CN108130369A - A kind of primer, kit and detection method for detecting the short vertebra syndrome of cow - Google Patents
A kind of primer, kit and detection method for detecting the short vertebra syndrome of cow Download PDFInfo
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- CN108130369A CN108130369A CN201711477596.4A CN201711477596A CN108130369A CN 108130369 A CN108130369 A CN 108130369A CN 201711477596 A CN201711477596 A CN 201711477596A CN 108130369 A CN108130369 A CN 108130369A
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- 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
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- 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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Abstract
The invention discloses a kind of primer, kit and detection methods for detecting the short vertebra syndrome of cow, belong to biology field.The primer is by nucleotide sequence such as SEQ ID NO:Forward primer and nucleotide sequence such as SED ID NO shown in 1:Reverse primer composition shown in 2；The forward primer is marked with biotin；The reverse primer is marked with digoxin.The present invention is using the nucleic acid amplification technologies of recombinase polymerase and Sidestream chromatography technology, it is only necessary to by isothermal duplication after clinical sample progress DNA extractions, not needing to PCR instrument and carrying out thermal cycle reaction, testing result naked eyes can directly be read from Sidestream chromatography test strips.The short vertebra syndrome high specificity of primer detection cow of the present invention, amplification efficiency are high.The kit and detection method response procedures of the present invention is simple, can be effective, and rapidly to cow, short vertebra syndrome is detected, and suitable for cattle farm simplicity, quickly, accurately carries out the diagnosis of the disease.
Description
Technical field
The present invention relates to biology fields, and in particular to a kind of primer, reagent for detecting the short vertebra syndrome of cow
Box and detection method.
Background technology
Short vertebra syndrome (Brachyspina syndrome, BS) quilt in Denmark Holstein cow group for the first time in 2006
Report, is a kind of critical defect of congenital recessive heredity in cow body, can lead to embryo's deformity or even dead or stillbirth, and
The calf of short vertebra syndrome defect, in spite of the normal or long-term pregnancy period, it would still be possible to seriously kick the beam with weight, hypoevolutism
The phenomenon that.When detecting stillbirth calf, it is found that have the backbone of serious curtailment, four limbs are long and thin, the comprehensive characteristics such as internal organ exception.This
The molecular genetic mechanism that symptom is formed has lacking for 3329bp segments for the FANC1 genes on No. 21 chromosome (BTA21) of ox
Lose mutation (Deletion), cause 25,26,27 exons lose, and 877 amino acids C-terminal by containing only 26
Residual peptide substitution 451 amino acid peptide chains of script of amino acid.Parental generation both sides must carry the allele of short vertebra syndrome,
The just offspring of this negative homozygous genetic defect of meeting output.Therefore, the gene genetic detection before breeding is extremely important.
The short vertebra syndrome of cow (FANC1 gene delections) common method, predominantly PCR methods detection blood are examined at present
In FANC1 genes, this method specificity and high sensitivity, but the time need at least 60-90 minutes, and need complex instrument, not
(point-of-care test are detected suitable for point-of-care；POCT it) uses.Therefore, it is necessary to one kind can quick and precisely, letter
Easily, the detection kit without expensive instrument, available for the short vertebra syndrome of Site Detection cow (FANC1 gene delections) and inspection
Survey method.
Based on recombinase-polymerase nucleic acid amplification technologies (Recombinase-polymerase amplification,
RPA intracellular nucleic acid synthesis mechanism) is simulated, in the range of steady temperature, by recombinase, polymerase and single strand binding protein
DNA double chain is made to untwist, and DNA fragment specific is promoted to expand, to achieve the effect that nucleic acid in vitro expands.Whole process is rapid
(10-20 minutes) and in (37-42 DEG C) progress of low constant temperature.At present still is in for the research of RPA technologies the starting stage, and domestic
The outer report that RPA combination Sidestream chromatographies technology is applied to the short vertebra syndrome of cow (FANC1 gene delections) detection not yet.
The present invention uses the nucleic acid amplification technologies of recombinase-polymerase and combines Sidestream chromatography technology, provides one simply, quickly, no
Must expensive device can scene carry out the screening mode of the short vertebra syndrome of cow (FANC1 gene delections), have it is highly sensitive,
The characteristics of specific, suitable for cattle farm rapid screening.
