CN107653333A - A kind of ox Babesia nest-type PRC specific primer and detection kit and nested PCR detection method - Google Patents
A kind of ox Babesia nest-type PRC specific primer and detection kit and nested PCR detection method Download PDFInfo
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Abstract
A kind of ox Babesia nest-type PRC specific primer and detection kit and nested PCR detection method, the simple expense of template DNA preparation process of this method is low, this method detection is very convenient quick, and the specificity and sensitiveness of detection method can be improved, the minigene group DNA of the portion of tissue organ extraction of single tick worm even tick worm can be used as template and be identified, compared to existing microscopic examination of perpheral blood smear diagnosis of technique babesiasis, the present invention only need to gather the epizoic tick of ox, by the DNA for extracting tick, utilize the ox Babesia outer primer and inner primer of design, nested PCR amplification target gene, can be accurate, quickly judge whether ox has infected ox Babesia.
Description
Technical field
Present invention relates particularly to biology techniques field, and in particular to a kind of ox Babesia nest-type PRC specific primer
And detection kit and nested PCR detection method.
Background technology
Ox Babesia is that one kind is propagated through tick, the entozoic protozoon of red blood cell, belongs to multiple top subphylum, by such parasite
Caused Babesia Gibsoni, in the world many areas occur and popular, also often there is generation China various regions, to animal husbandry and its people
Economy brings about great losses.After the cattle infected worm, often there are the symptoms such as anaemia, heating, hemoglobinuria, incoordination, seriously
Shi Yinqi is dead.The disease has had resulted in huge economic loss, and expands trend in world wide, thus causes domestic and international
The extensive concern of person.Babesia be through a kind of tick-borne Blood protozoan, parasitize mammalian erythropoietin, lymphocyte and
In other cells, it can also colonize in the various histocytes of tick.At present, regular-PCR detection technique is still that diagnosis colonizes in
The most frequently used method of Babesia in tick polypide, but this method has a very big shortcoming-low specificity, during amplification
Easily produce false positive.And found during ox Babesia is detected, between ox Babesia and two-pressure humidity generator
Often there is cross reaction.The higher method of Sensitivity and Specificity, such as reverse linear spot hybridization test（RBL）And PCR-
ELISA etc. has also been seen in report, but these methods all have the shortcomings that false positive rate is high.In addition, for economical and real
Reason, most of methods such as border application are not suitable for the laboratory diagnosis of epidemiology.Molecule diagnosis based on DNA level,
Its sensitiveness and the number of target gene amplification are closely bound up.Therefore the gene of one high copy number in genome of selection is made
The sensitiveness of existing PCR method will be greatly improved for target gene.
The content of the invention
In order to solve the defects of prior art and deficiency, the invention provides a kind of ox Babesia nest-type PRC specificity
Primer and detection kit and nested PCR detection method, establish a kind of easy, efficiently, practical DNA detection methods.This method
Available for babesiasis epidemiology survey and quick detection.
The technical solution that the present invention uses is：A kind of ox Babesia nest-type PRC specific primer, including design
Two pairs of specific primers, it is specially：
Primers F 1: 5'-CGA-GGA-AGG-AAC-TAC-CGA-TG-3'；
Primer R1: 5'-GCA-TAA-CGA-CGT-GCA-AAC-TT-3'`；
Primers F 2: 5'-ACC-GAT-GTT-GAA-TAT-CTT-G-3'；
Primer R2: 5'-CTT-GGA-AAG-AGT-TGG-AAT-CT-3'.
A kind of ox Babesia nest type PCR detection reagent, described nest type PCR detection reagent include following components：
（One）Two pairs of specific primers described in claim 1；
（Two）Positive control：Described positive control is the recombinant plasmid containing target gene fragment, and the plasmid contains Niu Babeisi
Worm 1092bp genetic fragments；
（Three）Negative control：Described negative control is deionized water；
（Four）Taq enzyme.
