CN109593826A - A kind of Mohs Babesia detection kit - Google Patents
A kind of Mohs Babesia detection kit Download PDFInfo
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- CN109593826A CN109593826A CN201910033593.4A CN201910033593A CN109593826A CN 109593826 A CN109593826 A CN 109593826A CN 201910033593 A CN201910033593 A CN 201910033593A CN 109593826 A CN109593826 A CN 109593826A
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- 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
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- 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
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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Abstract
The present invention discloses a kind of kit and application method detected using cross primer constant-temperature amplification combination immunochromatography technique and identify Mohs Babesia.It include five primers for constant-temperature amplification in Mohs Babesia detection kit of the invention, in which: a cross primer that sequence is SEQ ID No.3, the displacement primer that sequence is SEQ ID No.1 and sequence is SEQ ID No.2, sequence SEQ ID No.4 are the probe primer of the probe primer of FITC label and biotin labeling that sequence is SEQ ID No.5.Kit further includes having immuno-chromatographic test paper strip.The present invention includes the advantages of easy to operate, quick, high sensitivity, specificity are high, use scope is wide and testing result can with the naked eye be read.
Description
Technical field
For the purpose of being diagnosed the present invention relates to a kind of non-disease, for detecting and identifying whether animal has cause of disease helminth
Method and kit, exactly the present invention relates to it is a kind of using cross primer constant-temperature amplification combination immunochromatography technique detection with
Identify the method and kit of Mohs Babesia.
Background technique
The Babesia Gibsoni (Ovis Babesiosis) of sheep is a kind of to parasitize the red of tender goat by medium is tick-borne
Bloodprotozoonoses caused by intracellular, infection animal show as fever, jaundice, hemoglobinuria, and whens severe infections can cause to move
Object death (Uilenberg, 2006).Whole world report causes the cause of disease of sheep Babesia Gibsoni to have Mohs Babesia
It is (Babesia motasi), sheep Babesia (Babesia ovis), coarse Babesia (Babesia crassa), lobate
Babesia (Babesiafoliata) and Tai Shi Babesia (Babesia taylori).In the Babesia of China's prevalence
There are two kind, i.e., Mohs Babesia and Babesia U sp Xinjiang Strain (Guan et al., 2002,2009；Liu et
al.,2007；Niu et al.,2009).Utilize the bases such as ITS, HSP90, COI, COB, COX3, RPS8, TRAP, AMA1, RON2
Show that Mohs Babesia is divided into two subbranches, i.e. Hebei because the Babesia separation strains to China carry out sort research
Strain/Ning County strain (Mohs Babesia HN subspecies) and Lintan strain/God blessings strain (Mohs Babesia LT subspecies) (Bai et al.,
2002；Guan et al.,2002；Liu et al.,2007b；Niu et al.,2009a).
The Babesia Gibsoni of sheep is wide in China's Epidemic Scope, and harm is serious, makes to the healthy and sustainable development of China's sheep husbandry
At huge threat.Close that your congruence and Wang Jinming etc. are utilized respectively with Mohs Babesia LT subspecies and Babesia U sp
The indirect ELISA method that merozoite soluble antigen is established, to the Epidemic status of the 22 province sheep Babesia Gibsonis in China
It has been shown that, the average positive rate of Mohs Babesia are 43.5%, and sheep Babesia U sp is 31.66%, and the disease is in national model
Enclose it is interior have it is popular (Guan et al, 2012；Wang et al,2013).
Method currently used for the infection detection of sheep Babesia has a classical blood film decoration method, this method Infestation rat very
It is difficult to detect cause of disease in the case where low, and higher to the skill requirement of operator.Babe based on molecular biology exploitation
This worm, which identifies detection method, real-time fluorescence quantitative PCR, reverse linear trace, multiplex PCR etc., these method high sensitivities, spy
It is anisotropic also higher, but real-time fluorescence quantitative PCR and multiplex PCR need expensive instrument；Reverse linear blotting procedure is cumbersome, consumes
Duration.And the above method is unfavorable for the application of field quick detection.It would therefore be highly desirable to develop a kind of quick, highly sensitive and special
The anisotropic reliable method that can be used for clinical sites detection.
Summary of the invention
This hair provides one kind and can overcome the shortage of prior art, and identifies the cross primer constant temperature of detection for Mohs Babesia
Expand the method and kit of the detection of combined immunization chromatographic test paper.
