CN104293903A - Primer combination, kit and detection method for detecting babeisa canis - Google Patents
Primer combination, kit and detection method for detecting babeisa canis Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and a detection method for detecting babeisa canis, and belongs to the technical field of biological detection. A nested PCR primer combination is designed for 18s RNA gene sequence of babeisa canis. The primer combination has high primer specificity, is not interfered by other non-target genes, and can determine a transcriptional level of babeisa canis rapidly, efficiently and accurately. The method for qualitatively detecting babeisa canis is simple and accurate, can accurately detect DNA samples with the minimum copy number of 10 within 4 hours, and has high detection sensitivity. The sensitivity of the nested PCR is 1,000 times higher than that of common PCR in gradient detections for a standard sample under the same experimental conditions; and the sensitivity of the nested PCR is 3-10% higher than that of the common PCR in actual clinical detections.
Description
Technical field
The present invention is specifically related to a kind of combination of primers for detecting dog babesia, and the test kit containing this combination of primers, also relates to the method adopting this combination of primers to detect dog babesia simultaneously, belongs to technical field of biological.
Background technology
Babesiosis is a kind of through tick-borne haematogenous protozoal disease, extensively betides various domestic animal.Single or parasitize in pairs in red corpuscle in its carcass of being in, likely cause serious anaemia and hemoglobinuria.The pathogenic agent of the babesiosis of dog is caused to have 3 kinds, i.e. dog babesia (Babeisa canis), Webster babesia (B.vitlli) and Ji Shi babesia (B.gibsoni).Dog babesia is distributed in Europe, America and India, and Webster babesia is distributed in South America, and Ji Shi babesia is distributed in Asia.The worm kind of China's Major Epidemic is Ji Shi babesia, widely distributed at home at present, and have the place that tick grows, spring, summer, autumn all can fall ill.
At present, Blood protozoan detection method mainly contains smear staining microscopy, enzyme linked immunoassay, indirect fluorescent, LAMP, polymerase chain reaction (PCR) etc.The requirement of smear staining Microscopical Method For Detection to test set is lower, easy and simple to handle, but verification and measurement ratio is lower.LAMP technology detects simple and quick, and naked eyes get final product direct observations, but are easy to occur false positive, and not easily identify the kind of polypide.And round pcr is considered to its higher specificity and susceptibility the most effectual way detecting babesia.
Summary of the invention
The object of this invention is to provide a kind of combination of primers for detecting dog babesia.
Meanwhile, the present invention also provides a kind of test kit for detecting dog babesia.
Finally, the invention provides a kind of method detecting dog babesia.
In order to realize above object, the technical solution adopted in the present invention is:
For detecting a combination of primers for dog babesia, be made up of outer primer and inner primer, outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ' (as shown in SEQ ID NO:1),
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ' (as shown in SEQ ID NO:2);
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ' (as shown in SEQ ID NO:3),
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 ' (as shown in SEQ ID NO:4).
For detecting a test kit for dog babesia, mainly comprise the combination of primers be made up of outer primer and inner primer, outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
Concrete, for detecting the test kit of dog babesia, Taq enzyme, Mg can also be comprised
2+, mix dNTP, damping fluid (as 10 × PCR Buffer), DNA Marker(standard substance) and test kit reaction conditions specification sheets etc.
More specifically, for detecting the test kit of dog babesia, comprising: Taq enzyme 100U, 10 × PCR Buffer500 μ L, 25mmol.L
-1mg
2+salt 500 μ L, 10mmol.L
-1mix dNTP100 μ L, 10pmol μ L
-1the each 100 μ L of combination of primers (comprising interior outer primer forward and reverse).
Detect a method for dog babesia, comprise the following steps:
(1) with DNA sample to be detected for template, adopt combination of primers carry out sleeve type PCR amplification;
(2) get amplified production and carry out gel electrophoresis, whether judge in sample containing dog babesia according to electrophoresis result;
In described step (1), combination of primers is made up of outer primer and inner primer, and outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
The reaction system adopting combination of primers to carry out sleeve type PCR amplification in described step (1) is:
First time PCR:50 μ L system, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/L Mg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L outer primer, DNA sample to be detected 1 μ L, add ddH
2o to 50 μ L;
Second time PCR:50 μ L system, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/L Mg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L inner primer, first time pcr amplification product 1 μ L, add ddH
2o to 50 μ L.
The response procedures of described first time PCR is: 94 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 25 circulations, 72 DEG C extend 10min, and cooling terminates reaction.
The response procedures of described second time PCR is: 95 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 30 circulations, 72 DEG C extend 7min, and cooling terminates reaction.
