CN103088149A - Kit for detecting anaplasma bovis - Google Patents
Kit for detecting anaplasma bovis Download PDFInfo
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- CN103088149A CN103088149A CN2013100627367A CN201310062736A CN103088149A CN 103088149 A CN103088149 A CN 103088149A CN 2013100627367 A CN2013100627367 A CN 2013100627367A CN 201310062736 A CN201310062736 A CN 201310062736A CN 103088149 A CN103088149 A CN 103088149A
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Abstract
The invention discloses a kit for detecting anaplasma bovis. The kit for detecting anaplasma bovis at least contains the following three primer probe sequences: a forward primer SEQ No.1, a reverse primer SEQ No.2 and a probe SEQ No.3, wherein a fluorescent luminophore FAM is combined to a fifth end of the probe sequence, and a non-fluorescent quencher connected with an MGB (minor groove binder) is combined to a third end of the probe sequence. The application of the kit provides a real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting anaplasma bovis by using a 16srRNA gene. The kit has good sensitivity, specificity and repeatability, and the sensitivity of the kit is more than 10 times than that of nested PCR, so that the kit can be used for quick quantitative detection of the anaplasma bovis.
Description
Technical field
The present invention relates to a kind of test kit for detection of the animal blood pathogenic bacteria, the present invention relates to exactly a kind of for detection of the test kit of ox without slurry.
Background technology
Without slurry (anaplasma) be a class through tick-borne special sexual cell endophyte, main parasitic is in the hemocyte of the ruminating animals such as cattle and sheep, its disease that causes is called as anaplasmosis.Found first the ox anaplasmosis in north African in 1910, found afterwards that this disease was distributed widely in all over the world.Anaplasmosis causes that the weight of animals descends, miscarriage, and milk yield descends, and severe patient causes death, causes serious financial loss.Given this, OIE (OIE) classifies it as must circulate a notice of epidemic disease.Cause the cause of disease of ox anaplasmosis certified have the edge without slurry (anaplasma marginale), central authorities without slurry (anaplasma centrale), ox without slurry (anaplasma bovis) and Anaplasma phagocytophilum (anaplasma phagocytophilum).
Ox is to belong to an important member of (Anaplasma) without slurry without slurry (Anaplasma bovis), ox has host widely without slurry, verified it can parasitize in the body of the ruminating animals such as ox, sheep, ox parasitizes after arthropods-tick is propagated without slurry in the monocyte of blood of host animal.Ox is widely current all over the world without slurry, do not have in the past the report that ox infects without slurry in the ox anaplasmosis cause of disease of China, but find to exist ox without slurry in the many areas of China in our investigation recently, and be high positive rate state in certain areas, should attract great attention, but at present for the detection method of this cause of disease, blood smear staining and Nested Polymerase Chain Reaction only be arranged.The blood smear staining is very low or be difficult to go out cause of disease at test under microscope when early stage infecting at infection rate, and the skilled operation degree will directly affect the accuracy of diagnostic result.The sleeve type PCR detection method, referring to: Kawahara M, et al. 2006, Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp.in wild deer and ticks on two major islands in Japan, Appl. Environ. Microbiol, 2006. 72:1102-1109, but easily cause environmental pollution in the sleeve type PCR testing process, and waste time and energy.Therefore, set up a kind of fast, sensitive, accurately easy to operate detection method is very necessary again.
Summary of the invention
The invention provides a kind of overcome the prior art deficiency for detection of the test kit of ox without slurry.
Has three following primer probe sequences: forward primer SEQ № 1 at least in the test kit of detection ox of the present invention without slurry, reverse primer SEQ № 2 and probe SEQ № 3,5 ends of probe sequence wherein are combined with fluorescence radiation group FAM, and 3 ends are combined with the non-fluorescent quenching group that connects MGB.
