CN109337972A - The method of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease - Google Patents
The method of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease Download PDFInfo
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- CN109337972A CN109337972A CN201811411395.9A CN201811411395A CN109337972A CN 109337972 A CN109337972 A CN 109337972A CN 201811411395 A CN201811411395 A CN 201811411395A CN 109337972 A CN109337972 A CN 109337972A
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- pyrosequencing
- primer
- deficiency disease
- uridine monophosphate
- pcr
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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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
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a kind of methods of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, including the following steps: (1) extracts the genomic DNA of milk cow to be detected；(2) polymerase chain reaction；(3) the single-stranded sample preparation of pyrosequencing；(4) pyrosequencing and interpretation of result.The pyrosequencing techniques that the present invention establishes absorb advantage of both PCR and DNA sequencing technology, on the basis of PCR specificity, measurement using very short one section conservative/distinguished sequence can meet the needs to molecular diagnosis, so that subtype identification result is relatively reliable, method is faster.
Description
Technical field
The present invention relates to a kind of methods of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease.
Background technique
Bovine uridine monophosphate synthase deficiency disease (Deficiency of uridine monophophate synthase,
It DUMPS), is Robinson etc. (1983) discovery, due to the intracorporal uridine acid synthase (Uridine of part milk cow
Monophophate synthase, UMPS) lack and causes the level of orotic acid in cream very high (more than 300 μ g/ml, average value is
81.8 μ g/ml), and speculate that its genetic development follows autosomal recessive monogenic inheritance.It is active according to UMPS in red blood cell
Just, Holstein cow group is divided into two kinds of phenotypes, wherein the UMPS activity of normal phenotype ox is twice of defect phenotype ox, by
In not finding the third phenotype, thus it is speculated that recessive homozygous genotype has lethal.Shanks etc. (1989) confirms recessive base
It is dead at maternal gestational 40-60 days because of homozygous embryo.
Schober etc. (1992) cloning and sequencing normal type UMPS gene cDNA sequence of ox[4].By will be normal
The mRNA of type UMPS gene is compared with the sequence of heterozygote UMPS gene, and Schwenger etc. (1993) discovery is miscellaneous
There are a point mutation (transversion of C → T), original encoding essence ammonia for codon at the end C- 405 of zygote UMPS gene
The codon CGA of acid is changed into the TGA of terminator codon.
Pyrosequencing techniques are the new technologies that developed recently gets up, it is the currently the only skill for obtaining Quantitative Sequence result
Art has high accuracy, and reproducibility is splendid, is a kind of universal technology platform, vdiverse in function, application field is extremely wide.
Have the characteristics that high-throughput, low cost, PCR product can be directly used for being sequenced, is not required to carry out the secondary treatments such as product purification, behaviour
Make extremely simplicity, required sample size is small, meets the requirement of inspection and quarantining for import/export.In view of the seriousness of DUMPS harm, reinforce
The monitoring of DUMPS carrier improves accuracy and the detection efficiency of detection method, has weight to the propagation of effective control DUMPS
The meaning wanted.Therefore, the method that this project establishes pyrosequencing method detection DUMPS, and on this basis in Shandong
The milk cow sample of area acquisition is detected.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of pyrosequencing techniques detection holstein cow uridylic acid
The method of synthase deficiency disease.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
The present invention provides the primers of one group of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, and sequence is such as
Under:
Upstream primer: 5 '-AGGCAGCAGTGCAAATGG-3 '；
Downstream primer: 5 '-GTACTGCTGGCCGAGATTATCA-3 '；
Sequencing primer: 5 '-TTGGTTTTATTTCTGGC-3 '.
The present invention also provides a kind of method of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, packets
Include the following steps:
(1) genomic DNA of milk cow to be detected is extracted.
(2) polymerase chain reaction: gained genomic DNA is template, nucleotides sequence shown in claim 1 in step (1)
It is classified as primer, PCR amplification UMPS genetic fragment.
Prepare 50 μ l PCR amplification systems, include: 10 × PCR buffer, 5.0 5.0 μ l of μ l, dNTP, upstream amplification draw
0.5 μ l of object, downstream amplification primer 0.5 μ l, rTaq 0.5 μ l, 33.5 μ l of water, 5.0 μ l of template；
Cyclic program are as follows: after 94 DEG C of 5 min of initial denaturation, into 94 DEG C of 30 s of denaturation, annealing, the circulation of 72 DEG C of extension 30s is total
Circulation 50 times；Then 10 min, 4 DEG C of end are re-extended for 72 DEG C；Obtain amplified production.
