CN105734158A - Fluorescent PCR (polymerase chain reaction) detection kit for babesia caballi disease - Google Patents
Fluorescent PCR (polymerase chain reaction) detection kit for babesia caballi disease Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and in particular relates to a babesia caballi disease detection kit based on a fluorescent quantitative PCR (polymerase chain reaction) scalar quantity positive control, which is used for clinical diagnosis and daily monitoring of equine animal infected babesia caballi disease. By designing detection primers as shown by SEQ ID No.1 and SEQ ID No.2 and a fluorescent probe as shown by SEQ ID No.3, combining absolute quantification analysis of a fluorescent quantitative PCR system, and optimizing a reaction system and cycling conditions, a fluorescent PCR detection method for the babesia caballi disease is established. Moreover, the fluorescent PCR detection kit for the babesia caballi disease based on a fluorescent quantitative PCR scalar quantity is established, and is suitable for clinical diagnosis and epidemiological monitoring of the babesia caballi disease.
Description
Technical field
The invention belongs to Biological Detection technical field, be specifically related to a kind of horse Babesia caballi disease detection kit set up based on quantitative fluorescent PCR scalar positive control, infect the sick clinical diagnosis of horse Babesia caballi and daily monitoring for equus.
Background technology
Horse Babesia caballi disease is to be parasitized a kind of bloodprotozoonoses caused in equus erythrocyte by horse Babesia caballi (Babesiacaballi), and ill domestic animal shows as anemia more, jaundice, high heat are delaied, anorexia, the clinical symptoms such as become thin.Primary disease is especially serious to entry and exit animal injury, is classified as B class epidemic disease by OIE.At present, the effect of drugs of the treatment horse Babesia caballi disease circulated on the market is general, and due to the geographical environmental condition that Xinjiang is special, Tick victor distribution is extensively many, the sickness rate of this disease in ascendant trend year by year, has had a strong impact on the development of horse industry in my district (Xinjiang).Therefore, in order to this Bing Wo district of integrated control occurs and popular, must first set up high specificity, diagnostic method that recall rate is high quarantines horse in time, therefore the Shi Wo district veterinarian that develops of horse Babesia caballi disease fluorescent PCR quick detection kit makes great efforts one of research, new problem urgently to be resolved hurrily.
At present, horse Babesia caballi disease diagnostic method is broadly divided into pathogen microscopic detecting method, serological diagnostic method and Protocols in Molecular Biology diagnostic method.The diagnosis of this disease of Disease monitor department of basic unit remains in traditional Microscopical Method For Detection, and it is subject to the impact of the many factors such as film-making and staining technique, easily causing and fail to pinpoint a disease in diagnosis and mistaken diagnosis, particularly the Differential Diagnosis of similar morphology polypide and large-scale Epidemiological study are comparatively difficult.Conventional serological diagnostic method also exists the problems such as antigenic source deficiency, Sensitivity and Specificity be poor, and horse Babesia caballi In vitro culture is not yet successful.So in my district, 3 annual～July, a large amount of Ma Yinma Babesia caballis are died of illness and are died, therefore unique feasible, it is use for reference epidemiological study, creates practical, easy, economic, highly sensitive new diagnosis system.Fluorescent PCR detection technique is as a kind of newer detection method, very extensive in the application of Animal diseases research field especially at present.Compared with regular-PCR technology, the advantage of Real-Time Fluorescent Quantitative PCR Technique is very notable: easy and simple to handle, it is not necessary to carry out gel electrophoresis imaging after amplification；Due to eliminate uncap after occur secondary pollution so that this technology occur false-positive probability be substantially reduced；By the intervention of computer system, amplification procedure can be carried out real-time quantitative monitoring by the method, amplified production can be carried out quantitative analysis accurately.Quantitative fluorescent PCR principle is by fluorescent material or has specific binding by with target gene of the specific probe of fluorescence signal, the PCR reaction of instrument each closing of monitor in real time, systems soft ware draws response curve automatically according to the power of fluorescence signal, thus the increase and decrease of the fluorescence signal of whole PCR course of reaction is monitored in real time.In fluorescent quantitative PCR technique, it should be understood that Ct value, implication representated by C therein is Cycle, implication representated by t is threshold, represent the period that each amplified reaction experiences when reaching threshold value, the initial copy number of each sample is linear to Ct value relevant, so when Ct value is only small, meaning that starting copy number is a lot.Therefore, when we utilize the standard substance determined can draw out the standard curve of these standard substance, owing to the reporter group fluorescence intensity sent of specific probe shows as linear correlation with pcr amplification product quantity, when after the Ct value knowing sample to be tested, just can from the starting copy number calculating this sample according to standard curve, it is achieved quantitative analysis.
