CN111534625A - PCR detection method for Babesia gibsoni - Google Patents
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- 241001466447 Babesia gibsoni Species 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 230000003321 amplification Effects 0.000 claims abstract description 20
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 20
- 238000001962 electrophoresis Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- 241000223836 Babesia Species 0.000 claims abstract description 8
- 201000008680 babesiosis Diseases 0.000 claims abstract description 8
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 239000007787 solid Substances 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 150000007523 nucleic acids Chemical group 0.000 claims description 9
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- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
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- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
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- 230000009286 beneficial effect Effects 0.000 abstract description 2
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Abstract
The invention provides a PCR detection method of Babesia gibsoni, which comprises the following steps: extracting DNA of a sample to be detected; secondly, PCR amplification is carried out by utilizing the Babesia gibsoni identification specific primer; and step three, carrying out electrophoresis detection on the amplification product. The method has the beneficial effect of quickly and accurately detecting whether the babesia infected by the pet is the babesia gibsoni or not.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a PCR detection method of Babesia gibsoni, which can quickly and accurately detect whether the Babesia gibsoni infected by pets is the Babesia gibsoni.
Background
The babesia gibsoni belongs to one of the babesia, the proportion of the babesia gibsoni in canine infection babesia is the highest, and the differences of pathogenicity, prognosis and drug sensitivity exist in different babesia, so that the identification of the species of the babesia gibsoni is necessary, and whether the babesia gibsoni is used as the one with the highest domestic babesia proportion can be accurately detected and is directly related to the subsequent clinical treatment.
The traditional detection method of the Babesia gibsoni is mainly under-microscope detection, namely under-microscope detection after Reye staining of a blood smear. However, because the sample size of the blood smear detection is small, multiple microscopic examinations are often needed to determine the sample size, even if false negative possibility cannot be eliminated, and the microscopic observation needs to be distinguished from other hematoparasites, so that certain difficulty exists, and the detection result is often deviated.
Although a plurality of immunological methods are reported to be used for detecting the Babesia gibsoni at present, the specificity of Elisa or IFA is poor, and the antigen used in the kit is easy to generate cross reaction with other worms, so that false positive is generated.
Disclosure of Invention
The invention overcomes the defects in the prior art and provides a PCR detection method for Babesia gibsoni.
The purpose of the invention is realized by the following technical scheme.
A PCR detection method for Babesia gibsoni comprises the following steps:
extracting DNA of a sample to be detected;
step two, carrying out PCR amplification by using the Babesia gibsoni identification specific primer, wherein the sequence of the specific primer is as follows:
gi.BaB-1:5’-CTCGRCTACTTGCCTTGT-3’；
gi.BaB:5’-CGAARCTGAAAYAACGG-3’；
and step three, carrying out electrophoresis detection on the amplification product.
Further, the specific method of the first step comprises the following steps:
step one, adding proteinase K into a sample to obtain a mixture;
secondly, adding Buffer AL into the mixture obtained in the first step, mixing uniformly and then carrying out vortex oscillation;
a third step of incubating the mixture of the second step;
step four, adding ethanol into the mixture incubated in the step three, and fully and uniformly mixing to ensure that the liquid on the tube wall sinks to the tube bottom;
fifthly, transferring the liquid obtained in the fourth step to a nucleic acid purification column, and centrifuging to leave a solid;
sixthly, adding Buffer AW1 into the solid obtained by the centrifugation in the fifth step, and centrifuging to leave the solid;
seventhly, adding Buffer AW2 into the solid obtained after the centrifugation in the sixth step, and centrifuging to leave the solid;
eighth, centrifuging the solid obtained in the seventh step again;
and a ninth step of adding Buffer AE or sterilized water to the solid obtained in the eighth step, standing at room temperature for 1min, and centrifuging again to obtain DNA.
Further, the incubation temperature in the third step is 56 ℃ for 10 min.
Further, the rotating speed of the centrifugation in the fifth step and the sixth step is 8000rpm, and the duration is 1 min.
Further, the rotating speed of the centrifugation in the seventh step is 12000rpm, and the time duration is 3 min.
Further, the rotating speed of the centrifugation in the eighth step is 12000rpm, and the time duration is 1 min.
Further, the rotating speed of the centrifugation in the ninth step is 8000rpm, and the time duration is 1 min.
Further, the amplification system of the second step is:
the total volume of the reaction system is 25 mul, and the reaction components are as follows: 8. mu.l of sterilized water, 12.5. mu.l of rtaqMix, 1. mu.l of gi.BaB-1, 1. mu.l of gi.BaB, 2.5. mu.l of sample DNA extract;
gi.BaB-1:5’-CTCGRCTACTTGCCTTGT-3’；
gi.BaB:5’-CGAARCTGAAAYAACGG-3’；
further, the second step amplification reaction program is as follows:
the first step, pre-amplification is carried out at 94 ℃ for 5 min;
secondly, amplifying at 94 ℃ for 30 s;
step three, amplifying at 60 ℃ for 30 s;
fourthly, amplifying at 72 ℃ for 42 s;
the first to fourth steps were performed for 35 cycles before amplification at 72 ℃ for 5 min.