For convenience of using, also include following composition in without the test kit of slurry detection ox of the present invention: fluorescence quantitative PCR reaction solution, standard positive plasmid template pGEM-Teasy-16S rRNA, negative quality control standard product.Fluorescence quantitative PCR reaction solution wherein is by Premix Ex Taq
TM(2 *) 12.5 μ L, forward primer SEQ № 1 and reverse primer SEQ № 2(10 μ Μ) each 0.5 μ L, fluorescent probe solution (5 μ M) 1.0 μ L, ROX Reference Dye II (50 *) 0.5 μ L, sterile purified water 8.0 μ L form.In addition, the present invention detects the pGEM-Teasy recombinant plasmid that ox builds for the DNA fragmentation by forward primer EE1 and reverse primer EE2 amplification without the standard positive plasmid template pGEM-Teasy-16S rRNA in the test kit of slurry; And negative quality control standard product can be sterile purified waters.
The present invention is actually a kind of 16S of utilization rRNA gene test ox without the real time fluorescence quantifying PCR method of slurry.Real-time fluorescence quantitative PCR (real-time fluorescent quantitative polymerasechain reaction, real-time FQ-PCR) technology was released by U.S. Applied Biosystems company in 1996, because this technology has not only realized the leap of PCR from qualitative to quantitative, and compare with conventional round pcr high specificity that it has, highly sensitive, good reproducibility, quantitatively accurately, the advantages such as level of automation is high, speed is fast, totally-enclosed reaction, make it become very soon the hot spot technology of scientific research, clinical diagnosis.At present, all do not have both at home and abroad and detect ox without the report of slurry real time fluorescence quantifying PCR method, the present invention utilizes ox to detect target gene without slurry 16S rRNA gene as real-time fluorescence quantitative PCR, design is for Auele Specific Primer and the probe of ox without slurry 16S rRNA gene, thereby provide a kind of fast, sensitive, accurately easy to operate detection ox without the method for slurry, has been filled up technological gap again.In the present invention, the optimization by to real-time fluorescence quantitative PCR reaction bar condition makes the present invention have good susceptibility, specificity, repeatability, and its susceptibility is higher 10 times than sleeve type PCR, can be used for ox without the Quantitative detection of slurry.
Description of drawings
Fig. 1 is real-time fluorescence quantitative PCR typical curve of the present invention.
Fig. 2 is real-time fluorescence quantitative PCR sensitivity test of the present invention.
Be followed successively by from top to bottom 1.0 * 10
7-1.0 * 10
0The plasmid standard of copy/μ L and the amplification of negative control.
Fig. 3 sleeve type PCR detection method electrophorogram.
Wherein M is DNA molecular amount standard DL2000; 1,2,3,4,5,6,7, be respectively 1.0 * 10
6-1.0 * 10
0The plasmid standard of copy/μ L; 8 negative contrasts; 9 positive controls; 10 is normal ox whole blood genome.
Fig. 4 is real-time fluorescence quantitative PCR specific test of the present invention.
Baseline is above be ox without the amplification of slurry, below baseline be sheep without slurry, edge without slurry, Anaplasma phagocytophilum, sheep Taylor worm, Theileria luwenshuni, Theileria sinensis, sheep Babesia, ox Babesia,
Two bud Babesias, mycoplasma, chlamydozoan, spirochete, toxoplasma gondii and negative control amplification.
Embodiment
The present invention elaborates below in conjunction with drawings and Examples.
Most importantly following three primer probe sequences in test kit of the present invention, that is:
The ABRTF1(forward primer): 5 '-CACGCTGTAAACGATGAG-3 ' SEQ № 1
The ABRTR1(reverse primer): 5 '-CGTCAATTCCTTTGAGTTTTAG-3 ' SEQ № 2
The ABRTPb(fluorescent probe): 3 ' the SEQ № 3 of 5 ' (FAM)-ACCTCCGTGTTGTA-(NFQ)-(MGB)
Wherein 5 ends of fluorescent probe SEQ № 3 be combined with fluorescence radiation group FAM, 3 ends are combined with the non-fluorescent quenching group that connects MGB.The purpose clip size is 117 bp.