In described step (2), gel electrophoresis adopts the sepharose of 1.0%EB dyeing, so that observe electrophoresis result after electrophoresis under ultraviolet lamp.Contrasting with DNA Marker, when occurring in tiselius apparatus that size is the fragment of 658bp, can judge in sample containing dog babesia genetic material.
Beneficial effect of the present invention:
The present invention is directed to dog babesia 18s rna gene sequences Design sleeve type PCR combination of primers, the high specificity of this primer, not by the interference of other non-goal gene, the transcriptional level of quick, efficient, the accurate qualitative dog babesia of energy.
The present invention is easy to use for the test kit detecting dog babesia, is easy to carry, and is applicable to repeated detection.
The method of qualitative detection dog babesia of the present invention is simple, accurate, in 4 hours, accurately can detect that minimum is the DNA sample of 10 copies, detection sensitivity is high, in detecting the gradient of standard substance under equal experiment condition, the remolding sensitivity regular-PCR of sleeve type PCR exceeds 1000 times, and during actual clinical detects, the remolding sensitivity regular-PCR of sleeve type PCR is high 3 ~ 10 percentage points.
Accompanying drawing explanation
Fig. 1 is the electrophoretic image of amplified production in the embodiment of the present invention 3;
Fig. 2 is the electrophoretic image of specific amplification dog babesia in test example 1;
Fig. 3 is the electrophoretic image detecting the sensitivity of dog babesia Nested Polymerase Chain Reaction in test example 2;
Fig. 4 is the electrophoretic image detecting the sensitivity of dog babesia regular-PCR method in test example 2;
Fig. 5 is the electrophoretic image of sleeve type PCR and regular-PCR amplified production in test example 3.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
For detecting the combination of primers of dog babesia in the present embodiment, be made up of outer primer and inner primer, outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
Embodiment 2
For detecting the test kit of dog babesia in the present embodiment, comprise the combination of primers be made up of outer primer and inner primer, and Taq enzyme 100U, 10 × PCR Buffer500 μ l, 25mmol.L
-1mg
2+salt 500 μ L, 10mmol.L
-1mix dNTP100 μ L, 10pmol μ L
-1the each 100 μ L of combination of primers (comprising interior outer primer forward and reverse), standard substance 25 μ L and test kit reaction conditions specification sheets.
Described outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Described inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
Embodiment 3
Detect the method for dog babesia in the present embodiment, comprise the following steps:
(1) with DNA sample to be detected for template, adopt combination of primers carry out sleeve type PCR amplification, combination of primers is made up of outer primer and inner primer, and outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ',
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 ';
The reaction system of pcr amplification is for the first time: 50 μ L systems, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/LMg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L outer primer, DNA sample to be detected 1 μ L, add ddH
2o to 50 μ L, response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, and carry out 25 circulations, 72 DEG C extend 10min, and cooling terminates reaction;
The reaction system of second time pcr amplification is: 50 μ L systems, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/LMg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L inner primer, first time pcr amplification product 1 μ L, add ddH
2o to 50 μ L, response procedures is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, and carry out 30 circulations, 72 DEG C extend 7min, and cooling terminates reaction;
(2) electrophoresis on sepharose that 5 μ L sleeve type PCR amplified productions dye at 1.0%EB is got, electrophoretic voltage is 80V, time is 20 minutes, after electrophoresis under ultraviolet lamp observations, contrast with DNA Marker, find between 500 ~ 750bp, occur that size is that the characteristic band of 658bp is (see Fig. 1, in figure, M is DNA Marker, 1, positive product external primer amplification product, 2, positive product inner primer amplified production, 3, distilled water external primer amplification product, 4, distilled water inner primer amplified production), judge in this sample containing dog babesia genetic material.
Test example 1
Sleeve type PCR amplification is carried out under the negative control reaction system not adding template in positive for dog babesia product, sterilizing tri-distilled water, pasteurellosis bacillus, Eperythrozoon, toxoplasma gondii, suis and reaction solution is placed in identical conditions, specifically see step (1) in embodiment 3, second time pcr amplification product 5 μ L is used for carrying out agarose gel electrophoresis detection.Electrophoresis result is shown in Fig. 2, is followed successively by DNA Marker, dog babesia positive product sleeve type PCR result, negative control, sterilizing tri-distilled water, pasteurellosis bacillus, Eperythrozoon, toxoplasma gondii, suis and DNA Marker in figure from right-to-left.As can be seen from Figure 2, the characteristic band of dog babesia positive product sleeve type PCR result is between Marker750bp and Marker500bp band.There is not apparent position characteristic band in other negative controls, sterilizing tri-distilled water, pasteurellosis bacillus, Eperythrozoon, toxoplasma gondii, suis.