Described positive control converts DH5- α competent cells by the way that amplified production is cloned into pUCm-T, is reflected through digestion
The positive control for positive recombinant plasmid is obtained after fixed and sequencing.
A kind of ox Babesia nested PCR detection method, comprises the following steps：
（1）Tested specimen dna extraction：Taking boophilus microplus number, only alcohol washes, every boophilus microplus is put into single 1.5ml EP
Dof is managed, and 200 μ L PBS liquid submergence boophilus microplus, the polypide of tick is crushed with sample broke instrument, draws the solution after crushing, is added
Trizol reagents crack, and using phenol/chloroform of routine, ethanol precipitation, trishydroxymethylaminomethane-ethylenediamine are used after drying
Tetraacethyl dissolves, and -20 DEG C save backup；
（2）Expanded with outer primer F1, R1 in claim 1：Boophilus microplus sample in EP dof pipes after addition processing
DNA, while set positive and negative control, 10 × PCR Buffer, Mgcl2, dNTP, Taq enzyme, 25pmol/ μ L F1,25pmol/ μ L
R1, DNA profiling, ddH2First round PCR is carried out in the 50 μ L reaction systems that O is slightly centrifuged after mixing；
（3）Expanded with inner primer F2, R2 in claim 1：Using first round PCR primer DNA as template, using F2, R2 second
Primer is covered, in 10 × PCR Buffer 5 μ L, Mgcl22 μ L, dNTP 4 μ L, the μ L of 0.5 μ L, 25pmol/ μ L F2 of Taq enzyme 1,
The μ L of 25pmol/ μ L R2 1, DNA profiling 2 μ L, ddH2The 50 μ L reaction systems that the μ L of O 34.5 are slightly centrifuged after mixing carry out the second wheel
PCR；
（4）PCR primer is identified：The μ L of the second wheel PCR primer 2 are taken to carry out 1.5% agarose gel electrophoresis identification；
（5）Sequencing：Sequencing company is sent to carry out sequencing the second wheel PCR primer.
Described step（2）In first round PCR reaction condition be：
95 DEG C of preheating 5min；
95 DEG C of holding 30s, 55 DEG C of holding 1min, 72 DEG C keep 1min, and the step carries out totally 30 circulations；
Last extends to 72 DEG C, keeps 10min.
Described step（2）In second wheel PCR reaction conditions be：
95 DEG C of preheating 5min；
95 DEG C of holding 30s, 55 DEG C of holding 1min, 72 DEG C keep 1min, and the step carries out totally 30 circulations；
Last extends to 72 DEG C, keeps 10min.
Described step（2）Calf tick sample gene group DNA profiling amount is 5 μ l in middle first round PCR50 μ L reaction systems,
10 × PCR Buffer are 5 μ L, Mgcl2It is 4 μ L for 2 μ L, dNTP, Taq enzyme is that 0.5 μ L, 25pmol/ μ L F1 are 1 μ L,
25pmol/ μ L R1 are 1 μ L, ddH2O is 31.5 μ L.
Described step（4）In in the second wheel PCR50 μ L reaction systems first round PCR primer DNA profiling amount be 2 μ l, 10
× PCR Buffer are 5 μ L, Mgcl2It is 4 μ L for 2 μ L, dNTP, Taq enzyme is 0.5 μ L, 25pmol/ μ L F2 1 μ L, 25pmol/ μ
L R2 are 1 μ L, ddH2O is 34.5 μ L.
The beneficial effects of the invention are as follows：The invention provides a kind of ox Babesia nest-type PRC specific primer and detection
Kit and nested PCR detection method, the simple expense of template DNA preparation process of this method is low, and this method detection is very convenient
Fast, and the specificity and sensitiveness of detection method can be improved, single tick worm even the portion of tissue organ of tick worm extract micro
Genomic DNA can be used as template and be identified, compared to existing microscopic examination of perpheral blood smear diagnosis of technique babesiasis, this hair
Bright need to gather the epizoic tick of ox, by extracting the DNA of tick, using the ox Babesia outer primer and inner primer of design,
Nested PCR amplification target gene, it can accurately and rapidly judge whether ox has infected ox Babesia.