Invention content
A kind of drawing for detection short vertebra syndrome of cow is provided it is an object of the invention to overcome the deficiencies in the prior art
Object, kit and the method for inspection, solve that the detection of cow in the prior art short vertebra syndrome is cumbersome to be difficult to meet demand
The problem of.
To achieve the above object, the technical solution that the present invention takes is as follows：
A kind of primer for being used to detect the short vertebra syndrome of cow, the primer is by nucleotide sequence such as SEQ ID NO:1
Shown forward primer and nucleotide sequence such as SED ID NO:Reverse primer composition shown in 2；The forward primer is marked with
Biotin (Biotin)；The reverse primer is marked with digoxin (DIG).
The primer of the present invention is the pathogenic genes sequence and TwistDx that inventor refers to NCBI gene databases
Instruction manual and Primer-BLAST design of primers principles, design and have synthesized a plurality of primer.In design process
In, consider not only the formation that avoid primer dimer, hairpin structure, it is also contemplated that different target gene is anti-at one
With answering in system coamplification problem.Primer screening experiment shows that primer specificity of the invention is strong, amplification efficiency is high, can effectively,
The rapidly detection short vertebra syndrome of cow (FANC1 gene delections).
The present invention also provides a kind of for detecting the kit of the short vertebra syndrome of cow, including above-mentioned primer.
The kit and detection method response procedures of above-mentioned technical proposal are simple, can be effective, rapidly the short vertebra to cow
Syndrome is detected, and suitable for cattle farm simplicity, quickly, accurately carries out the diagnosis of the disease.
For the preferred embodiment of the kit of the present invention for being used to detect the short vertebra syndrome of cow, the reagent
Box further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and Sidestream chromatography detection examination
Agent.
For the preferred embodiment of the kit of the present invention for being used to detect the short vertebra syndrome of cow, the positive
Interior Quality Control contains by such as SEQ ID NO:3 and SED ID NO:The primer pair of nucleotide sequence composition shown in 4.
For the preferred embodiment of the kit of the present invention for being used to detect the short vertebra syndrome of cow, the recombination
Enzyme is T4uvsX/uvsY；The polymerase is Bsu archaeal dna polymerases；The DNA binding protein is T4gp32；The buffering is molten
Liquid contains following components:Tris-HCl buffer solutions, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphocreatine, flesh
Acid kinase and Carbowax20M..
For the preferred embodiment of the kit of the present invention for being used to detect the short vertebra syndrome of cow, the buffering
Liquid contains the component of following concentration:Tris-HCl buffer solutions 50mM, potassium acetate 100mM, magnesium acetate 14mM, dithiothreitol (DTT) 2mM,
200 μM of dNTPs, ATP 3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L, Carbowax20M 5wt%；The Tris-
The pH value of HCl buffer solutions is 7.9.
For the preferred embodiment of the kit of the present invention for being used to detect the short vertebra syndrome of cow, the effluent
It chromatographs detection reagent and includes Sidestream chromatography test strips and hybridization check buffer solution.
To be of the present invention for detecting the preferred embodiment of the kit of the short vertebra syndrome of cow, described is miscellaneous
Detection buffer solution is handed over to be made of the component of following concentration：BSA 5wt% and PBS buffer solution 10mM.
It is described as of the present invention for detecting the preferred embodiment of the kit of the short vertebra syndrome of cow
Quality Control is to insert the recombinant plasmid of people's GAPDH genes in the positive.
The preparation method of Quality Control is in the positive：Respectively with nucleotide sequence such as SEQ ID No.3 and SEQ ID No.4
Shown primer is upstream and downstream primer, and with people's GAPDH gene cDNAs, its PCR product genetic fragment is recombinated to plastid (pMD-
It in 20T), treats to filter out single bacterium colony with antibiotic (ampicillin), then with Plasmid DNA Preparation Mini
Kit (QIAGEN, Valencia, CA) extracts Plasmid DNA, and correct recombinant plasmid is sequenced as Quality Control in the positive.