Test example 2
By dog babesia positive criteria product 10
6~ 10
0copy adds in sleeve type PCR reaction system as template respectively, and system carries out sleeve type PCR amplification under being placed in identical conditions, is used for carrying out agarose gel electrophoresis detection, second time pcr amplification product 5 μ L with test example 1.Electrophoresis result is shown in Fig. 3, is followed successively by DNA Marker, (1 ~ 6 is followed successively by 10 for standard substance that positive dog DNA makes in figure from right-to-left
6~ 10
0copy sample sleeve type PCR result) and DNA Marker.As can be seen from Figure 3,10
6~ 10
1all there is specific band in copy sample sleeve type PCR result, between Marker750bp and Marker500bp band, and 10
0there is not apparent position characteristic band in copy sample sleeve type PCR result.
By dog babesia positive criteria product 10
8~ 10
1copy adds in common PCR reaction system as template respectively, and system carries out regular-PCR amplification under being placed in identical conditions (concrete operations are see number of patent application: 201110157247.0), are used for carrying out agarose gel electrophoresis detection by pcr amplification product 5 μ l.Electrophoresis result is shown in Fig. 4, is followed successively by DNAMarker, (1 ~ 8 is followed successively by 10 for standard substance that positive dog DNA makes in figure from right-to-left
8~ 10
1copy sample sleeve type PCR result).As can be seen from Figure 4,10
8~ 10
4all there is specific band in copy sample P CR result, between Marker250bp and Marker500bp band, and 10
3~ 10
1there is not apparent position characteristic band in copy sample P CR result.
Test example 3
1, incidence
The small-sized sleuth in somewhere, Henan one, male, 4 years old, press normal procedure immunity.Be admitted to hospital to being in a bad way, this dog of clinical observation only spirit is depressed, and eat less or do not eat, body temperature is up to 40.5 DEG C, and visual mucous membrane is anaemia height jaundice, and urine is strong tea water colour.Morbidity is once movable in mountain area for the last week, finds tick worm, doubtful haematogenous protozoan infection when being admitted to hospital at a dog basal part of the ear place.But do not find polypide through microscopy, adopt regular-PCR detection method do not occur yet positive findings (concrete operations are see number of patent application: 201110157247.0), and then adopt sleeve type PCR detection method, concrete steps are:
(1) preparation of DNA sample to be detected: get 300 μ L anticoagulations in dog forelimb saphena, adopt whole blood DNA to extract test kit (TAKATA company), by specification working method extracts DNA sample;
(2) with DNA sample to be detected for template, adopt combination of primers carry out sleeve type PCR amplification, combination of primers is made up of outer primer and inner primer, and outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ',
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 ';
The reaction system of pcr amplification is for the first time: 50 μ L systems, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/LMg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L outer primer, DNA sample to be detected 1 μ L, add ddH
2o to 50 μ L, response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, and carry out 25 circulations, 72 DEG C extend 10min, and cooling terminates reaction;
The reaction system of second time pcr amplification is: 50 μ L systems, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/LMg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L inner primer, first time pcr amplification product 1 μ L, add ddH
2o to 50 μ L, response procedures is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, and carry out 30 circulations, 72 DEG C extend 7min, and cooling terminates reaction;
(3) electrophoresis on sepharose that 5 μ L sleeve type PCR amplified productions dye at 1.0%EB is got, electrophoretic voltage is 80V, time is 20 minutes, after electrophoresis under ultraviolet lamp observations, contrast with DNA Marker, find between 500 ~ 750bp, occur that size is that the characteristic band of 658bp is (see Fig. 5, in figure, M is DNA Marker, 1, DNA sample sleeve type PCR detected result, 2, DNA sample regular-PCR detected result, 3, distilled water contrasts, 4, negative control), the nucleotide sequence of amplified production is as shown in SEQ ID NO:2, judge in this sample containing dog babesia genetic material.
2, Clinics and Practices
According to epidemiology and clinicing symptom observation, because microscopy does not find polypide, in conjunction with sleeve type PCR detected result, this dog of preliminary judgement infected dogs babesia.
Test example 4
Get area, west, Henan (Luoyang, the Sanmenxia Gorge, tafelberg) dog blood sample and carry out the detection of dog babesia, adopt sleeve type PCR detection method and regular-PCR detection method (detecting step is with test example 3) respectively, detected result refers to following table 1.
Dog babesia detected result in the western regional dog blood sample in table 1 Henan
As known from Table 1, in clinical detection, dog babesia sleeve type PCR detection method is compared with highly sensitive 3 ~ 10 percentage points of regular-PCR detection method.