Brief description of the drawings
Fig. 1 is the inventive method to the RAP-1 gene first round（By external primer amplification）The wheels of PCR and second（Expanded by inner primer
Increase）The gel electrophoresis figure of PCR primer, wherein 1 is F1 and R1 PCR amplifications；2 be F2 and R2 PCR amplifications；3 are
Negative control.
Fig. 2 be RAP-1 gene nested PCR products sequencing result, common 297bp.
Fig. 3 is the digestion qualification result of recombinant plasmid.
Fig. 4 is the PCR qualification results of recombinant plasmid.
Embodiment
Nest type PCR detection reagent is set, and the kit includes：
(1) EP dof are managed：Reaction tube includes 10 × PCR Buffer, deoxyribonucleoside triphosphate（dNTP）, magnesium chloride
（Mgcl2）.
(2) primers F 1, primer R1, primers F 2, primer R2.Primers F 1, primer R1, primers F 2, primer R2 are pressed respectively
State the DNA fragmentation that base sequence is synthesized by DNA synthesizer：
Primers F 1: 5'-CGA-GGA-AGG-AAC-TAC-CGA-TG-3'；
Primer R1: 5'-GCA-TAA-CGA-CGT-GCA-AAC-TT-3'`；
Primers F 2: 5'-ACC-GAT-GTT-GAA-TAT-CTT-G-3'；
Primer R2: 5'-CTT-GGA-AAG-AGT-TGG-AAT-CT-3'.
(3) positive control：The control is the recombinant plasmid containing target gene fragment, is built by this laboratory.Its method
It is：Amplified production is cloned into pUCm-T conversion DH5- α competent cells, positive restructuring is obtained after digestion is identified and is sequenced
Plasmid, the plasmid Babesia containing ox 1092bp genetic fragments.
(4) negative control, the control are deionized water（ddH2O）；
(5) Taq enzyme.
Operation sequence：
(1) it is detected specimen dna extraction：Taking boophilus microplus number, only 75% alcohol washes, every boophilus microplus is put into individually
1.5ml EP dof are managed, and 200 μ L PBS liquid submergence tick, the polypide of tick are crushed with sample broke instrument, the solution after crushing is drawn, adds
Enter the cracking of Trizol reagents, using phenol/chloroform of routine, ethanol precipitation, trishydroxymethylaminomethane-second two is used after drying
Amine tetraacethyl（Tris-EDTA TE）Dissolving, -20 DEG C save backup；
(2) outer primer F1, R1 is expanded：Specimen dna in EP dof pipes after addition processing, while set positive and negative control, 50 μ
L reaction systems are：10 × PCR Buffer 5 μ L, Mgcl2（25mM）2 μ L, dNTP (2.5mM) 4 μ L, Taq enzyme（5U/μL）0.5μ
1 μ L, 25pmol/ μ L R1 of L, 25pmol/ μ L F1 1 μ L, DNA profiling 5 μ L, ddH2The μ L of O 31.5, are slightly centrifuged after mixing.
Amplification condition is：
95℃, 5min;
95℃, 30sec;
55℃, 1min;30×
72℃, 1min;
72℃, 10min
(3) inner primer F2, R2 is expanded：Using first round PCR primer DNA as template (1 μ L), using F2, R2 as second set of primer, 50 μ
L reaction systems are：10 × PCR Buffer 5 μ L, Mgcl2（25mM）2 μ L, dNTP (2.5mM) 4 μ L, Taq enzyme（5U/μL）0.5μ
1 μ L, 25pmol/ μ L R2 of L, 25pmol/ μ L F2 1 μ L, DNA profiling 2 μ L, ddH2The μ L of O 34.5, are slightly centrifuged after mixing.
Amplification condition is：
95℃, 5min;
95℃, 30sec;
55℃, 1min;30×
72℃, 1min;
72℃, 10min
(4) PCR primer is identified
The μ L of the second wheel PCR primer 2 are taken to carry out 1.5% agarose gel electrophoresis identification.