In recent years, horse Babesia caballi diagnostic techniques is progressively innovated, and observes polypide applied molecular biology means detection horse Babesia caballi till now from microscope the earliest.China hole in 1993 is all brave has carried out horse giardiasis pathogen microscopic examination to horse giardiasis.60 dry goods are carried out IFAT detection by Camacho etc. (2005), and horse Babesia caballi positive rate is 28.3%.Kappmeyer etc. (1999) establish the bar-shaped Protein reconstitution antigen ELISA method of horse Babesia caballi, and the positive rate of cELISA method reaches 49.5%.YohTamaki etc. (2003) apply the bar-shaped albumen of Babesiacaballi and clone and have expressed recombiant protein BC134, can be used for ELISA detection.Bashiruddin etc. (1999) establish PCR detection method, and 23 parts of Portugal horses have been detected by this test, and result shows that sickness rate is 34.8%.The Tick victor insect infection horse giardiasis application PCR method propagating horse giardiasis is detected by Badgar etc. (2001), and result shows that in 54 samples, the horse babesia caballi rate of catching an illness reaches 12.9%.Alhassan (2005) has gone out can detect dual-PCR method two kinds sick according to 18SrRNA gene sequential design simultaneously.Xue Shujiang etc. (2007) utilize the design of horse Babesia caballi Bc48 gene order, synthesis specific primer, establish horse Babesia caballi disease PCR detection method, 35 parts of collecting samples carry out detection and finds that positive rate is 20%.Horse giardiasis is suffered from Pitel etc. (2010) extraction 35 does not but have the horses bone marrow of obvious clinical disease to carry out pcr amplification to detect whether to infect horse giardiasis, and testing result display regular-PCR detects and detects under the microscope coincidence rate and reaches 100%.From blood smear microscopy PCR diagnostic techniques till now, the sick detection technique of horse Babesia caballi progressively development innovation.Based on previous work, we separate local worm strain (Xinjiang worm strain), to its Bc-48 gene clone, expression.On above-mentioned basis, we primer, the design of probe, reagent dosage, Establishing, condition optimization and supporting assembling test kit etc. in inquire into, finally have developed horse Babesia caballi disease fluorescent PCR diagnostic kit.With its quick, special, inexpensive diagnostic reagent, carry out detecting and anti-horse-controlling Babesia caballi disease, thus purifying herds of horses, eradicating the harm to horse keeping industry of this disease, ensureing the sound development of animal husbandry.
Summary of the invention
The purpose of the present invention: it is an object of the invention to provide a kind of horse Babesia caballi quick diagnosis technology and test kit solves conventional sense test kit costliness, poor specificity, the shortcoming such as consuming time.For solving the present stage horse Babesia caballi detection present invention by designing detection primer and fluorescent probe, combined with fluorescent quantitative PCR system absolute quantification analysis, by optimizing reaction system and cycling condition, set up horse Babesia caballi disease fluorescence PCR detecting method.And set up a kind of horse Babesia caballi disease fluorescence PCR detection reagent kit based on quantitative fluorescent PCR scalar, it is adaptable to the clinical diagnosis of horse Babesia caballi disease and epidemiological surveillance.
Technical scheme is as follows: one is used for detecting the genetic marker of horse Babesia caballi sick (Babesiacaballi), and it has SEQIDNO.4 the sequence shown in or its specific fragment.