Claims (9)
1. for detecting a combination of primers for dog babesia, it is characterized in that: be made up of outer primer and inner primer, outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
2. for detecting a test kit for dog babesia, it is characterized in that: mainly comprise the combination of primers be made up of outer primer and inner primer, outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
3. the test kit for detecting dog babesia according to claim 2, is characterized in that: also comprise Taq enzyme, Mg
2+, mix dNTP and damping fluid.
4. the test kit for detecting dog babesia according to claim 3, is characterized in that: comprising: Taq enzyme 100U, 10 × PCR Buffer500 μ l, 25mmol.L
-1mg
2+salt 500 μ L, 10mmol.L
-1the each 100 μ L of mix dNTP, 10pmol μ L
-1the each 100 μ L of combination of primers.
5. detect a method for dog babesia, it is characterized in that: comprise the following steps:
(1) with DNA sample to be detected for template, adopt combination of primers carry out sleeve type PCR amplification;
(2) get amplified production and carry out gel electrophoresis, whether judge in sample containing dog babesia according to electrophoresis result;
In described step (1), combination of primers is made up of outer primer and inner primer, and outer primer is:
Forward primer: 5 '-GGCTTTCGGTGATTCATA-3 ',
Reverse primer: 5 '-CCTTCCGTCAATTCCTTT-3 ';
Inner primer is:
Forward primer: 5 '-TGGACCATTCAAGTTTCTG-3 ',
Reverse primer: 5 '-TCGTCTTCGATCCCCTA-3 '.
6. the method for detection dog babesia according to claim 5, is characterized in that: the reaction system adopting combination of primers to carry out sleeve type PCR amplification in described step (1) is:
First time PCR:50 μ L system, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/L Mg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L outer primer, DNA sample to be detected 1 μ L, add ddH
2o to 50 μ L;
Second time PCR:50 μ L system, Taq enzyme 1U, 10 × PCR Buffer5 μ L, 25mmol/L Mg
2+the each 1 μ L of 4 μ L, 10mmol/L mix dNTP1 μ L, the forward and reverse primer of 10pmol/ μ L inner primer, first time pcr amplification product 1 μ L, add ddH
2o to 50 μ L.
7. the method for detection dog babesia according to claim 6, it is characterized in that: the response procedures of described first time PCR is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 25 circulations, 72 DEG C extend 10min, and cooling terminates reaction.
8. the method for detection dog babesia according to claim 6, it is characterized in that: the response procedures of described second time PCR is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 30 circulations, 72 DEG C extend 7min, and cooling terminates reaction.
9. the method for detection dog babesia according to claim 5, is characterized in that: in described step (2), gel electrophoresis adopts the sepharose of 1.0%EB dyeing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861651A (en) * | 2016-04-01 | 2016-08-17 | 南京农业大学 | Babesia canis PCR detection kit containing internal amplification control and use method of Babesia canis PCR detection kit |
| CN106893785A (en) * | 2017-05-08 | 2017-06-27 | 河南科技大学 | Fluorescence quantification PCR primer combination, kit and detection method for quick detection dog babesia |
| US10494680B2 (en) | 2017-08-15 | 2019-12-03 | Delta Electronics Int'l (Singapore) Pte Ltd | Primer pair, kit and method for detecting Babesia gibsoni |
| CN112251521A (en) * | 2020-10-28 | 2021-01-22 | 龙岩学院 | Universal primer and kit for detecting babesia within tiger or tick |
-
2014
- 2014-03-20 CN CN201410105100.0A patent/CN104293903B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 尚泽松等: "犬巴贝西虫病PCR检测方法的建立", 《河南农业科学》, vol. 41, no. 5, 31 December 2012 (2012-12-31), pages 158 - 160 * |
| 简子健等: "牛巴贝斯虫巢式PCR诊断方法的建立", 《中国兽医学报》, vol. 30, no. 3, 31 March 2010 (2010-03-31), pages 356 - 358 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861651A (en) * | 2016-04-01 | 2016-08-17 | 南京农业大学 | Babesia canis PCR detection kit containing internal amplification control and use method of Babesia canis PCR detection kit |
| CN106893785A (en) * | 2017-05-08 | 2017-06-27 | 河南科技大学 | Fluorescence quantification PCR primer combination, kit and detection method for quick detection dog babesia |
| US10494680B2 (en) | 2017-08-15 | 2019-12-03 | Delta Electronics Int'l (Singapore) Pte Ltd | Primer pair, kit and method for detecting Babesia gibsoni |
| CN112251521A (en) * | 2020-10-28 | 2021-01-22 | 龙岩学院 | Universal primer and kit for detecting babesia within tiger or tick |
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| CN104293903B (en) | 2016-08-24 |
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