CN110317888A - A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer - Google Patents
A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer Download PDFInfo
- Publication number
- CN110317888A CN110317888A CN201910267851.5A CN201910267851A CN110317888A CN 110317888 A CN110317888 A CN 110317888A CN 201910267851 A CN201910267851 A CN 201910267851A CN 110317888 A CN110317888 A CN 110317888A
- Authority
- CN
- China
- Prior art keywords
- sybr green
- primer
- blood
- cat
- dna
- 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
- 210000004369 blood Anatomy 0.000 title claims abstract description 59
- 239000008280 blood Substances 0.000 title claims abstract description 59
- 241000282326 Felis catus Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 40
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 241001661918 Bartonia Species 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 238000013461 design Methods 0.000 claims abstract description 6
- 238000010790 dilution Methods 0.000 claims abstract description 6
- 239000012895 dilution Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 12
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 10
- 239000008346 aqueous phase Substances 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
- 239000012498 ultrapure water Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 239000007983 Tris buffer Substances 0.000 claims description 6
- -1 Tris saturated phenol Chemical class 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 6
- 230000004087 circulation Effects 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- 238000005457 optimization Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 5
- 244000052769 pathogen Species 0.000 claims description 4
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 241000963347 Mycoplasma haemocanis Species 0.000 claims description 3
- 241000579205 Mycoplasma suis Species 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 238000002844 melting Methods 0.000 claims description 3
- 230000008018 melting Effects 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- MVMXJBMAGBRAHD-UHFFFAOYSA-N picoperine Chemical compound C=1C=CC=NC=1CN(C=1C=CC=CC=1)CCN1CCCCC1 MVMXJBMAGBRAHD-UHFFFAOYSA-N 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 241000546176 Mycoplasma haemomuris Species 0.000 claims 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims 1
- 239000000284 extract Substances 0.000 claims 1
- 235000017281 sodium acetate Nutrition 0.000 claims 1
- 239000001632 sodium acetate Substances 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 6
- 238000001502 gel electrophoresis Methods 0.000 abstract description 4
- 108020004711 Nucleic Acid Probes Proteins 0.000 abstract description 3
- 239000002853 nucleic acid probe Substances 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 32
- 239000000523 sample Substances 0.000 description 21
- 230000003321 amplification Effects 0.000 description 16
- 238000003199 nucleic acid amplification method Methods 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 241000204031 Mycoplasma Species 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108010034145 Helminth Proteins Proteins 0.000 description 1
- 241001148639 Mycoplasma haemofelis Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000606651 Rickettsiales Species 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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
- C12Q1/68—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]
-
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to technical field of gene detection, it is related to a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer, the following steps are included: complete genome DNA in cat blood is extracted, and design primer, primer dilution, optimize reaction condition, solubility curve is drawn, standard curve, detection specificity are drawn, measure the coefficient of variation, verifying and interpretation of result.Detection method advantage is to establish the round pcr that can be detected without nucleic acid probe, simply, quickly, it is cheap, it can be realized in general laboratory, compared with traditional regular-PCR method, this method is not necessarily to carry out gel electrophoresis analysis to PCR product, avoidable product causes template to pollute, and can carry out interpretation result by observing response in real time, avoids the false positive results that normal PCR is easy to produce to a certain extent.
Description
Technical field
The invention belongs to technical field of gene detection, are related to a kind of cat blood bartonia bodies SYBR Green I fluorescent PCR inspection
Survey method and primer.
Background technique
Haemobartonella felis disease is a kind of disease as caused by helminth, the also known as infectious anemia of cat.Its micro- life of causing a disease
Object is generally similar to mycoplasma form, and different from Haemobartonella.Cat blood bartonia bodies is between bacterium and Richettsia
Between a kind of microorganism, be Rickettsiales, no slurry section, Haemobartonella.Pathogen in spherical, rod-shaped or cyclic annular,
It is not of uniform size.Generally in erythrocyte surface, it is free in blood plasma sometimes.In the Blood piece of Wright's staining, thallus is in black-and-blue or purple
Color.
SYBR Green I fluorescent PCR: refer to the fuel of specific binding double-stranded DNA, in conjunction with fluorescence enhancement after double-stranded DNA
1000 times or so, and there is stabilization to DNA double chain structure, pcr amplification product can be continuously monitored.