For expanding the specific primer pair of above-mentioned genetic marker, its nucleotides sequence is classified as shown in SEQIDNo.1 and SEQIDNo.2:
Forward primer: 5 '-CGCATTTGCAGTCAGGTCC-3 '
Downstream primer: 5 '-ACTGGTAGGGCTCAGGAAGG-3 '.
With above-mentioned primer with the use of fluorescent probe, its nucleotides sequence is classified as shown in SEQIDNo.3:
5’-AGGGGAGTAACTGCAGTGCTTCCGT-3’。
A kind of detect the test kit that horse Babesia caballi is sick, containing above-mentioned specific primer to above-mentioned fluorescent probe.Also including quantitative fluorescent PCR reaction reagent, positive reference substance and negative controls, described positive reference substance is the standard positive template that horse Babesia caballi is sick, and negative controls is sterilizing deionized water.
Mentioned reagent box, adopts fluorescence PCR method to detect, formulates standard positive template, and its 25 μ L reaction system is:
Primer, fluorescence probe PCR working procedure in test kit be: 95 DEG C of denaturation 5min, 1 circulation；95 DEG C of degeneration 15s, 60 DEG C of degeneration 45s, 40 circulations.
Mentioned reagent box adopts fluorescence quantifying PCR method to detect, and the quantitative fluorescent PCR reaction system after optimization is 25 μ L:
Above-mentioned specific primer is detected the application in horse Babesia caballi disease test kit to fluorescent probe in preparation.
Implementation, inventor downloads the country variant reported in GenBank data base and the horse Babesia caballi Bc-48 gene order in area, homology by DNAMAN software analysis download sequence, find out specific conservative target sequence, its nucleotide sequence is such as shown in SEQIDNo.4, using this target sequence as detecting the genetic marker that horse Babesia caballi is sick.Additionally, it will be appreciated by those skilled in the art that the specific fragment of this sequence is only as the genetic marker that detection horse Babesia caballi is sick.
Design detection primer, fluorescent probe and acquisition place positive genes of interest fragment
1. by the comparison GenBank horse Babesia caballi Bc-48 gene conserved sequence logged in, use PrimerPrimier5 and PrimerExpress3.0 software design pair of primers and a probe, purpose clip size is 157bp, Beijing Ding Guo Bioisystech Co., Ltd synthesize.
2. extract test kit operation instruction according to whole blood DNA and extract the whole blood DNA containing worm strain, the worm strain whole blood DNA extracted is carried out standard PCR amplification, PCR primer uses glue to reclaim test kit after 1.2% agarose gel electrophoresis detection and reclaims and purification, PCR primer after recovery, purification is connected with pEASY-T1Vector, build plasmid vector and deliver to Beijing Ding Guo biotech company and carry out sequencing analysis, identifying that correct plasmid is measured as standard substance and to its concentration.Reaction system is 25 μ L:10 × PCRBuffer (Mg2+Plus) 2.5 μ L, dNTP (2.5mM) 2 μ L, Taq DNA polymerase (5U/ μ L) 0.15 μ L, each 1 μ L of upstream and downstream primer (10pM), template DNA 1.5 μ L, ddH2O supplies 25 μ L.Amplification condition is: 94 DEG C of denaturation 5min；94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations；72 DEG C extend 5min, the genes of interest fragment of amplification 157bp.
The clone of genes of interest, order-checking
1. PCR primer uses glue to reclaim test kit recovery and purification after 1.2% agarose gel electrophoresis detection, the PCR primer after recovery, purification is connected with pEASY-T1Vector, then proceeds in escherichia coli Trans1-T1 competent cell, through Amp+After/LB culture medium amplification culture, use PlasmidPurificationKit to extract thalline plasmid, and plasmid is carried out PCR qualification.Recombiant plasmid and clone bacterium that 2. PCR is accredited as the positive deliver to Beijing DingGuo ChangSheng Biology Technology Co., Ltd's order-checking, and sequencing result and Bc-48 gene (accession number: AB731211) sequence carry out similarity comparison.