The present invention also provides it is a kind of using above-mentioned kit detection the short vertebra syndrome of cow method, including with
Lower step:
(1) DNA of extraction sample to be tested is as detection template；
(2) detection template is expanded, obtains DNA amplification sequence：Reaction system is prepared, shakes mixing reaction system,
Sample reacts 20 minutes in 37~42 DEG C, is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using Sidestream chromatography test strips：Take a certain number of Sidestream chromatography test strips, needle
Different samples to be tested are marked, each sample takes 50 μ L hybridization check buffer solutions, adds in reaction tube；Take 20 μ L amplification productions
Object mixing in centrifuge tube, amplified production and hybridization check buffer solution totally 70 μ L；Above-mentioned reaction solution is added in into test strips sample application zone,
It is incubated 5 minutes；
(4) point is presented according to test strips reaction zone and judges testing result：After incubation, naked eyes are observed immediately；If examination
Paper slip reaction zone shows test paper quality control point (Marker) and Quality Control point (Internal Control), then this reaction is effectively anti-
It should；If test strips reaction zone shows FANC1 gene delection points, sample is detected as the positive；If test strips reaction zone is not shown
Show FANC1 gene delections point, then detect sample as feminine gender.
Above-mentioned detection method is combined using the nucleic acid amplification technologies of recombinase-polymerase with Sidestream chromatography technology, simple,
Quickly, there is highly sensitive, specificity.
Sample to be tested derives from cow sample, such as whole blood (using EDTA as anticoagulant), oral mucosa cell, Skin Cell
Or spermatoblast.
Compared with prior art, the beneficial effects of the present invention are：
The nucleic acid amplification technologies of recombinase-polymerase are combined by the present invention with Sidestream chromatography technology, provide one it is simple,
Quickly, being not necessary to expensive device can the live screening mode for carrying out the short vertebra syndrome of cow (FANC1 gene delections).Without heat
Circular response and special instrument, testing result can directly be observed by vision, have the characteristics that highly sensitive, specificity,
Suitable for instant quick diagnosis, screening can be carried out in a non-laboratory environment, it is possible to provide the quick detection of cattle farm First Line.
Description of the drawings
Fig. 1 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect the short vertebra syndrome of cow
The test strips diagram of (FANC1 gene delections)；In figure, test strips include sample application zone, reaction zone and siphon area；Wherein reaction zone packet
The point of quality control containing test paper, FANC1 gene delections point and Quality Control point.
Fig. 2 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect the short vertebra syndrome of cow
The test strips reaction icon of (FANC1 gene delections)；In figure, effecting reaction can identify the homozygous base of wild type (Wild Type)
Because of type (Homozygous) reaction, deletion form (Deletion) heterozygous genotypes (Heterozygous) and deletion form
(Deletion) homozygous genotype (Homozygous)；Abortive response is no test paper quality control point and Quality Control point.
Fig. 3 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect the short vertebra syndrome of cow
(FANC1 gene delections), and for the negative findings figure that different detection samples are marked.
Fig. 4 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect the short vertebra syndrome of cow
(FANC1 gene delections), and for the positive findings figure that different detection samples are marked.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with the drawings and specific embodiments pair
The present invention further illustrates.It will be appreciated by those skilled in the art that specific embodiment described herein is only explaining this hair
It is bright, it is not intended to limit the present invention.
Test method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.
Embodiment 1:Primer and kit
1st, design of primers
According to the respiratory tract infection pathogenic genes sequence of NCBI gene databases, with reference to TwistDx instruction
Manual and Primer-BLAST design of primers principles design primer.Primer length is about 30-35nt, due to currently without needle
It is designed to the primer-design software of RPA, during previous work of the present invention and has synthesized a large amount of primers, therefrom filter out high sensitivity
And specific good primer is used for (table 1) of the invention.
2nd, primer synthesizes
According to the primer sequence shown in table 1, sequent synthesis is carried out.It is characterized in that, its FANC1 gene forward primer sequence
Such as SEQ ID NO:Shown in 1, and modify and add in Biotin；Reverse primer sequences such as SEQ ID NO:Shown in 2, and modify addition
DIG.Its interior Quality Control forward primer sequence such as SEQ ID NO:Shown in 3, and modify and add in Biotin；Reverse primer sequences such as SEQ
ID NO:Shown in 4, and modify and add in TAMRA.