Currently, having regular-PCR and fluorescent probe PCR detection for cat blood bartonia bodies cause of disease detection technique.Commonly
PCR sensibility is lower than fluorescent probe PCR, and cannot quantify the copy number of DNA.Fluorescent probe PCR, it is by probe technique and PCR
Technology organically combines, and detects target DNA or mRNA in sample by the DNA content in PCR product come quantitative analysis
Content, but probe is expensive, is unfavorable for common lab conventional detection, is unable to satisfy actual demand.
Summary of the invention
It is a kind of simple, quick, cheap the purpose of the invention is to provide, it can be realized in general laboratory,
Compared with traditional regular-PCR method, without carrying out gel electrophoresis analysis to PCR product, avoidable product causes template dirty
Dye, and interpretation result can be carried out by observing response in real time, it can largely avoid the false positive that normal PCR is easy to produce
As a result detection method.
In order to achieve the above object, the invention adopts the following technical scheme:
A kind of cat blood bartonia bodies SYBR Green I fluorescent PCR detecting primer, the primer sequence are as follows:
F-2a:5'-GAA TAA GTG ACA GCW AAC TAT-3'；
R-2b:5'-ATA TCT ACR CAT TCC ACC GCT-3'；
A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method, comprising the following steps:
(1) complete genome DNA in cat blood is extracted；
(2) for cat blood bartonia bodies pathogen 16S rRNA gene in cat blood, it is conservative design primer: to find it
Area designs SYBR Green I amplified fluorescence primer；
(3) primer in step (2) is diluted using sterilizing ultrapure water；
(4) optimize reaction condition；
(5) result of reaction condition after circulation terminates is optimized according to step (4) and draws solubility curve；
(6) standard curve is drawn；
(7) detection specificity: positive, haemobartonella canis and mouse blood Ba Ertong to eperythrozoon suis microscopy respectively
Family name's body synthesizes its 16S rRNA sequence and detects, to evaluate its specificity；
(8) it measures the coefficient of variation: 2 repeating pipes being set to same positive criteria product, with the real-time fluorescence quantitative PCR of optimization
Condition detects positive criteria product, evaluates its repeatability, the coefficient of variation in calculating group；The standard positive sample is placed in-
20 DEG C of refrigerators save, the 1st after building on preservation of cat blood bartonia bodies SYBR Green I fluorescent quantitation PCR detection method
Week, 2 weeks and the 3rd Zhou Chongjian calculate its between-group variation coefficient to evaluate its reproducibility；
(9) verifying of SYBR Green I fluorescence PCR detecting method: by the cat anticoagulated blood sample extraction nucleic acid of collection
After carry out with SYBR Green I fluorescence PCR method detection blood in cat blood bartonia bodies it is whether positive.
Preferably, in the step (1), complete genome DNA step in cat blood is extracted:
1. the blood sample of frost is dissolved at room temperature；
2. 1ml blood is taken to be transferred in a sterile 2ml centrifuge tube, isometric PBS phosphate buffer is added, it is sufficiently mixed
12000rpm is centrifuged 5min after even, abandons supernatant；
3. 667 μ l STE, 20% SDS of 24 μ l, 37 DEG C of water-bath 1h are added；
4. plus the Proteinase K mixing of the 20mg/ml of 10 μ l, 55 DEG C of water-baths digest overnight (10~14h)；5. by the sample of digestion
Isometric Tris saturated phenol is added in product, sufficiently shakes up, and 12000rpm is centrifuged 10min；
6. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；Isometric Tris saturated phenol is added, is sufficiently shaken
Even, 12000rpm is centrifuged 10min；
7. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And it is added isometric phenol: chloroform: isoamyl alcohol (25:
24: 1), vortex is vibrated to mixing well, and 12000rpm is centrifuged 10min；
8. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And isometric chloroform: isoamyl alcohol (24: 1) is added,
Sufficiently oscillation mixes, and 12000rpm is centrifuged 10min；
9. supernatant is transferred in another sterile 2ml centrifuge tube；The ice ethyl alcohol of 2 times of volumes, 1/10 volume acetic acid is added
Sodium level is shaken, until visible cotton-shaped DNA is precipitated；
10. sample is set -20 DEG C of refrigerator freezing 30min or 15~20min of ice bath, 12000rpm is centrifuged again after taking-up
10min precipitates DNA；
DNA is chosen in another sterile centrifugation tube with pipette tips, with suitable 70% ethanol washing and is shaken, to wash out
The impurity of DNA；
Ethyl alcohol is poured out, by DNA vacuum drying or naturally dry, suitable TE buffer back dissolving, -20 DEG C of guarantors are added
It deposits spare.