Include five primers for constant-temperature amplification in Mohs Babesia detection kit of the invention: sequence is
A cross primer of SEQ ID No.3, sequence are SEQ ID No.1 and sequence is displacement primer, the FITC of SEQ ID No.2
The probe primer SEQ ID No.4 of the label and probe primer SEQ IDNo.5 of biotin labeling.Kit further include have it is immune
Chromatograph test strip.
Preferably, it uses for convenience, in Mohs Babesia detection kit of the invention further include: 10 ×
IsothermalAmplification Buffer、100mM MgSO4, 2.5mM dNTP mix, Bst archaeal dna polymerase.
Using application method of the aforementioned Mohs Babesia detection kit of the invention for non-disease diagnosis is: to mention
The genome of sample to be tested DNA taken is template, the cross primer isothermal amplification reactions established with the primer and probe in kit
System is expanded, then amplified production is added dropwise to immuno-chromatographic test paper strip, according to the nature controlling line of test strips and detection line whether
Whether the determining measuring samples that develop the color simultaneously have Mohs Babesia.If nature controlling line and detection line develop the color, result judgement is
It is positive；If developing the color only at nature controlling line, result judgement is feminine gender.
Preferably, the reaction condition of kit application method of the invention most preferably expanded are as follows: 63 DEG C, 50min；80 DEG C,
2min。
Nucleic acid detection test strip used in the present invention is purchased from Twistdx company.
Present invention utilizes cross primer amplification techniques.Cross primer amplification is a kind of novel constant-temperature amplification diagnosis detection
A kind of method, archaeal dna polymerase (Bst archaeal dna polymerase) and a plurality of primer with strand-displacement activity, under constant temperature conditions can be real
Existing target area efficiently, quickly expands.The key points and difficulties of this method are the design of primer sets and label probe primer, whole
A detection process only needs a thermostat water bath that detection reaction, judgement combination chromatograph test strip as a result, with meat can be completed
The visible mode of eye is completed, so being particularly suitable for having the clinical detection in the non-laboratory place of a large amount of samples to use.
The invention has the following advantages that
(1) operation of the present invention is easy, quick: entire detection process is fully completed within a hour, to operator's element
Matter requires low, it is only necessary to which the personnel for having regular-PCR operation can carry out this operation.
(2) high sensitivity: method established by the present invention can detecte the Mohs Babesia genomic DNA of 0.06pg,
Being equivalent to when Infestation rat is 0.00006% so can detecte positive sample, be 100 times of multiplex PCR sensitivity.
(3) present invention specificity is high: two label probe primer LT-2a, LT-3a and cross primer in cross primer amplification
LT-2a1s identifies the special sequence of Mohs Babesia, ensure that the high degree of specificity of this method；The detection product of constant-temperature amplification
Both ends are marked with FITC and biotin respectively, for tying with the specificity of FITC monoclonal antibody and biotin monoclonal antibody on chromatograph test strip
It closes, further ensures the specificity of the detection method.
(4) use scope is wide: cross primer amplification does not need expensive fluorescence quantitative PCR instrument or PCR instrument, as long as being provided with
The equipment of constant temperature, such as water-bath, hot air drying oven can carry out this detection, detection and epidemiology suitable for base's clinical sample
Investigation.
(5) testing result can with the naked eye be read: result is read, no after test strips develop the color in a manner of macroscopic
Need to carry out special technical training with regard to achievable.
Detailed description of the invention
The optimization of Fig. 1 Mohs Babesia cross primer amplification optimal reaction temperature.1-4 test strips reaction temperature point
It Wei not be 58 DEG C, 61 DEG C, 63 DEG C, 68 DEG C.
The screening of Fig. 2 Mohs Babesia cross primer amplification optimum reacting time.The 1-5 test strips reaction time point
It Wei not 35min, 40min, 45min, 50min, 60min.
Fig. 3 Mohs Babesia cross primer specific detection.1 is the strain of Mohs Babesia Lintan, and 2 be Mohs babe
The strain of this worm Hebei, 3 be the strain of Mohs Babesia Ning County, and 4 be Babesia U sp Xinjiang Strain, and 5 be T.uilenbergi, and 6 be silk floss
Theileria SP, 7 be Theileria luwenshuni, and 8 be negative control.