Set up horse Babesia caballi disease real-time fluorescence quantitative PCR (Real-TimePCR) detection method
1. identify that correct plasmid is measured as standard substance and to its concentration.With the standard positive recombiant plasmid of preparation for template, matrix method is adopted to optimize best concentration ratio, finally set up following reaction system: 2 × Real-TimePCRMix, primer (10 μm of ol) each 1 μ L, probe (10 μm of ol) 1 μ L, fluorescence correction liquid 0.5 μ L, template DNA 1 μ L, add sterilizing distilled water to 25 μ L volumes.Reaction condition is: 95 DEG C of 2min；95 DEG C of 10s, 56 DEG C of 40s, 45 circulations；Each circulation extends collection fluorescence signal when terminating.2. TEBuffer is used to carry out ten times of doubling dilutions (5.17 × 10 the standard positive plasmid having measured concentration10-5.17×101Copies/ μ L), in-20 DEG C of preservations.By the system optimized and condition, the positive plasmid of doubling dilution is carried out real-time fluorescence PCR, drawing standard curve.With 10 times of graded series concentration (5.17 × 1010Copies/ μ L～5.17 × 101Copies/ μ L) positive recombiant plasmid carry out real-time fluorescence PCR reaction as template.Template concentrations and Ct value are good linear relationship, and slope is-3.109, and intercept is 42.01, such that it is able to draw standard curve regression equation: Ct=-3.109 × log copy number+42.01, coefficient R 2=0.9996.And the Ct value of testing sample can read from instrument；The Ct value of testing sample is substituted into expression formula and just can calculate its initial copy number.3. the positive plasmid of doubling dilution is carried out real-time fluorescence PCR test, observe the positive plasmid copy number that its most mental retardation detects, the positive plasmid simultaneously utilizing the primer pair doubling dilution of this experimental design carries out standard PCR amplification, observe the minimum positive copy number that Standard PCR can be detected by, and the result of the two is contrasted.Result shows, when concentration is 5.17 × 101During copies/ μ L, fluorescent PCR still has amplification curve, and Standard PCR detectable concentration is 1.75 × 103Copies/ μ L, illustrates that Standard PCR detectable concentration is far below quantitative fluorescent PCR.4. specific test: extract the DNA of B.Caballi, Theileriaequi, Babesiabigemina, Theilerdiaannulata, Theilerdiasergenti and healthy horse blood as template, with distilled water as blank, carry out fluorescent quantitative PCR, carry out specific test, the specificity of evaluation test.Result shows that only B.caballi has specificity curve, Theileriaequi, Babesiabigemina, Theilerdiaannulata, Theilerdia.sergenti and healthy horse blood are all without curve, it was shown that B.caballi is had good specificity by the fluorescence quantifying PCR method of foundation.5. 5.17 × 10 are chosen6Copies/ μ L～5.17 × 103The positive template of copies/ L4 concentration of μ carries out in group and replica test, the Ct value according to amplified reaction, the calculating coefficient of variation (CV) between group, tests CV < 1.12% in result group；Replica test CV < 1.25% between group, it was shown that the method set up has good stability.6. the totally 90 parts of suspected case horse whole bloods being collected in area, bar state and Ili Prefecture are applied real-time fluorescence PCR, Standard PCR and blood smear respectively to detect, result shows, quantitative fluorescent PCR 48 parts of positives of detection, Standard PCR 27 parts of positives of detection, blood smear 10 parts of positives of detection, the positive fluorescent PCR of Standard PCR detection is illustrated as the positive, the detection positive rate of real-time fluorescence PCR and Standard PCR respectively 53% and 30%, it was shown that this quantitative fluorescent PCR has good clinical sensitivity and practicality.
Beneficial effect: use this detection kit to monitor my district (Xinjiang region) equus horse Babesia caballi sick, in conjunction with eliminating the measure suffering from poultry thus purifying herds of horses, eradicate the harm to horse keeping industry of this disease, ensure the sound development of animal husbandry, for ensureing that I haves laid a good foundation in the sound development of pastoral industry.