Preferably, in the step (3), the method for sterilizing ultrapure water dilution primer is used are as follows: dilute using sterilizing ultrapure water
Primer is released, upstream and downstream primer concentration is 50pmol/ μ L, dispenses -20 DEG C of postposition preservations.
Preferably, optimize reaction condition in the step (4) are as follows: use 2 × Super Real PreMix Plus
(with SYBR Green I) recommend 25 μ L reaction systems, with occur highest fluorescent value Δ Rn, the smallest Ct value and
Not occurring non-specific peak in melting curve is index, is optimized to annealing temperature (55~65 DEG C).
Preferably, the optimization reaction condition is best are as follows: 95 DEG C of initial denaturation 2min；(95 DEG C of denaturation 1min；60 DEG C of annealing
30sec (collecting fluorescence)) × 40 circulations.
Preferably, it is method that standard curve is drawn in the step (6) are as follows: measures positive matter with ultraviolet specrophotometer
Grain standard items 16S rRNA gene order calculates DNA copy number, then using the concentration of trace dna analyzer measurement plasmid
The progress of standard positive sample is serially diluted (10 for 10 times-1~10-9), it is expanded with optimizing reaction condition in step (4), with
Copy number is abscissa, Ct value is that ordinate draws standard curve.
Experiment effectiveness analysis:
The setting of interpretation of result condition, threshold value setting principle are adjusted, just with threshold line according to noise of instrument situation
Subject to highest point more than negative control sample amplification curve.It is successively that high concentration is bent to low concentration amplification from left to right in Fig. 3
Line when expanding to the template DNA solution that 2uL dilution is respectively 10-1~10-9, obtains a S as can be seen from Figure
Shape curve, and to dilution be 10-9 template DNA solution expand when, amplification curve substantially with blank control maintain an equal level, show
16S rRNA gene cannot effectively be detected under the concentration, the minimum inspection of the SYBR Green I fluorescence PCR detecting method
36.8 × 10 are limited out-8ng/uL。
Quality control standard:
Negative control Ct value is less than 38 or without amplification curve, and characteristic amplification curve should occur in positive control, and Ct value is small
In 25-30, such as negative and positive control is unsatisfactory for conditions above, and experiment is invalid, must reform.
Result judgement:
Negative: Ct value shows in sample less than 38 or without Ct value and atypism amplification curve without cat blood Ba Ertongshi
Body DNA；It is positive: characteristic amplification curve occur, and Ct value indicates to contain cat blood bartonia bodies DNA in sample less than 34.
The beneficial effects of the present invention are:
The method of the present invention can be detected PCR in specific detection without nucleic acid probe, and simply, quick, price is low
It is honest and clean, than more easily realized in general laboratory in the past, compared with traditional regular-PCR method, this method without pair
PCR product carries out gel electrophoresis analysis, can avoid template caused by product and pollutes, and can carry out interpretation knot by observing response in real time
Fruit can effectively avoid the false positive results that normal PCR is easy to produce.