Fig. 4 Mohs Babesia cross primer expands sensitivity technique.1 template concentrations are 4ng/ μ L, and 2 be 800pg/ μ L,
3 be 160pg/ μ L, and 4 be 32pg/ μ L, and 5 be 6.4pg/ μ L, and 6 be 1.28pg/ μ L, and 7 be 0.3pg/ μ L, and 8 be 0.06pg/ μ L, and 9 are
0.012pg/μL.。
Specific embodiment
Primer in kit of the present invention is as follows:
It replaces primer LT-5a (SEQ ID No.1):
5'-GCTAATTGTAGGGCTAATACAAG-3'SEQ ID No.1
Replace primer LT-4s (SEQ ID No.2): 5'-CTTGAATGGAACATCGCTAA-3'
Cross primer LT-2a1s (SEQ ID No.3):
5'-CGATGCCTTTTGGCGGCGCGATTCGCAAGTTTATTATG-3'
Probe primer LT-2a (SEQ ID No.4): 5'-FITC-CGATGCCTTTTGGCGGCG-3'
Probe primer LT-3a (SEQ ID No.5): 5'-Bio-GCTTTTAAACCAATTGTTGG-3'
Kit the primer sequent synthesis of the present invention and examining order are completed by Nanjing Jin Sirui limited liability company,
Nucleic acid detection test strip is purchased from Twistdx company.
Application method of the invention are as follows:
(1) measuring samples Whole Blood Genomic DNA is extracted, 200 μ L of animal's whole blood to be checked is taken, is mentioned using poba gene group DNA
Kit is taken to extract genomic DNA (kit is purchased from QIAGEN company, article No. 51306), operating method is carried out referring to specification.
(2) cross primer amplification reaction system
(3) amplification reaction condition: preferably, the cross primer amplification reaction condition is 58~71 DEG C, 50min, 85 DEG C,
2min.It is highly preferred that the cross primer amplification reaction condition is 63 DEG C, 50min, 85 DEG C, 2min.
(4) it is detected
It takes 10 μ L of constant-temperature amplification product to drop on the absorption pad of test strips, then immunity test strip is inserted into containing 100 μ
In the 1.5mL centrifuge tube of L chromatography buffer, 3~5min is stood, determines testing result.
(5) testing result determines
When immunity test strip nature controlling line develops the color, detection line also develops the color, then is determined as the positive；
When immunity test strip nature controlling line develops the color, detection line does not develop the color, then is determined as feminine gender；
When immunity test strip nature controlling line does not develop the color, then testing result is invalid.
The following are the actually detected embodiments of the present invention.
Embodiment 1: the optimization of cross primer constant-temperature amplification optimal reaction temperature and time
1. poba gene group DNA is extracted
The extraction of blood DNA, concrete operation step ginseng are carried out with QIAamp DNAMini Kit (Qiagen, Germany)
Book carries out as directed.
2. design of primers
Mohs Babesia, Babesia U sp Xinjiang Strain, continuous Theileria SP, the You Shi announced according to GenBank is safe
The 18S rRNA gene order of worm etc. is strangled, conservative in type after comparing analysis, the sequence design of inter-species variation is intersected amplification and drawn
Object, target sequence selection are located at 18S rRNA 5 ' and hold, amplification length 150bp.
Replace primer LT-5a:5'-GCTAATTGTAGGGCTAATACAAG-3'
Replace primer LT-4s:5'-CTTGAATGGAACATCGCTAA-3'
Cross primer LT-2a1s:
5'-CGATGCCTTTTGGCGGCGCGATTCGCAAGTTTATTATG-3'
Probe primer LT-2a:5'-FITC-CGATGCCTTTTGGCGGCG-3'
Probe primer LT-3a:5'-Bio-GCTTTTAAACCAATTGTTGG-3'
By constant-temperature amplification, the nucleotide fragments that both ends have FITC and biotin labeling are ultimately formed, are added dropwise in test paper
After on item, the FITC mouse monoclonal antibody and nucleic acid fragment of colloid gold label form compound, and when being diffused into detection line, compound is given birth to
The capture of object element ligand；It is not captured on the nucleic acid complexes nature controlling line of biotin labeling by sheep anti-mouse antibody, is shown at nature controlling line
Color.
3. cross primer constant-temperature amplification
Reaction system:
Above-mentioned reaction tube is subjected to reaction 50min, 80 DEG C of 2min inactivations in 58 DEG C, 61 DEG C, 63 DEG C, 68 DEG C respectively.Selection
After optimal reaction temperature, carry out the optimization in reaction time, i.e., at a temperature of the peak optimization reaction of selection react 20min, 30min,
40min, 50min, 70min, 80 DEG C of 2min inactivations.