Accompanying drawing explanation
Accompanying drawing 1 is: recombiant plasmid PCR qualification result, wherein: M.DNA molecular mass standard；1. recombiant plasmid pcr amplification product；2. positive control；3. negative control；Accompanying drawing 2 is: pcr amplification product sequence alignment result；Accompanying drawing 3 is: the electrophoretic analysis of PCR primer, wherein: M.DNA molecular mass standard；The amplified production of 1～2. horse Babesia caballi positive DNA；Accompanying drawing 4 is: quantitative fluorescent PCR kinetic curve and standard curve, wherein: 1-10:5.17 × 1010Copies/ μ L～5.17 × 101copies/μL；Accompanying drawing 5 is: quantitative fluorescent PCR specific test amplification curve.
Detailed description of the invention
Embodiment 1, design detection primer, fluorescent probe and acquisition place positive genes of interest fragment, inventor designs specific primer such as shown in SEQIDNo.1 and SEQIDNo.2 according to the Bc48 gene order delivered, the specific primer of design utilizes round pcr amplification place, Xinjiang worm pnca gene group to obtain purpose antigen gene, as specific purpose antigen gene, its nucleotide sequence is such as shown in SEQIDNo.4, using this purpose antigen gene sequences as detecting the genetic marker that horse Babesia caballi is sick.Additionally, it will be appreciated by those skilled in the art that the specific fragment of this sequence is only as the genetic marker that detection horse Babesia caballi is sick.
Obtain purpose antigen gene: (1) is according to the Bc48 gene order design specific primer delivered such as shown in SEQIDNo.1 and SEQIDNo.2 and probe such as shown in SEQIDNo.3；(2) round pcr amplification purpose antigen fragment gene (referring to accompanying drawing 1) from local worm pnca gene group is utilized；(3) cloning and expression of horse Babesia caballi Xinjiang Strain Bc48 gene: building strains Bc48-ZY protokaryon cloned plasmids, (referring to accompanying drawing 2) is identified in order-checking.
Embodiment 2, horse Babesia caballi fluorescence PCR detecting method foundation: (1) application specific primer carries out conventional amplification (referring to accompanying drawing 3)；(2) application specific primer and probe carry out amplified fluorescence；(3) optimization of fluorescent PCR amplification system；(4) comparison (participating in accompanying drawing 4, accompanying drawing 5, accompanying drawing 2) of the susceptiveness of fluorescence PCR method, specificity, stability；(5) fluorescence PCR method is compared with other detection, diagnostic result, detects its coincidence rate.
Embodiment 3, horse Babesia caballi disease fluorescence PCR detection reagent kit development
(1) test kit purposes: this test kit is used for detecting whether horse suffers from horse Babesia caballi disease, being whether contain horse Babesia caballi in detection equus blood sample, this detection method may be used for epidemiological study and formulates " purifying horse " measure that reduction horse Babesia caballi is popular in herds of horses.
(2) principle of test kit: this test kit adopts MGB fluorescence PCR detecting method, TaqManMGB probe is to improve on the basis of TaqMan probe, maximum with TaqMan probe it is different in that its length by length can shorten to 13bp, the distance of fluorophor and quenching group closer to, quenching effects is better, marked self non-luminous quenching group at probe 3 ' end, autofluorescent background reduces, and is that the enough resolution of fluorescent tube is greatly promoted and reduces background signal interference；Next to that conjugate held by probe 3 ', in conjunction with double-stranded DNA ditch position, probe being made to have better specificity, sensitivity, the Tm value of probe improves so that it is more stability.