For convenience of using, also can be provided with in the test kit of practical application: fluorescence quantitative PCR reaction solution, standard positive plasmid template pGEM-Teasy-16S rRNA, negative quality control standard product.Fluorescence quantitative PCR reaction solution is by Premix Ex Taq in the present embodiment
TM(2 *) 12.5 μ L, the forward primer SEQ № 1 of 10 μ Μ and reverse primer SEQ № 2 each 0.5 μ L, fluorescent probe SEQ № 3 solution (5 μ M) 1.0 μ L, fluorescence correction liquid ROX Reference Dye II (50 *) 0.5 μ L, sterile purified water 8.0 μ L.
Primer and probe above-mentioned in embodiments of the invention entrust U.S. Life Technologies company synthetic.
Test kit application example of the present invention
1. construction recombination plasmid standard substance.
(1) reference literature Barlough JE, et al. 1996, Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). the primer EE1 in Vet. Parasitol. 63:319 – 329. and EE2 amplification 16s rRNA gene, primer is synthetic by Sangon Biotech (Shanghai) Co., Ltd..
(2) ox that preserves take the laboratory as template increases, is adopted the PCR reaction system of 50 μ L without slurry bacterial strain DNA, and reaction conditions is 94 ℃ of denaturation 3min, then 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ of 30s, and 35 circulations, 72 ℃ are extended 5min.Getting 5 μ L amplified productions identifies with agarose gel electrophoresis.Be accredited as positive PCR product and reclaim purification kit (Transgen with sepharose, Beijing) carrying out purifying reclaims, to reclaim product and be connected to pGEM-Teasy carrier (Promega, America) be transformed into competent escherichia coli cell JM-109(TaKaRa after, Dalian) in, carry out blue hickie screening, picking positive colony (hickie) carries out PCR and order-checking is identified.
(3) extract plasmid to being accredited as positive clone, use NanoDrop 2000/2000C(Thermo Scientific, America) ultramicrospectrophotometer measures plasmid concentration, and it is converted into copy/μ L, with this as plasmid standard.
With the plasmid standard that builds, serial dilution is that final concentration is 1.0 * 10
8-1.0 * 10
0Copy/μ L, then carry out quantitative pcr amplification, the quantitative PCR reaction system is 25 μ L:Premix Ex Taq
TM(2 *) 12.5 μ L, forward primer SEQ № 1 and reverse primer SEQ № 2(10 μ Μ) each 0.5 μ L, fluorescent probe SEQ № 3(5 μ M) 1.0 μ L, ROX Reference Dye II (50 *) 0.5 μ L, DNA profiling 2 μ L, sterile purified water 8.0 μ L.Reaction conditions is: 95 ℃ of denaturation 30s, then 95 ℃ of 5s, 59 ℃ of 15s, 72 ℃ of 20s, 40 circulations.Setting fluorescent signal collection point is detected in real time when the elongating temperature of each circulation finishes, and each template concentrations is done 3 repetitions, the calculating variation coefficient of averaging, and negative control is all established in experiment.The real-time fluorescence quantitative PCR typical curve of setting up is referring to Fig. 1, and the template amount is linear dependence with corresponding Ct value, and relation conefficient is 0.998, and amplification efficiency is 101.7%, and the typical curve equation is y=-3.281x+41.43.
The real time fluorescence quantifying PCR method specificity analysis.
(1) sensitivity analysis
Get concentration 1.0 * 10
7-1.0 * 10
0The plasmid standard of copy/μ L carries out sensitivity test, and result shows that the real time fluorescence quantifying PCR method minimum detectability is 10
1Copy/μ L, referring to Fig. 2, and the minimum detectability of sleeve type PCR is 10
2Copy/μ L is referring to Fig. 3, and obviously the present invention is than sleeve type PCR detection method highly sensitive 10 times.