(5) it is sequenced
Sequencing company is sent to carry out sequencing the second wheel PCR primer.
The invention provides a kind of ox Babesia nest-type PRC specific primer and detection kit to detect with nest-type PRC
Method, it can quickly and accurately detect the ox Babesia in tested sample, it can also be used to the Molecular Epidemic of ox Babesia
Learn investigation and curative effect monitoring.The simple expense of template DNA preparation process of this method is low, and conventional method need to be through lysozyme, albumen
Enzyme K, SDS（Lauryl sodium sulfate）、CTAB（CTAB）Etc. agent treatment, time length is costly.This
Method detection is very convenient quick, and can improve the specificity and sensitiveness of detection method.The masterplate of inner primer amplification is outer
The product of side primer amplification, can second stage reaction be carried out, and react the first stage identification of correctness, ensure that whole
Change the accuracy and feasibility of reaction, the nested PCR detection method of foundation can meet the needs of clinical detection, and application prospect is wide
It is wealthy.
In existing technology, microscopic examination of perpheral blood smear technology is still to diagnose the most suitable method of babesiasis, but this method
It may not apply to the very low animal of periphery hematozoic parasite content and the resistance to detection with echiuran later of infection.And gather ox body
The parasitic boophilus microplus of table, by extracting the DNA of tick, using the ox Babesia outer primer and inner primer of design, nest-type PRC expands
Increase target gene, can accurately and rapidly judge whether ox has infected ox Babesia.
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (8)
1. a kind of ox Babesia nest-type PRC specific primer, it is characterised in that two pairs of specific primers including design, tool
Body is：
Primers F 1: 5'-CGA-GGA-AGG-AAC-TAC-CGA-TG-3'；
Primer R1: 5'-GCA-TAA-CGA-CGT-GCA-AAC-TT-3'`；
Primers F 2: 5'-ACC-GAT-GTT-GAA-TAT-CTT-G-3'；
Primer R2: 5'-CTT-GGA-AAG-AGT-TGG-AAT-CT-3'.
2. a kind of ox Babesia nest type PCR detection reagent, it is characterised in that described nest type PCR detection reagent includes
Following components：
（One）Two pairs of specific primers described in claim 1；
（Two）Positive control：Described positive control is the recombinant plasmid containing target gene fragment, and the plasmid contains Niu Babeisi
Worm 1092bp genetic fragments；
（Three）Negative control：Described negative control is deionized water；
（Four）Taq enzyme.
3. ox Babesia nest type PCR detection reagent according to claim 2, it is characterised in that described is positive right
DH5- α competent cells are converted according to by the way that amplified production is cloned into pUCm-T, are obtained after digestion is identified and is sequenced as the positive
The positive control of recombinant plasmid.
4. a kind of ox Babesia nested PCR detection method of specific primer using described in claim 1, its feature exist
In comprising the following steps：
（1）Tested specimen dna extraction：Taking boophilus microplus number, only alcohol washes, every boophilus microplus is put into single 1.5ml EP
Dof is managed, and 200 μ L PBS liquid submergence boophilus microplus, the polypide of tick is crushed with sample broke instrument, draws the solution after crushing, is added
Trizol reagents crack, and using phenol/chloroform of routine, ethanol precipitation, trishydroxymethylaminomethane-ethylenediamine are used after drying
Tetraacethyl dissolves, and -20 DEG C save backup；
（2）Expanded with outer primer F1, R1 in claim 1：Boophilus microplus sample in EP dof pipes after addition processing
DNA, while set positive and negative control, 10 × PCR Buffer, Mgcl2, dNTP, Taq enzyme, 25pmol/ μ L F1,25pmol/ μ L
R1, DNA profiling, ddH2First round PCR is carried out in the 50 μ L reaction systems that O is slightly centrifuged after mixing；
（3）Expanded with inner primer F2, R2 in claim 1：Using first round PCR primer DNA as template, using F2, R2 second
Primer is covered, in 10 × PCR Buffer, Mgcl2, dNTP, Taq enzyme, 25pmol/ μ L F2,25pmol/ μ L R2, DNA profiling,
ddH2The 50 μ L reaction systems that O is slightly centrifuged after mixing carry out the second wheel PCR；
（4）PCR primer is identified：The μ L of the second wheel PCR primer 2 are taken to carry out 1.5% agarose gel electrophoresis identification；
（5）Sequencing：Sequencing company is sent to carry out sequencing the second wheel PCR primer.