(3) composition of test kit and preparation of reagents
| Test kit composition (48T/kit) | Specification | Quantity |
| Description | 1 part | |
| 2×Real-Time PCR Mix | 1ml | 1 pipe 5--> |
| Fluorescence correction liquid | 0.5ml | 1 pipe |
| DEPC water | 1ml | 1 pipe |
| Horse Babesia caballi positive control | 0.1ml | 1 pipe |
| Horse Babesia caballi negative control | 0.1ml | 1 pipe |
(4) preservation condition of test kit and effect duration: this test kit is in-20 DEG C of preservations, and effect duration is 6 months.It is suitable for instrument: ABI7500,9600 series；The quantitative real time PCR Instruments such as Bio-Rad；Provide material for oneself: quantitative real time PCR Instrument；Accurate pipettor and disposable tip；Eight connecting legs；Ice chest；PCR pipe frame etc..
(5) sample requires: the horses of heating, anemia, jaundice, the associated clinical symptoms such as become thin or the horses blood crossed by Ticks insect stings are chosen in sample collection.DNA extraction should be carried out after blood collection in time, and carry out fluorescent PCR detection.If condition does not allow, fail to extract in time DNA, first blood can be placed in-20 DEG C of Refrigerator stores, and extract DNA in time.
(6) test kit operation and points for attention
1. fluorescent PCR is highly sensitive, therefore very easily occurs polluting, therefore should be equipped with independent liquor room and upper specimen chamber in process of the test, it is ensured that PCR reagent and sample separate application of sample, to avoid positive to pollute reagent.2., after fluorescent PCR needs the fluorescence letter collecting in eight unions or 96 orifice pipe lids and plate lid, therefore in operation, must ensure that Guan Gai and Ban Gai shows plot, in order to avoid error occurs in the collection of fluorescence signal.3. PCR reagent, primer and Taq enzyme are easily degraded at normal temperatures and are caught fire, therefore in application of sample process, it is necessary to whole process completes on ice chest.4. after sample pipetting volume, concussion should be carried out and make reagent mix completely, simultaneously brief centrifugation, with the reagent sticking to tube wall under centrifugal and Guan Gai shows, and be immediately placed in fluorescent PCR instrument and expand.
Embodiment 4, experimental technique: 2 × Real-TimePCRMix, DEPC water, 10pM forward primer, 10pM downstream primer, 10pM fluorescent probe, fluorescence correction liquid are put equilibrium at room temperature by (1), namely each test reaction system is formulated as: each 1 μ l+ fluorescence correction liquid 0.5 μ l+DEPC water 8 μ l of 2 × Real-TimePCRMix12.5 μ l+ forward primer, downstream primer, fluorescent probe；(2) adding template: the nucleic acid extractive being saved in-20 DEG C is put thaw at RT, positive and negative comparison uses preposition thaw at RT.Each PCR reaction tube is separately added into template or positive and negative compare each 1 μ l, build pipe lid, be placed in PCR instrument, record respective sample number；(3) upper machine amplification: reaction condition is 95 DEG C: 5min, 1 circulation；95 DEG C: 15s, 60 DEG C: 45s (signal collection), 40 circulations.Reaction system is 50 μ l.FAM fluorescein it is set as during signals collecting；(4) result judges: when a detects sample Ct value≤35.0, the report positive；B detects sample Ct value>35.0 and Ct value<when 40, need duplicate detection once, if Ct value still<40, but curve has obvious logarithmic growth characteristic, is reported as the positive, is otherwise reported as feminine gender；When c sample does not show Ct value, report feminine gender.
Embodiment 5, horse Babesia caballi disease fluorescence PCR detection reagent kit Clinical practice: horse Babesia caballi fluorescence PCR detecting method 90 parts of horse blood DNA that Yili of Xinjiang is gathered that application is set up detect, and Babesia caballi positive rate of must going into action reaches 53.3% (48/90).
Table 2 quantitative fluorescent PCR replica test
Abbreviation and Key Term definition: following abbreviations is applicable to this standard: Bc-ZY: Xinjiang Strain Bc48 gene order procaryotic clone carrier；MGB probe: novel fluorescence probe；PCR: polymerase chain reaction；Taq: thermus aquaticus's archaeal dna polymerase；DNTP: deoxyribose triphosphoric acid；MGB: minor groove binding molecule.