4. amplified production test strips detect
Above-mentioned 10 μ L of reaction product is taken, is added dropwise on the absorption pad of chromatograph test strip, insertion is equipped with 100 μ L chromatography buffers
1.5ml centrifuge tube in, stand 3-5min, read experimental result.
5. testing result determines
When nature controlling line develops the color, detection line also develops the color, then is determined as the positive；
When nature controlling line develops the color, detection line does not develop the color, then is determined as feminine gender；
When nature controlling line does not develop the color, then testing result is invalid.
Experimental result is the result shows that (Fig. 1), influence of the reaction temperature to amplification are little, it is contemplated that Bst polymerize enzyme activity
Property optimum temperature, amplified reaction temperature select 63 DEG C；Reaction time selects 50min (Fig. 2).
Embodiment 2: the specific detection of cross primer augmentation detection
1. ovine Piroplasma molitor genomic dna extracts
Take the Mohs Babesia Lintans strain of -20 DEG C of preservations is positive, the strain of Mohs Babesia Hebei is positive, Mohs babe this
The strain of worm Ning County is positive, Babesia U sp Xinjiang Strain is positive, continuous Theileria SP is positive, T.uilenbergi is positive, pyriform worm yin
The extraction that the 200 μ L of Blood In Sheep of property is carried out with QIAamp DNA Mini Kit (Qiagen, Germany), concrete operation step
It is carried out referring to specification.
2. cross primer constant-temperature amplification
Reaction system:
Above-mentioned reaction tube is subjected to reaction 50min, 80 DEG C of 2min inactivations at 63 DEG C respectively.
3. amplified production test strips detect
Above-mentioned 10 μ L of reaction product is taken, is added dropwise on the absorption pad of chromatograph test strip, insertion is equipped with 100 μ L chromatography buffers
1.5ml centrifuge tube in, stand 1min, read experimental result.
4. testing result determines
When nature controlling line develops the color, detection line also develops the color, then is determined as the positive；
When nature controlling line develops the color, detection line does not develop the color, then is determined as feminine gender；
When nature controlling line does not develop the color, then testing result is invalid.
Experimental result is shown in Fig. 3, the results showed that, amplimer group and detection probe used in the present invention to Mohs babe this
Worm has good specificity, and there is no cross reactions with other pyriform worms of infection sheep.
Embodiment 3: the sensitivity test of cross primer amplification
1. Mohs Babesia merozoite DNA is extracted
Take Mohs Babesia Lintan strain 100 μ L of the merozoite QIAamp DNA Mini Kit of the purifying of -80 DEG C of preservations
The extraction that (Qiagen, Germany) is carried out, concrete operation step are carried out referring to specification.
2. the preparation of genomic DNA gradient dilution sample
The Mohs Babesia merozoite genome DNA sample of extraction is measured with trace dna analyzer
(NanoDrop 2000, Thermo Scientific, USA).Concentration is 20ng/ μ L, using ultrapure water by genome DNA sample
Gradient dilution is carried out, 4ng/ μ L, 800pg/ μ L, 160pg/ μ L, 32pg/ μ L, 6.4pg/ μ L, 1.28pg/ μ L, 0.3pg/ are followed successively by
μL、0.06pg/μL、0.012pg/μL。
3. cross primer constant-temperature amplification
Reaction system:
Above-mentioned reaction tube is subjected to reaction 50min, 80 DEG C of 2min inactivations at 63 DEG C respectively.
4. amplified production is analyzed
Above-mentioned 10 μ L of reaction product is taken, is added dropwise on the absorption pad of chromatograph test strip, insertion is equipped with 100 μ L chromatography buffers
1.5mL centrifuge tube in, stand 3-5min, read experimental result.
When nature controlling line develops the color, detection line also develops the color, then is determined as the positive；
When nature controlling line develops the color, detection line does not develop the color, then is determined as feminine gender；
When nature controlling line does not develop the color, then testing result is invalid.
Experimental result is shown in Fig. 4, the results showed that, amplimer group and detection probe used in the present invention to Mohs babe this
Worm has high sensitivity, can detecte the sample of the 0.06pg/ μ of DNA concentration containing polypide L, suitable 50 μ L red blood cell Infestation rat is
0.00006% genomic DNA amount.
Embodiment 4: the detection of Mohs Babesia in goat or Blood In Sheep sample
1. poba gene group DNA is extracted
Pick up from 200 parts of Blood In Sheep sample QIAamp DNA Mini Kit of Gansu Province Tibetan Autonomous Prefecture of Gannan
(Qiagen, Germany) carries out the extraction of blood DNA, and concrete operation step is carried out referring to specification.