Detailed description of the invention
Fig. 1 is the dissolution of a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method of the present invention and primer
Curve graph；
Fig. 2 is the standard of a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method of the present invention and primer
Curve graph；
Fig. 3 is the sensitive of a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer of the invention
It writes music line chart；
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
With reference to Fig. 1~3,
A kind of cat blood bartonia bodies SYBR Green I fluorescent PCR detecting primer, the primer sequence are as follows:
F-2a:5'-GAA TAA GTG ACA GCW AAC TAT-3'；
R-2b:5'-ATA TCT ACR CAT TCC ACC GCT-3'；
A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method, which is characterized in that including following step
It is rapid:
(1) complete genome DNA in cat blood is extracted；
(2) for cat blood bartonia bodies pathogen 16S rRNA gene in cat blood, it is conservative design primer: to find it
Area designs SYBR Green I amplified fluorescence primer；
(3) primer in step (2) is diluted using sterilizing ultrapure water；
(4) optimize reaction condition；
(5) result of reaction condition after circulation terminates is optimized according to step (4) and draws solubility curve；
(6) standard curve is drawn；
(7) detection specificity: positive, haemobartonella canis and mouse blood Ba Ertong to eperythrozoon suis microscopy respectively
Family name's body synthesizes its 16S rRNA sequence and detects, to evaluate its specificity；
(8) it measures the coefficient of variation: 2 repeating pipes being set to same positive criteria product, with the real-time fluorescence quantitative PCR of optimization
Condition detects positive criteria product, evaluates its repeatability, the coefficient of variation in calculating group；The standard positive sample is placed in-
20 DEG C of refrigerators save, the 1st after building on preservation of cat blood bartonia bodies SYBR Green I fluorescent quantitation PCR detection method
Week, 2 weeks and the 3rd Zhou Chongjian calculate its between-group variation coefficient to evaluate its reproducibility；
(9) verifying of SYBR Green I fluorescence PCR detecting method: by the cat anticoagulated blood sample extraction nucleic acid of collection
After carry out with SYBR Green I fluorescence PCR method detection blood in cat blood bartonia bodies it is whether positive.
Further, in the step (1), complete genome DNA step in cat blood is extracted:
1. the blood sample of frost is dissolved at room temperature；
2. 1ml blood is taken to be transferred in a sterile 2ml centrifuge tube, isometric PBS phosphate buffer is added, it is sufficiently mixed
12000rpm is centrifuged 5min after even, abandons supernatant；
3. 667 μ l STE, 20% SDS of 24 μ l, 37 DEG C of water-bath 1h are added；
4. plus the Proteinase K mixing of the 20mg/ml of 10 μ l, 55 DEG C of water-baths digest overnight (10~14h)；5. by the sample of digestion
Isometric Tris saturated phenol is added in product, sufficiently shakes up, and 12000rpm is centrifuged 10min；
6. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；Isometric Tris saturated phenol is added, is sufficiently shaken
Even, 12000rpm is centrifuged 10min；
7. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And it is added isometric phenol: chloroform: isoamyl alcohol (25:
24: 1), vortex is vibrated to mixing well, and 12000rpm is centrifuged 10min；
8. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And isometric chloroform: isoamyl alcohol (24: 1) is added,
Sufficiently oscillation mixes, and 12000rpm is centrifuged 10min；
9. supernatant is transferred in another sterile 2ml centrifuge tube；The ice ethyl alcohol of 2 times of volumes, 1/10 volume acetic acid is added
Sodium level is shaken, until visible cotton-shaped DNA is precipitated；
10. sample is set -20 DEG C of refrigerator freezing 30min or 15~20min of ice bath, 12000rpm is centrifuged again after taking-up
10min precipitates DNA；
DNA is chosen in another sterile centrifugation tube with pipette tips, with suitable 70% ethanol washing and is shaken, to wash out
The impurity of DNA；
Ethyl alcohol is poured out, by DNA vacuum drying or naturally dry, suitable TE buffer back dissolving, -20 DEG C of guarantors are added
It deposits spare.
Further, in the step (3), the method for sterilizing ultrapure water dilution primer is used are as follows: use sterilizing ultrapure water
Primer is diluted, upstream and downstream primer concentration is 50pmol/ μ L, dispenses -20 DEG C of postposition preservations.
Further, optimize reaction condition in the step (4) are as follows: use 2 × Super Real PreMix Plus
(with SYBR Green I) recommend 25 μ L reaction systems, with occur highest fluorescent value Δ Rn, the smallest Ct value and
Not occurring non-specific peak in melting curve is index, is optimized to annealing temperature (55~65 DEG C).
Further, the optimization reaction condition is best are as follows: 95 DEG C of initial denaturation 2min；(95 DEG C of denaturation 1min；60 DEG C of annealing
30sec (collecting fluorescence)) × 40 circulations.