(3) the single-stranded sample preparation of pyrosequencing.
The PCR product and the 200 coated magnetic beads of μ g Streptavidin of biotin are marked with using 50 μ l, at 25 DEG C of room temperature
It is incubated for 20 minutes.The PCR product after in conjunction with magnetic bead is picked up with vacuum prep tool, is then cleaned in 70% ethyl alcohol
5s；Denatureation buffer washes 5s；Finally to cleaning 10s in washing buffer；vaccum prep tool
It is put into the plate containing sequencing primer, shakes, discharge magnetic bead.Sample is put into 80 DEG C of baking ovens 2 minutes, is cooled back to room temperature.
(4) pyrosequencing and interpretation of result.
Reaction detects on PYROMARK ID instrument automatically at 28 DEG C, and sample-adding uses the sample-adding of 600mbar 8-msec
Pressure and time, 65 seconds every wheel reaction time.Primer strand extends with the addition of different dNTP.With the combination of nucleic acid, CCD
Video camera detects the optical signal of sending.
The present invention also provides a kind of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease kit,
Including following component:
Upstream primer, downstream primer and sequencing primer, primer sequence are above-mentioned shown；The PCR reaction solution of amplimer, PCR reaction
Liquid includes: 10 × PCR buffer, dNTP, rTaq.
The beneficial effects of the present invention are:
The pyrosequencing techniques that the present invention establishes absorb advantage of both PCR and DNA sequencing technology, in PCR specificity
On the basis of, the measurement of one section of very short conservative/distinguished sequence of application can meet the needs to molecular diagnosis, so that hypotype is reflected
It is relatively reliable to determine result, method is faster.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is amplimer specific test electrophoretogram.M. DNA marker DL2000；The amplification of 1-2. cow genome produces
Object；
Fig. 2 A/A purified genes formed coke phosphoric acid sequencing result.
Fig. 3 A/a hydridization genotype pyrosequencing result.
Fig. 4 a/a hydridization genotype pyrosequencing result.
Specific embodiment
Embodiment 1
A kind of method of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, including the following steps:
(1) genomic DNA of milk cow to be detected is extracted.
(2) polymerase chain reaction: gained genomic DNA is template, nucleotides sequence shown in claim 1 in step (1)
It is classified as primer, PCR amplification UMPS genetic fragment.
Prepare 50 μ l PCR amplification systems, include: 10 × PCR buffer, 5.0 5.0 μ l of μ l, dNTP, upstream amplification draw
0.5 μ l of object, downstream amplification primer 0.5 μ l, rTaq 0.5 μ l, 33.5 μ l of water, 5.0 μ l of template；
Cyclic program are as follows: after 94 DEG C of 5 min of initial denaturation, into 94 DEG C of 30 s of denaturation, annealing, the circulation of 72 DEG C of extension 30s is total
Circulation 50 times；Then 10 min, 4 DEG C of end are re-extended for 72 DEG C；Obtain amplified production.
(3) the single-stranded sample preparation of pyrosequencing.
The PCR product and the 200 coated magnetic beads of μ g Streptavidin of biotin are marked with using 50 μ l, at 25 DEG C of room temperature
It is incubated for 20 minutes.The PCR product after in conjunction with magnetic bead is picked up with vacuum prep tool, is then cleaned in 70% ethyl alcohol
5s；Denatureation buffer washes 5s；Finally to cleaning 10s in washing buffer；vaccum prep tool
It is put into the plate containing sequencing primer, shakes, discharge magnetic bead.Sample is put into 80 DEG C of baking ovens 2 minutes, is cooled back to room temperature.
(4) pyrosequencing and interpretation of result.
Reaction detects on PYROMARK ID instrument automatically at 28 DEG C, and sample-adding uses the sample-adding of 600mbar 8-msec
Pressure and time, 65 seconds every wheel reaction time.Primer strand extends with the addition of different dNTP.With the combination of nucleic acid, CCD
Video camera detects the optical signal of sending.Such as Fig. 2-4 is shown in the sequencing analysis result of different genotype.
The primer specificity of 2 pyrosequencing method of embodiment is tested
Structure milk cow gene nucleic acid is extracted, is expanded using the PCR amplification primer of pyrosequencing detection method, it is special as the result is shown
It is anisotropic good, it amplifies and expected band in the same size and single.Such as Fig. 1.