Claims (9)
1. being used for detecting a genetic marker of horse Babesia caballi sick (Babesiacaballi), it has the sequence shown in SEQIDNo.4 or its specific fragment.
2., for expanding the specific primer pair of genetic marker described in claim 1, its nucleotides sequence is classified as shown in SEQIDNo.1 and SEQIDNo.2:
Forward primer: 5 '-CGCATTTGCAGTCAGGTCC-3 '
Downstream primer: 5 '-ACTGGTAGGGCTCAGGAAGG-3 '.
3. with described in claim 2 primer with the use of fluorescent probe, its nucleotides sequence is classified as shown in SEQIDNo.3:
5’-AGGGGAGTAACTGCAGTGCTTCCGT-3’。
4. one kind is detected the test kit that horse Babesia caballi is sick, it is characterised in that containing the specific primer described in claim 2 to the fluorescent probe described in claim 3.
5. test kit as claimed in claim 4, it is characterised in that also including quantitative fluorescent PCR reaction reagent, positive reference substance and negative controls, described positive reference substance is the standard positive template that horse Babesia caballi is sick, and negative controls is sterilizing deionized water.
6. test kit as claimed in claim 4, it is characterised in that adopting fluorescence PCR method to detect, formulate standard positive template, its 25 μ L reaction system is:
7. according to the arbitrary described test kit of claim 4-6, it is characterised in that primer, fluorescence probe PCR working procedure in test kit be: 95 DEG C of denaturation 5min, 1 circulation；95 DEG C of degeneration 15s, 60 DEG C of degeneration 45s, 40 circulations.
8. test kit as claimed in claim 4, it is characterised in that it adopts fluorescence quantifying PCR method to detect, and the quantitative fluorescent PCR reaction system after optimization is 25 μ L:
9. specific primer according to claim 2 detects the application in horse Babesia caballi disease test kit to the fluorescent probe described in claim 3 in preparation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755392A (en) * | 2016-12-16 | 2017-05-31 | 国家开发投资公司 | The Quantitative detection qPCR methods of algae culture lumen wheel animalcule |
| CN106755393A (en) * | 2016-12-16 | 2017-05-31 | 国家开发投资公司 | The Quantitative detection qPCR methods of Brachionus in algae culture |
| CN108486223A (en) * | 2018-04-18 | 2018-09-04 | 华中农业大学 | A kind of Ji Shi Babesias RPA molecular detecting methods |
| CN109633160A (en) * | 2018-11-13 | 2019-04-16 | 新疆农业大学 | Horse Babesia caballi disease colloidal gold colloidal gold detection test paper strip and its preparation and application method |
| CN110564882A (en) * | 2019-07-09 | 2019-12-13 | 沈阳农业大学 | Dual TaqMAN probe fluorescent quantitative PCR detection method for equine piroplasmosis |
-
2016
- 2016-04-28 CN CN201610274783.1A patent/CN105734158A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 张杨等: "驽巴贝斯虫二温式PCR检测方法的建立及初步应用", 《动物医学进展》 * |
| 张杨等: "驽巴贝虫和马泰勒虫双重PCR检测方法的建立", 《中国寄生虫学与寄生虫病杂志》 * |
| 薛书江等: "驽巴贝斯虫PCR检测方法的建立及应用", 《河南农业科学》 * |
| 赵祥平等: "驽巴贝斯虫病巢式PCR检测方法的研究", 《中国动物检疫》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755392A (en) * | 2016-12-16 | 2017-05-31 | 国家开发投资公司 | The Quantitative detection qPCR methods of algae culture lumen wheel animalcule |
| CN106755393A (en) * | 2016-12-16 | 2017-05-31 | 国家开发投资公司 | The Quantitative detection qPCR methods of Brachionus in algae culture |
| CN106755393B (en) * | 2016-12-16 | 2020-12-18 | 国投生物科技投资有限公司 | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting Brachionus brachialis in algae culture |
| CN106755392B (en) * | 2016-12-16 | 2020-12-18 | 国投生物科技投资有限公司 | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting coelomacter in algae culture |
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