Further, it is method that standard curve is drawn in the step (6) are as follows: is measured with ultraviolet specrophotometer positive
Plasmid standard 16S rRNA gene order calculates DNA copy number using the concentration of trace dna analyzer measurement plasmid, with
Standard positive sample is carried out 10 times afterwards and is serially diluted (10-1~10-9), it is expanded with optimizing reaction condition in step (4),
It is that ordinate draws standard curve using copy number as abscissa, Ct value.
Experiment effectiveness analysis:
Threshold value setting principle: the setting of interpretation of result condition is adjusted, just with threshold line according to noise of instrument situation
Subject to highest point more than negative control sample amplification curve.It is successively that high concentration is bent to low concentration amplification from left to right in Fig. 3
Line is respectively as can be seen from Figure 10 to 2uL dilution-1~10-9Template DNA solution amplification when, obtain a S-shaped
Curve, and be 10 to dilution-9Template DNA solution amplification when, amplification curve substantially with blank control maintain an equal level, show dense at this
16S rRNA gene cannot effectively be detected under degree, the minimum detectability of the SYBR Green I fluorescence PCR detecting method
Position 36.8 × 10-8ng/uL。
Quality control standard:
Negative control Ct value is less than 38 or without amplification curve, and characteristic amplification curve should occur in positive control, and Ct value is small
In 25-30, such as negative and positive control is unsatisfactory for conditions above, and experiment is invalid, must reform.
Result judgement:
Negative: Ct value shows in sample less than 38 or without Ct value and atypism amplification curve without cat blood Ba Ertongshi
Body DNA；It is positive: characteristic amplification curve occur, and Ct value indicates to contain cat blood bartonia bodies DNA in sample less than 34.
In conclusion a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method of the present invention and primer, mention
It is simple, quick, cheap for a kind of round pcr that can be detected without nucleic acid probe, it can be real in general laboratory
Existing, compared with traditional regular-PCR method, this method is not necessarily to carry out gel electrophoresis analysis to PCR product, can avoid product
Cause template to pollute, and interpretation result can be carried out by observing response in real time, avoids what normal PCR was easy to produce to a certain extent
False positive results.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Claims (7)
1. a kind of cat blood bartonia bodies SYBR Green I fluorescent PCR detecting primer, which is characterized in that the primer sequence is such as
Under:
F-2a:5'-GAA TAA GTG ACA GCW AAC TAT-3'；
R-2b:5'-ATA TCT ACR CAT TCC ACC GCT-3'。
2. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method, which comprises the following steps:
(1) complete genome DNA in cat blood is extracted；
(2) design primer: for cat blood bartonia bodies pathogen 16S rRNA gene in cat blood, finding its conserved region, if
Count SYBR Green I amplified fluorescence primer；
(3) primer in step (2) is diluted using sterilizing ultrapure water；
(4) optimize reaction condition；
(5) result of reaction condition after circulation terminates is optimized according to step (4) and draws solubility curve；
(6) standard curve is drawn；
(7) detection specificity: positive, haemobartonella canis and haemobartonella muris to eperythrozoon suis microscopy respectively
It synthesizes its 16S rRNA sequence to be detected, to evaluate its specificity；
(8) it measures the coefficient of variation: 2 repeating pipes being set to same positive criteria product, with the real-time fluorescence quantitative PCR condition of optimization
Positive criteria product is detected, its repeatability, the coefficient of variation in calculating group are evaluated；The standard positive sample is placed in -20 DEG C
Refrigerator saves, the 1st week, 2 weeks after building on preservation of cat blood bartonia bodies SYBR Green I fluorescent quantitation PCR detection method
Its between-group variation coefficient is calculated with the 3rd Zhou Chongjian to evaluate its reproducibility；
(9) verifying of SYBR Green I fluorescence PCR detecting method: the cat anticoagulated blood sample extraction nucleic acid of collection is laggard
Whether row is positive with cat blood bartonia bodies in SYBR Green I fluorescence PCR method detection blood.
3. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method according to claim 2, feature
It is: in the step (1), extracts complete genome DNA step in cat blood:
1. the blood sample of frost is dissolved at room temperature；
2. 1ml blood is taken to be transferred in a sterile 2ml centrifuge tube, isometric PBS phosphate buffer is added, after mixing well
12000rpm is centrifuged 5min, abandons supernatant；
3. 667 μ l STE, 20% SDS of 24 μ l, 37 DEG C of water-bath 1h are added；
4. plus the Proteinase K mixing of the 20mg/ml of 10 μ l, 55 DEG C of water-baths digest overnight (10~14h)；5. the sample of digestion is added
Enter isometric Tris saturated phenol, sufficiently shake up, 12000rpm is centrifuged 10min；
6. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；Isometric Tris saturated phenol is added, is sufficiently shaken up,
12000rpm is centrifuged 10min；
7. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And it is added isometric phenol: chloroform: isoamyl alcohol (25: 24:
1), vortex is vibrated to mixing well, and 12000rpm is centrifuged 10min；
8. upper strata aqueous phase is transferred in another sterile 2ml centrifuge tube；And isometric chloroform: isoamyl alcohol (24: 1) is added, sufficiently
Oscillation mixes, and 12000rpm is centrifuged 10min；
9. supernatant is transferred in another sterile 2ml centrifuge tube；The ice ethyl alcohol of 2 times of volumes, 1/10 volume sodium acetate water is added
Yawing is dynamic, until visible cotton-shaped DNA is precipitated；
10. sample is set -20 DEG C of refrigerator freezing 30min or 15~20min of ice bath, 12000rpm is centrifuged 10min again after taking-up,
Precipitate DNA；
DNA is chosen in another sterile centrifugation tube with pipette tips, with suitable 70% ethanol washing and is shaken, to wash out DNA's
Impurity；
Ethyl alcohol is poured out, by DNA vacuum drying or naturally dry, suitable TE buffer back dissolving is added, -20 DEG C of preservations are standby
With.
4. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method according to claim 2, feature
It is: in the step (3), uses the method for sterilizing ultrapure water dilution primer are as follows: primer is diluted using sterilizing ultrapure water, on
Downstream primer concentration is 50pmol/ μ L, dispenses -20 DEG C of postposition preservations.
5. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method according to claim 2, feature
It is: optimizes reaction condition in the step (4) are as follows: uses 2 × Super Real PreMix Plus (with SYBR
Green I) recommend 25 μ L reaction systems, to there is highest fluorescent value Δ Rn, the smallest Ct value and in melting curve
Not occurring non-specific peak is index, is optimized to annealing temperature (55~65 DEG C).
6. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method according to claim 5, feature
Be: the optimization reaction condition is best are as follows: 95 DEG C of initial denaturation 2min；(95 DEG C of denaturation 1min；60 DEG C of annealing 30sec (are collected glimmering
Light)) × 40 circulations.
7. a kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method according to claim 2, feature
Be: it is method that standard curve is drawn in the step (6) are as follows: measures positive plasmid standard items 16S with ultraviolet specrophotometer
RRNA gene order calculates DNA copy number, then by standard positive sample using the concentration of trace dna analyzer measurement plasmid
10 times of this progress are serially diluted (10-1~10-9), it is expanded with optimizing reaction condition in step (4), with copy number for horizontal seat
Mark, Ct value are that ordinate draws standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910267851.5A CN110317888A (en) | 2019-04-03 | 2019-04-03 | A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910267851.5A CN110317888A (en) | 2019-04-03 | 2019-04-03 | A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110317888A true CN110317888A (en) | 2019-10-11 |
Family
ID=68112934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910267851.5A Pending CN110317888A (en) | 2019-04-03 | 2019-04-03 | A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110317888A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111719005A (en) * | 2020-06-29 | 2020-09-29 | 天津拓瑞医药科技有限公司 | PCR detection method for mycoplasma feline haemophilus |
| CN112410445A (en) * | 2020-11-17 | 2021-02-26 | 武汉大学 | Method for detecting croton body by TaqMan probe fluorescence quantitative PCR and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935696A (en) * | 2010-04-01 | 2011-01-05 | 中国农业科学院上海兽医研究所 | Fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira as well as primer and detection kit thereof |