(2) specificity analyses
Utilize real time fluorescence quantifying PCR method that this paper sets up, detected respectively ox without slurry, sheep without slurry, edge without slurry, Anaplasma phagocytophilum, sheep Taylor worm, Theileria luwenshuni, Theileria sinensis, sheep Babesia, ox Babesia,
Two bud Babesias, mycoplasma, chlamydozoan, spirochete and toxoplasma gondii, result shows to only have ox to be positive without slurry, other pathogenic agent all are negative, referring to Fig. 4.
(3) stability analysis
Getting concentration is 1.0 * 10
6-1.0 * 10
1The standard substance of 5 concentration of copy/μ L carry out stability analysis.Replica test in first criticizing, to the standard substance of same batch of dilution, each concentration duplicate detection three times, the Ct value variation coefficient is at 0.10%-1.01%, less than 5% by analysis.Then replica test between criticizing, the standard substance that 3 different batches are diluted detect, and the Ct value variation coefficient is at 0.61%-1.88%, less than 10% by analysis.Illustrate that thus the present invention has stability preferably.
Show a collection of interior replica test result
| Standard substance copy/μ L | 1.0×10 1 | 1.0×10 2 | 1.0×10 3 | 1.0×10 4 | 1.0×10 5 | 1.0×10 6 |
| Ct1 | 37.77 | 34.95 | 31.69 | 28.34 | 24.76 | 21.69 |
| Ct2 | 37.67 | 34.61 | 31.94 | 28.21 | 24.74 | 21.71 |
| Ct3 | 37.88 | 35.15 | 31.87 | 27.92 | 24.72 | 22.08 |
| CV（%） | 0.29 | 0.77 | 0.41 | 0.78 | 0.10 | 1.01 |
Replica test result between table two batch
| Standard substance copy/μ L | 1.0×10 1 | 1.0×10 2 | 1.0×10 3 | 1.0×10 4 | 1.0×10 5 | 1.0×10 6 |
| Ct1 | 37.76 | 34.84 | 31.79 | 28.70 | 24.79 | 21.32 |
| Ct2 | 38.16 | 34.62 | 31.61 | 27.99 | 24.82 | 21.25 |
| Ct3 | 38.87 | 34.28 | 30.79 | 27.67 | 24.2 | 21.50 |
| CV（%） | 1.46 | 0.81 | 1.69 | 1.88 | 1.42 | 0.61 |
Above embodiment is with a specific embodiment of the present invention, but protection scope of the present invention is not limited to above-described embodiment.
＜110〉Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
＜120〉detect ox without the test kit of slurry
<160> 3
<210> 1
<211> 18
<212> DNA
＜213〉artificial sequence forward primer (ABRTF1)
<400>
cacgctgtaa acgatgag 18
<210> 2
<211> 22
<212> DNA
＜213〉artificial sequence reverse primer (ABRTR1)
<400>
cgtcaattcc tttgagtttt ag 22
<210> 3
<211> 14
<212> DNA
＜213〉artificial sequence fluorescent probe (ABRTPb)
<400>
Claims (4)
1. detect ox without the test kit of slurry, it is characterized in that test kit has three following primer probe sequences: forward primer SEQ № 1 at least, reverse primer SEQ № 2 and probe SEQ № 3,5 ' end of probe sequence wherein is combined with fluorescence radiation group FAM, and 3 ' end is combined with the non-fluorescent quenching group that connects MGB.
2. detection ox claimed in claim 1 without the test kit of slurry, is characterized in that also including in test kit following composition: fluorescence quantitative PCR reaction solution, standard positive plasmid template pGEM-Teasy-16S rRNA, negative quality control standard product.
3. detection ox claimed in claim 2 without the test kit of slurry, is characterized in that fluorescence quantitative PCR reaction solution is by Premix Ex Taq
TM(2 *) 12.5 μ L, the forward primer SEQ № 1 of 10 μ Μ and reverse primer SEQ № 2 each 0.5 μ L, the fluorescent probe SEQ № 3 solution 1.0 μ L of 5 μ M, ROX Reference Dye II (50 *) 0.5 μ L, sterile purified water 8.0 μ L form.