Table 1
Include described in the present embodiment for the kit of the short vertebra syndrome of cow (FANC1 gene delections) quick diagnosis
Sense primer and downstream primer are stated, the kit further includes recombinase, polymerase, DNA binding protein, buffer solution, the positive
Interior Quality Control, ddH2O and Sidestream chromatography detection reagent.The recombinase is T4uvsX/uvsY, and the polymerase gathers for Bsu DNA
Synthase, the DNA binding protein are T4gp32；The buffer solution contains the component of following concentration:Tris-HCl buffer solutions 50mM,
Potassium acetate 100mM, magnesium acetate 14mM, 200 μM of dithiothreitol (DTT) 2mM, dNTPs, ATP 3mM, phosphocreatine 50mM, creatine swash
Enzyme 100ng/ μ L, Carbowax20M 5wt%；The pH value of the Tris-HCl buffer solutions is 7.9.The Sidestream chromatography detection examination
Agent includes Sidestream chromatography test strips and hybridization check buffer solution, and the hybridization check buffer solution is made of the component of following concentration：
BSA 5wt% and PBS buffer solution 10mM.
Embodiment 2 detects the short vertebra syndrome of cow (FANC1 gene delections)
The present embodiment uses the kit detection short vertebra syndrome of cow (FANC1 gene delections) described in embodiment 1, detection
The method used is recombination enzymatic polymerization enzyme constant temperature gene amplification method and combines Sidestream chromatography technology, is as follows:
1st, clinical sample prepares
Cow whole blood sample is collected, collection condition is:It is confirmed as having after laboratory examination (fluorescence quantitative RT-RCR)
The positive clinical sample (10) of the short vertebra syndrome of cow (FANC1 gene delections) and to be confirmed as no cow after inspection short
The negative clinical sample (10) of vertebra syndrome (FANC1 gene delections).Clinical sample is placed in -80 DEG C of preservations.
2nd, the nucleic acid extraction of sample
The present embodiment is extracted or is automated sample nucleic acid extraction apparatus using whole blood DNA col-umn chromatography, carries out clinical sample
Nucleic acid extraction.
3rd, prepared by positive reference product nucleic acid
Respectively with SEQ ID NO:3 and SEQ ID NO:Primer shown in 4 is upstream and downstream primer, with people's GAPDH genes
CDNA recombinates its PCR product genetic fragment into plastid (pMD-20T), treats to filter out list with antibiotic (ampicillin)
One bacterium colony, then Plasmid DNA is extracted with Plasmid DNA Preparation Mini Kit (QIAGEN, Valencia, CA), it surveys
The correct recombinant plasmid of sequence is Quality Control in the positive.
4th, reaction system is configured
By 2 proportional arrangement reaction system of table:
Table 2
5th, it shakes made from mixing step 4 after reaction system, sample carried out recombinase and gather in 42 DEG C of isothermal reactions 20 minutes
Synthase isothermal duplication.
6th, a certain number of Sidestream chromatography test strips are taken, are marked for different detection samples.Each detection sample
50 μ L hybridization checks buffer solutions (5wt%BSA in PBS) are taken, are added in reaction tube.The amplification obtained by 20 μ L steps (5) is taken to produce
Object mixing in centrifuge tube, amplified production and hybridization check buffer solution totally 70 μ L.Above-mentioned reaction solution is added in into test strips sample application zone,
It is incubated 5 minutes.
7th, after being incubated, naked eyes are observed immediately.If test strips reaction zone shows test paper quality control point (Marker) and matter
Point (Internal Control) is controlled, then this reaction is effecting reaction；If test strips reaction zone shows FANC1 gene delections
Point then detects sample as the positive；If test strips reaction zone does not show FANC1 gene delections point, sample is detected as feminine gender.
8th, result interpretation
The short vertebra syndrome of cow (FANC1 gene delections), institute are used for quickly detecting with the primer sets designed by the present invention
There is the sample of the short vertebra syndrome of the positive cow identified through laboratory RT-PCR method (FANC1 gene delections) in Sidestream chromatography
The short vertebra syndrome of cow (FANC1 gene delections) missing point is shown in test strips, all negative samples do not show this point
(Fig. 3-4, table 3).