The repeated experiment of 3 detection method of embodiment
The detection of pyrosequencing repeatability is carried out to 2 parts of PCR-RFLP sample for being detected as A/A and portion A/a, repeats to examine respectively
It surveys three times, the sequencing result of reading is compared with practical sequencing result carries out nucleotide homology.Three times testing result with reality
Sequencing result is consistent, and this method has repeatability well.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, the variations, modifications, additions or substitutions made all should belong to the present invention
Protection scope.
Claims (2)
1. the primer sets of pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, which is characterized in that sequence is such as
Under:
Upstream primer: 5 '-AGGCAGCAGTGCAAATGG-3 '；
Downstream primer: 5 '-GTACTGCTGGCCGAGATTATCA-3 '；
Sequencing primer: 5 '-TTGGTTTTATTTCTGGC-3 '.
2. the method for pyrosequencing techniques detection holstein cow uridine monophosphate synthase deficiency disease, which is characterized in that including following
Step:
(1) genomic DNA of milk cow to be detected is extracted；
(2) polymerase chain reaction: gained genomic DNA is template in step (1), and nucleotides sequence shown in claim 1 is classified as
Primer, PCR amplification UMPS genetic fragment；
50 μ l PCR amplification systems are prepared, include: 10 × PCR buffer, 5.0 5.0 μ l of μ l, dNTP, upstream amplification primer 0.5
μ l, downstream amplification primer 0.5 μ l, rTaq 0.5 μ l, 33.5 μ l of water, 5.0 μ l of template；
Cyclic program are as follows: after 94 DEG C of 5 min of initial denaturation, into 94 DEG C of 30 s of denaturation, annealing, the circulation of 72 DEG C of extension 30s is total
Circulation 50 times；Then 10 min, 4 DEG C of end are re-extended for 72 DEG C；Obtain amplified production；
(3) the single-stranded sample preparation of pyrosequencing；
(4) pyrosequencing and interpretation of result.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705306A (en) * | 2009-12-01 | 2010-05-12 | 华中农业大学 | Method for detecting bovine uridine monophosphate synthase deficiency disease by using AS-PCR |
| CN103627804A (en) * | 2013-11-29 | 2014-03-12 | 华南农业大学 | Primer composition for detecting harmful gene of deficiency of uridine monophosphate synthase of cattle, kit with primer composition and application of kit |
| CN103710442A (en) * | 2013-12-18 | 2014-04-09 | 广东出入境检验检疫局检验检疫技术中心 | Detection primer set, detection kit and detection method of related SNP (single-nucleotide polymorphism) for causing DUMPS (Deficiency of Uridine Monophophate Synthase) |
| CN105925674A (en) * | 2015-12-10 | 2016-09-07 | 中国农业大学 | Detection product for detecting nucleic acid mass spectra of four genetic diseases of dairy cow and application of detection product |
| CN107653316A (en) * | 2017-10-24 | 2018-02-02 | 中北大学 | For detecting the primer pair and its application of three kinds of recessive inheritance defect diseases of milk cow |
-
2018
- 2018-11-24 CN CN201811411395.9A patent/CN109337972A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705306A (en) * | 2009-12-01 | 2010-05-12 | 华中农业大学 | Method for detecting bovine uridine monophosphate synthase deficiency disease by using AS-PCR |
| CN103627804A (en) * | 2013-11-29 | 2014-03-12 | 华南农业大学 | Primer composition for detecting harmful gene of deficiency of uridine monophosphate synthase of cattle, kit with primer composition and application of kit |
| CN103710442A (en) * | 2013-12-18 | 2014-04-09 | 广东出入境检验检疫局检验检疫技术中心 | Detection primer set, detection kit and detection method of related SNP (single-nucleotide polymorphism) for causing DUMPS (Deficiency of Uridine Monophophate Synthase) |
| CN105925674A (en) * | 2015-12-10 | 2016-09-07 | 中国农业大学 | Detection product for detecting nucleic acid mass spectra of four genetic diseases of dairy cow and application of detection product |
| CN107653316A (en) * | 2017-10-24 | 2018-02-02 | 中北大学 | For detecting the primer pair and its application of three kinds of recessive inheritance defect diseases of milk cow |
Non-Patent Citations (3)
| Title |
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| 王群等: "荷斯坦奶牛脊椎畸形综合征焦磷酸测序检测方法建立与应用", 《中国动物检疫》 * |
| 王群等: "荷斯坦牛瓜氨酸血症焦磷酸测序检测方法的", 《中国奶牛》 * |
| 苏锐等: "荷斯坦牛隐性遗传缺陷病检测技术及应用", 《中国奶牛》 * |
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Application publication date: 20190215 |