5. ox Babesia nested PCR detection method according to claim 4, it is characterised in that described step（2）In
First round PCR reaction condition be：
95 DEG C of preheating 5min；
95 DEG C of holding 30s, 55 DEG C of holding 1min, 72 DEG C keep 1min, and the step carries out totally 30 circulations；
Last extends to 72 DEG C, keeps 10min.
6. ox Babesia nested PCR detection method according to claim 4, it is characterised in that described step（2）In
Second wheel PCR reaction conditions be：
95 DEG C of preheating 5min；
95 DEG C of holding 30s, 55 DEG C of holding 1min, 72 DEG C keep 1min, and the step carries out totally 30 circulations；
Last extends to 72 DEG C, keeps 10min.
7. ox Babesia nested PCR detection method according to claim 4, it is characterised in that described step（2）In
Boophilus microplus sample gene group DNA profiling amount is that 5 μ l, 10 × PCR Buffer are 5 μ L in first round PCR50 μ L reaction system,
Mgcl2It is 4 μ L for 2 μ L, dNTP, Taq enzyme is that 0.5 μ L, 25pmol/ μ L F1 are that 1 μ L, 25pmol/ μ L R1 are 1 μ L, ddH2O
For 31.5 μ L.
8. ox Babesia nested PCR detection method according to claim 4, it is characterised in that described step（4）In
First round PCR primer DNA profiling amount is that 2 μ l, 10 × PCR Buffer are 5 μ L, Mgcl in second wheel PCR50 μ L reaction systems2
It is 4 μ L for 2 μ L, dNTP, Taq enzyme is that μ L, the 25pmol/ μ L R2 of 0.5 μ L, 25pmol/ μ L F2 1 are 1 μ L, ddH2O is 34.5 μ
L。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486223A (en) * | 2018-04-18 | 2018-09-04 | 华中农业大学 | A kind of Ji Shi Babesias RPA molecular detecting methods |
| CN110551837A (en) * | 2019-07-09 | 2019-12-10 | 沈阳农业大学 | Nested PCR (polymerase chain reaction) detection method for equine piroplasmosis |
| CN110951903A (en) * | 2020-01-20 | 2020-04-03 | 龙岩学院 | Universal primer for detecting blood protozoa in tiger or tick |
| CN113215293A (en) * | 2021-06-23 | 2021-08-06 | 成都大熊猫繁育研究基地 | Primer for detecting panda-derived babesia as well as kit and detection method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486223A (en) * | 2018-04-18 | 2018-09-04 | 华中农业大学 | A kind of Ji Shi Babesias RPA molecular detecting methods |
| CN110551837A (en) * | 2019-07-09 | 2019-12-10 | 沈阳农业大学 | Nested PCR (polymerase chain reaction) detection method for equine piroplasmosis |
| CN110951903A (en) * | 2020-01-20 | 2020-04-03 | 龙岩学院 | Universal primer for detecting blood protozoa in tiger or tick |
| CN113215293A (en) * | 2021-06-23 | 2021-08-06 | 成都大熊猫繁育研究基地 | Primer for detecting panda-derived babesia as well as kit and detection method thereof |
| CN113215293B (en) * | 2021-06-23 | 2022-09-13 | 成都大熊猫繁育研究基地 | Primer for detecting panda-derived babesia as well as kit and detection method thereof |
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Application publication date: 20180202 |