Further, the method for detecting the amplification product by electrophoresis in the second step comprises the following steps: and (3) adopting 8 mu l of the three-step PCR amplification product, adopting a 1.5% agarose platform, carrying out electrophoresis at 5V/cm for 30min, stopping when bromophenol blue reaches the bottom, and observing a 671bp nucleic acid fragment in an ultraviolet detector, thus determining that the detected sample is positive to the Babesia gibsonii nucleic acid.
The invention has the beneficial effects that:
the existing national standard does not apply to the canine Babesia gibsoni detection method, but the existing Babesia gibsoni detection method aiming at other animals also mainly depends on the serological detection method, and the method is an effective supplement for corresponding detection methods and different detection objects.
PCR belongs to an exponential amplification method, and the nucleic acid of target pathogens with multiple indexes can be finally obtained by simulating in vivo amplification in vitro.
In the conventional PCR experiment and reagents selected in part of laboratories, polymerase, dntp, loading buffer and buffer solution are often added into a system separately, and the detection method in the patent selects a PCR mix reagent and simultaneously adds the reagents into the system in one step, so that cross contamination caused by aerosol generated by using a pipettor for many times is avoided, the risk of contamination is reduced, and the detection accuracy is improved.
The detection primers adopted by the method are specific, so that cross reaction can not occur to other types of babesia or other hematozootic production, and the possible false positive risk in serological detection is reduced.
Drawings
FIG. 1 shows the electrophoresis of the assay in example 1, wherein A shows the electrophoresis result (671bp) of the standard positive fragment and B shows the electrophoresis result of DL 2000 Marker.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
The invention uses PCR method to determine the specificity DNA fragment of Babesia gibsoni in the sample, uses DNA nucleic acid extraction reagent to extract the total genome DNA of blood sample for suspected case, after heating denaturation of Babesia gibsoni body DNA, two artificially synthesized specificity primers are respectively denatured with the wing sequence of the Babesia gibsoni body DNA, under proper condition, the heat-resistant DNA polymerase catalyzes the synthesis (namely extension) of the Babesia gibsoni body DNA guided by the primer, completes the PCR cycle of heat denaturation-renaturation-extension, through 35 times of cycle amplification, can check the specificity strip of the Babesia gibsoni body DNA by agarose gel electrophoresis, thus judges whether the Babesia gibsoni nucleic acid exists in the sample.
Example 1
PCR detection of Babesia gibsoni
Comprises the following steps:
1. sample DNA extraction
1) Add 20. mu.l proteinase K to a 1.5ml EP tube;
2) add 200. mu.l of sample to a 1.5ml EP tube. If the sample is insufficient, PBS may be added to make up the volume;
3) adding 200 mul Buffer AL into the sample, mixing uniformly, and performing vortex oscillation for 15 s;
4) incubating at 56 deg.C for 10 min;
5) adding 200 mul of ethanol into the sample, and fully and uniformly mixing to ensure that the liquid on the tube wall sinks to the bottom of the tube;
6) carefully transfer the liquid obtained in the previous step to a nucleic acid purification column (place on a 2ml collection tube) without wetting the edges. After capping, centrifuge at 1000rpm for 1 min. After the waste liquid is discarded, a new collecting pipe is replaced;
7) carefully open the nozzle and add 500. mu.l Buffer AW1 without wetting the edges. Centrifuge at 1000rpm for 1min with the cap closed. After the waste liquid is discarded, a new collecting pipe is replaced;
8) carefully open the nozzle and add 500. mu.l Buffer AW2 without wetting the edges. Cover the cover tightly and centrifuge at 12000rpm for 3 min;
9) after discarding the waste liquid, the collection tube was replaced with a new one. Centrifuging at 12000rpm for 1 min;
10) the nucleic acid purification column was placed on a new 1.5ml collection tube and the collection tube containing the waste liquid was discarded. Carefully open the tube, add 50. mu.l Buffer AE and let stand at room temperature for 1 min. Then, the DNA was obtained by centrifugation at 1000rpm for 1 min.
