, primer and probe for detecting bovine rotavirus |
| CN114381533A (en) * | 2021-11-23 | 2022-04-22 | 珠海国际旅行卫生保健中心(拱北海关口岸门诊部) | Method for field detection of salmonella and shigella and double-color iPCR kit |
| CN114540549A (en) * | 2022-03-02 | 2022-05-27 | 西南民族大学 | Primer, probe, kit and iPCR method for detecting African swine fever virus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005059174A1 (en) * | 2003-12-17 | 2005-06-30 | Universitat Autònoma De Barcelona | Method and kit for the genotypification of hla-drb based on real time pcr |
| CN102268488A (en) * | 2011-07-25 | 2011-12-07 | 武汉中博生物股份有限公司 | Fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for detecting bovine viral diarrhea virus and application of kit |
| CN104152579A (en) * | 2014-07-18 | 2014-11-19 | 魏锁成 | VP7 gene-based bovine rotavirus qPCR detecting kit |
| CN106191309A (en) * | 2016-07-19 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination simultaneously differentiating 8 kinds of cattle disease substances and GeXP detection method |
-
2017
- 2017-04-27 CN CN201710299035.3A patent/CN106868227A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005059174A1 (en) * | 2003-12-17 | 2005-06-30 | Universitat Autònoma De Barcelona | Method and kit for the genotypification of hla-drb based on real time pcr |
| CN102268488A (en) * | 2011-07-25 | 2011-12-07 | 武汉中博生物股份有限公司 | Fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for detecting bovine viral diarrhea virus and application of kit |
| CN104152579A (en) * | 2014-07-18 | 2014-11-19 | 魏锁成 | VP7 gene-based bovine rotavirus qPCR detecting kit |
| CN106191309A (en) * | 2016-07-19 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination simultaneously differentiating 8 kinds of cattle disease substances and GeXP detection method |
Non-Patent Citations (3)
| Title |
|---|
| 周芳等: ""牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用"", 《畜牧兽医学报》 * |
| 沈思思等: ""猪流行性腹泻病毒恒温隔绝式荧光PCR检测方法的建立"", 《中国兽医科学》 * |
| 范晴等: ""牛轮状病毒TaqMan 实时荧光RT-PCR快速检测方法的建立"", 《中国畜牧兽医》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988434A (en) * | 2017-12-22 | 2018-05-04 | 西南民族大学 | Infectious bovine rhinotrachetis virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108085417A (en) * | 2017-12-22 | 2018-05-29 | 西南民族大学 | A kind of yak coronavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108707696A (en) * | 2018-06-07 | 2018-10-26 | 西南民族大学 | A kind of gene C type duck hepatitis A virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108866156A (en) * | 2018-07-19 | 2018-11-23 | 北京世纪元亨动物防疫技术有限公司 | The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system |
| CN111719020A (en) * | 2020-06-19 | 2020-09-29 | 内蒙古农业大学 | Kit, primer and probe for detecting bovine rotavirus |
| CN111719020B (en) * | 2020-06-19 | 2022-11-11 | 内蒙古农业大学 | Kit, primer and probe for detecting bovine rotavirus |
| CN114381533A (en) * | 2021-11-23 | 2022-04-22 | 珠海国际旅行卫生保健中心(拱北海关口岸门诊部) | Method for field detection of salmonella and shigella and double-color iPCR kit |
| CN114540549A (en) * | 2022-03-02 | 2022-05-27 | 西南民族大学 | Primer, probe, kit and iPCR method for detecting African swine fever virus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868227A (en) | Yak rotavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform | |
| CN107022650A (en) | Pig Delta coronavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform | |
| CN107513584A (en) | A kind of five heavy fluorescence quantitative kits for detecting enterovirus | |
| CN105695631B (en) | Human immunodeficiency virus, hepatitis type B virus, Hepatitis C Virus Quick joint inspection kit and its preparation and application | |
| CN103409509B (en) | Group B streptococcus fluorescence PCR detection kit | |
| CN110453011A (en) | A kind of method and application based on CRISPR/Cas12a fast accurate detection African swine fever virus | |
| CN107365862A (en) | For detecting the primer and probe and its kit of Echinococcus granulosus in dog excrement | |
| CN108315490A (en) | Pig acute diarrhea syndrome coronavirus and Delta coronavirus RT-PCR diagnostic kits and its detection method | |
| CN109097499A (en) | It is a kind of for detecting the dual fluorescence quantification RT-PCR detection kit of PEDV-TGEV nucleic acid | |
| CN103031386B (en) | Triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus | |
| CN103898195A (en) | Helicobacter pylori drug resistance nucleic acid detection kit | |
| CN106148506A (en) | The antenatal detection kit of a kind of B race streptococcus quantitative fluorescent PCR and application thereof | |
| CN105385786B (en) | A kind of human papillomavirus fluorescence quantitative PCR detection kit and its application method | |
| CN105018647B (en) | A kind of kit and its detection method based on the accurate quantitative typing detection HPV16/18 of digital pcr | |
| CN106916907A (en) | The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid | |
| CN108085417A (en) | A kind of yak coronavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform | |
| CN103388033A (en) | Enterovirus type 71 (EV71) real-time fluorescent nucleic acid isothermal amplification detection kit | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN105274100A (en) | Human TWIST1/Vimentin gene methylation detection marker and kit | |
| CN103045762B (en) | Kit for detecting proviral DNA (deoxyribonucleic acid) of bovine leukemia virus (BLV) and application of kit | |
| CN101812503A (en) | Reagent kit for detecting live bifidobacteriums in dairy products | |
| CN103224998A (en) | Rotavirus PCR detection kit and detection method thereof | |
| CN103740860A (en) | PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and detection method of swine transmissible gastroenteritis virus | |
| CN111172301A (en) | PCR fluorescence detection kit for clostridium difficile toxin B and application thereof | |
| CN109971886A (en) | A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170620 |
|
| RJ01 | Rejection of invention patent application after publication |