4. detection ox claimed in claim 3 without the test kit of slurry, is characterized in that negative quality control standard product are sterile purified water.
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| CN2013100627367A CN103088149A (en) | 2013-02-28 | 2013-02-28 | Kit for detecting anaplasma bovis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293936A (en) * | 2014-09-18 | 2015-01-21 | 黑龙江八一农垦大学 | Use of loop-mediated isothermal amplification primer in preparing reagent for detecting bovine anaplasma marginale antigen |
| CN105154557A (en) * | 2015-09-22 | 2015-12-16 | 河南农业大学 | Dual PCR method for detecting theileria hirci and anaplasma |
| CN107815501A (en) * | 2016-09-12 | 2018-03-20 | 台达电子国际（新加坡）私人有限公司 | Detect primer pair, set group and the method for sheet side worm |
| CN113293223A (en) * | 2021-07-16 | 2021-08-24 | 吉林农业科技学院 | Application of primer combination in preparation of reagent for detecting bovine babesia ovale and anaplasma by adopting double PCR (polymerase chain reaction) method |
Citations (3)
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| US20090004654A1 (en) * | 2007-06-29 | 2009-01-01 | Instituto De Salud Carlos Iii | Method for the detection of bacterial species of the genera anaplasma/ehrlichia and bartonella |
| EP2267161A1 (en) * | 2005-06-17 | 2010-12-29 | Instituto de Salud Carlos III | Primers, probes and kits for the detection of bacterial species belonging to the genus bartonella |
| CN102634597A (en) * | 2012-05-04 | 2012-08-15 | 中国农业科学院兰州兽医研究所 | Kit for detecting anaplasma ovis |
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2013
- 2013-02-28 CN CN2013100627367A patent/CN103088149A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2267161A1 (en) * | 2005-06-17 | 2010-12-29 | Instituto de Salud Carlos III | Primers, probes and kits for the detection of bacterial species belonging to the genus bartonella |
| US20090004654A1 (en) * | 2007-06-29 | 2009-01-01 | Instituto De Salud Carlos Iii | Method for the detection of bacterial species of the genera anaplasma/ehrlichia and bartonella |
| CN102634597A (en) * | 2012-05-04 | 2012-08-15 | 中国农业科学院兰州兽医研究所 | Kit for detecting anaplasma ovis |
Non-Patent Citations (2)
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|---|
| 《畜牧兽医学报》 20121031 周作勇等 "牛无浆体16S rRNA基因的克隆测序及分析" 第41卷, 第10期 * |
| 周作勇等: ""牛无浆体16S rRNA基因的克隆测序及分析"", 《畜牧兽医学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293936A (en) * | 2014-09-18 | 2015-01-21 | 黑龙江八一农垦大学 | Use of loop-mediated isothermal amplification primer in preparing reagent for detecting bovine anaplasma marginale antigen |
| CN105154557A (en) * | 2015-09-22 | 2015-12-16 | 河南农业大学 | Dual PCR method for detecting theileria hirci and anaplasma |
| CN107815501A (en) * | 2016-09-12 | 2018-03-20 | 台达电子国际（新加坡）私人有限公司 | Detect primer pair, set group and the method for sheet side worm |
| CN113293223A (en) * | 2021-07-16 | 2021-08-24 | 吉林农业科技学院 | Application of primer combination in preparation of reagent for detecting bovine babesia ovale and anaplasma by adopting double PCR (polymerase chain reaction) method |
| CN113293223B (en) * | 2021-07-16 | 2022-07-12 | 吉林农业科技学院 | Application of primer combination in preparation of reagent for detecting bovine babesia ovale and anaplasma by adopting double PCR (polymerase chain reaction) method |
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Application publication date: 20130508 |