2. cross primer constant-temperature amplification
Reaction system:
By above-mentioned reaction tube in 58 DEG C of constant-temperature amplification 50min, 80 DEG C of 2min inactivations.
4. amplified production test strips detect
Above-mentioned 10 μ L of reaction product is taken, is added dropwise on the absorption pad of chromatograph test strip, insertion is equipped with 100 μ L chromatography buffers
1.5ml centrifuge tube in, stand 3-5min, read experimental result.
5. testing result determines
When nature controlling line develops the color, detection line also develops the color, then is determined as the positive；
When nature controlling line develops the color, detection line does not develop the color, then is determined as feminine gender；
When nature controlling line does not develop the color, then detection is invalid.
The results show that detecting 3 parts of (1.5%) Mohs Babesia senses in 200 parts of ovine genomes of Gansu acquisition
The sample of dye.These results and the result investigated using LAMP, RLB and Real-time method early period are substantially consistent.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of Mohs Babesia detection kit
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (displacement primer LT-5a)
<400> 1
gctaattgta gggctaatac aag 23
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (displacement primer LT-4s)
<400> 2
cttgaatgga acatcgctaa 20
<210> 3
<211> 38
<212> DNA
<213>artificial sequence (cross primer LT-2a1s)
<400> 3
cgatgccttt tggcggcgcg attcgcaagt ttattatg 38
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (probe primer LT-2a)
<400> 4
cgatgccttt tggcggcg 18
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (displacement primer LT-5a)
<400> 5
gcttttaaac caattgttgg 20
Claims (4)
1. a kind of this detection kit of Mohs babe, it is characterised in that include in detection kit: sequence is SEQ ID No.3
A cross primer, sequence be SEQ ID No.1 and sequence be SEQ ID No.2 displacement primer, with FITC mark spy
The probe primer SEQ ID No.5 and immuno-chromatographic test paper strip of needle primer SEQ ID No.4 and biotin labeling.
2. Mohs Babesia detection kit according to claim 1, it is characterised in that in detection kit further include:
10 × Isothermal Amplification Buffer, 100mM MgSO4,2.5mM dNTP mix, Bst archaeal dna polymerase.
3. Mohs Babesia detection kit as claimed in claim 1 or 2 is used for the application method of non-disease diagnosis, feature exists
In the genome of sample to be tested DNA using extraction as template, expanded with the cross primer constant temperature that the primer and probe in kit is established
Increase reaction system to be expanded, then amplified production is added dropwise to immuno-chromatographic test paper strip, according to the nature controlling line of test strips and detection
Simultaneously whether the determining measuring samples that develop the color have Mohs Babesia to line.
4. application method according to claim 3, it is characterised in that the reaction condition of amplification are as follows: 63 DEG C, 50min；80 DEG C,
2min。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913569A (en) * | 2019-04-12 | 2019-06-21 | 中国农业科学院兰州兽医研究所 | A kind of primer and kit can be used for Theileria SP detection |
| CN109913568A (en) * | 2019-04-12 | 2019-06-21 | 中国农业科学院兰州兽医研究所 | A kind of primer and kit can be used for hepatozoon canis detection |
| CN110551838A (en) * | 2019-07-26 | 2019-12-10 | 中国农业科学院兰州兽医研究所 | Method and kit for undetermined species identification and detection of babesia |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561343A (en) * | 2015-01-24 | 2015-04-29 | 中国农业科学院兰州兽医研究所 | Primer and kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi |
-
2019
- 2019-01-14 CN CN201910033593.4A patent/CN109593826A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561343A (en) * | 2015-01-24 | 2015-04-29 | 中国农业科学院兰州兽医研究所 | Primer and kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi |
Non-Patent Citations (1)
| Title |
|---|
| 黄梦琦等: "交叉引物恒温扩增技术（CPA）研究进展", 《民营科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913569A (en) * | 2019-04-12 | 2019-06-21 | 中国农业科学院兰州兽医研究所 | A kind of primer and kit can be used for Theileria SP detection |
| CN109913568A (en) * | 2019-04-12 | 2019-06-21 | 中国农业科学院兰州兽医研究所 | A kind of primer and kit can be used for hepatozoon canis detection |
| CN110551838A (en) * | 2019-07-26 | 2019-12-10 | 中国农业科学院兰州兽医研究所 | Method and kit for undetermined species identification and detection of babesia |
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Application publication date: 20190409 |