Table 3
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of primer for being used to detect the short vertebra syndrome of cow, which is characterized in that the primer is by nucleotide sequence such as SEQ
ID NO:Forward primer and nucleotide sequence such as SED ID NO shown in 1:Reverse primer composition shown in 2；The forward primer
It is marked with biotin；The reverse primer is marked with digoxin.
2. a kind of kit for being used to detect the short vertebra syndrome of cow, which is characterized in that the kit includes claim 1
The primer.
3. the kit according to claim 2 for being used to detect the short vertebra syndrome of cow, which is characterized in that the reagent
Box further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and Sidestream chromatography detection examination
Agent.
4. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the positive
Interior Quality Control contains by such as SEQ ID NO:3 and SED ID NO:The primer pair of nucleotide sequence composition shown in 4.
5. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the recombination
Enzyme is T4uvsX/uvsY；The polymerase is Bsu archaeal dna polymerases；The DNA binding protein is T4gp32；The buffering is molten
Liquid contains following components:Tris-HCl buffer solutions, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphocreatine, flesh
Acid kinase and Carbowax20M..
6. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the buffering
Liquid contains the component of following concentration:Tris-HCl buffer solutions 50mM, potassium acetate 100mM, magnesium acetate 14mM, dithiothreitol (DTT) 2mM,
200 μM of dNTPs, ATP 3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L, Carbowax20M 5wt%；The Tris-
The pH value of HCl buffer solutions is 7.9.
7. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the effluent
It chromatographs detection reagent and includes Sidestream chromatography test strips and hybridization check buffer solution.
8. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the hybridization
Detection buffer solution is made of the component of following concentration：BSA 5wt% and PBS buffer solution 10mM.
9. the kit according to claim 3 for being used to detect the short vertebra syndrome of cow, which is characterized in that the sun
Quality Control is to insert the recombinant plasmid of people's GAPDH genes in property.
10. a kind of method of detection short vertebra syndrome of cow using claim 2~8 any one of them kit,
It is characterized in that, includes the following steps:
(1) DNA of extraction sample to be tested is as detection template；
(2) detection template is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using Sidestream chromatography test strips；
(4) point is presented according to test strips reaction zone and judges testing result.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711477596.4A CN108130369A (en) | 2017-12-29 | 2017-12-29 | A kind of primer, kit and detection method for detecting the short vertebra syndrome of cow |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711477596.4A CN108130369A (en) | 2017-12-29 | 2017-12-29 | A kind of primer, kit and detection method for detecting the short vertebra syndrome of cow |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005118853A2 (en) * | 2004-06-01 | 2005-12-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
| WO2012155995A1 (en) * | 2011-05-13 | 2012-11-22 | Université de Liège | Detecting the brachyspina mutation |
| CN104419752A (en) * | 2013-08-28 | 2015-03-18 | 北京奶牛中心 | Method and kit for detecting bovine FANCI gene deletion |
| CN104862420A (en) * | 2015-06-01 | 2015-08-26 | 山东省农业科学院奶牛研究中心 | Primer, probe and kit for detecting foot-and-mouth disease virus in aerosol through RPA-lateral flow assay technology |
-
2017
- 2017-12-29 CN CN201711477596.4A patent/CN108130369A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005118853A2 (en) * | 2004-06-01 | 2005-12-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
| WO2012155995A1 (en) * | 2011-05-13 | 2012-11-22 | Université de Liège | Detecting the brachyspina mutation |
| CN104419752A (en) * | 2013-08-28 | 2015-03-18 | 北京奶牛中心 | Method and kit for detecting bovine FANCI gene deletion |
| CN104862420A (en) * | 2015-06-01 | 2015-08-26 | 山东省农业科学院奶牛研究中心 | Primer, probe and kit for detecting foot-and-mouth disease virus in aerosol through RPA-lateral flow assay technology |
Non-Patent Citations (1)
| Title |
|---|
| 郑秀芬: "《法医DNA分析》", 31 May 2002, 中国人民公安大学出版社 * |
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Application publication date: 20180608 |