| CN102994641A (en) * | 2012-12-14 | 2013-03-27 | 山东滨州沃华生物工程有限公司 | SYBR green I fluorogenic quantitative PCR (polymerase chain reaction) detecting method of pig proliferative enteropathy Lawsonia Intracellularis |
| US20170022519A1 (en) * | 2013-11-28 | 2017-01-26 | Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii (Cnic) | Stable episomes based on non-integrative lentiviral vectors |
-
2019
- 2019-04-03 CN CN201910267851.5A patent/CN110317888A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935696A (en) * | 2010-04-01 | 2011-01-05 | 中国农业科学院上海兽医研究所 | Fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira as well as primer and detection kit thereof |
| CN102994641A (en) * | 2012-12-14 | 2013-03-27 | 山东滨州沃华生物工程有限公司 | SYBR green I fluorogenic quantitative PCR (polymerase chain reaction) detecting method of pig proliferative enteropathy Lawsonia Intracellularis |
| US20170022519A1 (en) * | 2013-11-28 | 2017-01-26 | Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii (Cnic) | Stable episomes based on non-integrative lentiviral vectors |
Non-Patent Citations (2)
| Title |
|---|
| 冯育芳等: "应用实时荧光定量PCR方法检测实验用猫巴尔通体的感染", 《实验动物科学》 * |
| 吕艳等: "云南部分地区猫血巴尔通氏体感染状况的监测", 《畜牧兽医学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111719005A (en) * | 2020-06-29 | 2020-09-29 | 天津拓瑞医药科技有限公司 | PCR detection method for mycoplasma feline haemophilus |
| CN112410445A (en) * | 2020-11-17 | 2021-02-26 | 武汉大学 | Method for detecting croton body by TaqMan probe fluorescence quantitative PCR and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103275862B (en) | Fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) kit for detecting influenza A virus subtype H7N9 | |
| CN103642945B (en) | A kind of highly sensitive Epstein-Barr FLuorescent quantitative PCR kit containing internal reference | |
| Hosmillo et al. | Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses | |
| CN105624329A (en) | Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1 | |
| CN106435039A (en) | Primers, probe, kit and method for detecting PRRS (porcine reproductive and respiratory syndrome) NADC-30 strain | |
| CN110317888A (en) | A kind of cat blood bartonia bodies SYBR Green I fluorescence PCR detecting method and primer | |
| CN103866034A (en) | Multiple real-time fluorescence quantification PCR (polymerase chain reaction) detection kit and detection method for helicobacter pylori in gastric juice | |
| CN103509877A (en) | Fluorescence quantitative PCR kit used for detecting PRV, and application thereof | |
| CN101519697A (en) | Phytophthora sojae molecule detecting primer and detection reagent kit thereof | |
| CN108018379A (en) | A kind of prawn irido virus real-time fluorescence quantitative PCR detection kit | |
| CN103060452B (en) | Kit for detecting chlamydia trachomatis (CT) | |
| Couetil et al. | Equine respiratory viruses, airway inflammation and performance in thoroughbred racehorses | |
| CN106676198A (en) | High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5 | |
| CN103122396B (en) | Kit for detecting human cytomegalovirus (HCMV) | |
| CN101328506B (en) | Fluorescent quantitative PCR rapid diagnosis reagent kit for specific detection of classical swine fever virus wild virus infection and its application method | |
| CN101613751A (en) | The PCR kit for fluorescence quantitative of rapid detection toxoplasma gondii | |
| CN105779650B (en) | For identifying primer, probe and the kit of the triple fluorescent quantitative PCR of pseudorabies virus strain | |
| CN101705310A (en) | Fluorescent quantization PCR detection method for swine astrovirus | |
| CN110373496A (en) | Kit and detection method for the detection of 8 type aviadenovirus of I subgroup serum | |
| CN101831506A (en) | Kit for authenticating deletion of vaccine strain and wild strain of PRVgE | |
| Zhang et al. | Diagnostics and detection of African swine fever virus | |
| CN102994641A (en) | SYBR green I fluorogenic quantitative PCR (polymerase chain reaction) detecting method of pig proliferative enteropathy Lawsonia Intracellularis | |
| CN103074429B (en) | UU (ureaplasma urealyticum) detection kit | |
| CN106119421A (en) | Fluorescent quantitation detection primer, probe and the test kit of pig blue-ear disease QYYZ strain | |
| CN102154523B (en) | Primer for detecting human BK viral nucleic acid, fluorescent probe and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191011 |