2. PCR amplification
2.1, carrying out PCR amplification by using the Babesia gibsoni identification specific primer, wherein the sequence of the specific primer is as follows:
BaB-1:5 '-CTCGRCTACTTGCCTTGT-3' as shown in sequence table 1;
BaB:5 '-CGAARCTGAAAYAACGG-3' as shown in sequence Listing 2;
the amplification system is as follows: the total volume of the reaction system is 25 mul, and the reaction components are as follows: 8. mu.l of sterilized water, 12.5. mu.l of rtaqMix, 1. mu.l of gi.BaB-1, 1. mu.l of gi.BaB, 2.5. mu.l of sample DNA extract;
BaB-1:5 '-CTCGRCTACTTGCCTTGT-3' as shown in sequence table 1;
BaB:5 '-CGAARCTGAAAYAACGG-3' as shown in sequence Listing 2;
the second step of amplification reaction program is:
the first step, pre-amplification is carried out at 94 ℃ for 5 min;
secondly, amplifying at 94 ℃ for 30 s;
step three, amplifying at 60 ℃ for 30 s;
fourthly, amplifying at 72 ℃ for 42 s;
the first to fourth steps were performed for 35 cycles before amplification at 72 ℃ for 5 min.
3. Agarose gel electrophoresis
Prepare 1.5% agarose platform with TAE electrophoresis buffer (1. mu.l GelRed stock solution is needed for each 10ml of electrophoresis solution), place the plate in horizontal electrophoresis apparatus, make the electrophoresis buffer just over the gel surface, take 8. mu.l PCR product and add to the sample well. DNA standard molecular weight was set as a control during electrophoresis. Electrophoresis at 5V/cm for about 30min, stopping when bromophenol blue reaches the bottom, and observing the result in an ultraviolet detector.
4. Determination of results
Under the condition that the negative control and the positive control of the Babesia gibsoni are established, if a nucleic acid fragment with the size of 671bp is amplified from a sample to be detected, and the specific sequence is shown as a sequence table 3, the nucleic acid of the Babesia gibsoni is preliminarily judged to be positive; and if the sample to be detected has no amplified band or the amplified band size is not 671bp, judging that the nucleic acid of the Babesia gibsoni is negative.
While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Sequence listing
<110> Tianjin Tuorui medicine science and technology Co., Ltd
<120> PCR detection method for Babesia gibsoni
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<170>SIPOSequenceListing 1.0
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<213> sequence 1(2 Ambystoma latex x Ambystoma jeffersonia)
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ctcgrctact tgccttgt 18
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<212>DNA
<213> sequence 2(2 Ambystoma latex x Ambystoma jeffersonia)
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cgaarctgaa ayaacgg 17
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<213> sequence 3(2 Ambystoma latex x Ambystoma jeffersonia)
<400>3
ctcggctact tgccttgtct ggtttcgctt ttggggtttt ccccttttta ctttgagaaa 60
attagagtgt ttcaagcaga cttgtgtctt gaatacttca gcatggaata ataaagtagg 120
actttggttc tattttgttg gtttgtgaac cttagtaatg gttaatagga acggttgggg 180
gcattcgtat ttaactgtca gaggtgaaat tcttagattt gttaaagacg aactactgcg 240
aaagcatttg ccaaggacgt tttcattaat caagaacgaa agttagggga tcgaagacga 300
tcagataccg tcgtagtcct aaccataaac catgccgact agggattgga ggtcgtcatt 360
tttcgactcc ttcagcacct tgagagaaat caaagtcttt gggttctggg gggagcatgg 420
tcgcaaggct gaaacttaaa ggaattgacg gaagggcacc accaggcgtg gagcctgcgg 480
cttaatttga ctcaacacgg ggaaactcac caggtccaga caaagttagg attgacagat 540
tgatagctct ttcttgattc tttgggtggt ggtgcatggc cgttcttagt tggtggagtg 600
atttgtctgg ttaattccgt taacgaacga gaccttaacc tgctaactag ttgccgttat 660
ttcagtttcg g 671
Claims (10)
1. A PCR detection method of Babesia gibsoni is characterized in that: the method comprises the following steps:
extracting DNA of a sample to be detected;
step two, carrying out PCR amplification by using the Babesia gibsoni identification specific primer, wherein the sequence of the specific primer is as follows:
gi.BaB-1:5’-CTCGRCTACTTGCCTTGT-3’；
gi.BaB:5’-CGAARCTGAAAYAACGG-3’；
and step three, carrying out electrophoresis detection on the amplification product.
2. The method for PCR detection of Babesia gibsoni according to claim 1, wherein: the specific method of the first step comprises the following steps:
step one, adding proteinase K into a sample to obtain a mixture;
secondly, adding Buffer AL into the mixture obtained in the first step, mixing uniformly and then carrying out vortex oscillation;
a third step of incubating the mixture of the second step;
step four, adding ethanol into the mixture incubated in the step three, and fully and uniformly mixing to ensure that the liquid on the tube wall sinks to the tube bottom;
fifthly, transferring the liquid obtained in the fourth step to a nucleic acid purification column, and centrifuging to leave a solid;
sixthly, adding Buffer AW1 into the solid obtained by the centrifugation in the fifth step, and centrifuging to leave the solid;
seventhly, adding Buffer AW2 into the solid obtained after the centrifugation in the sixth step, and centrifuging to leave the solid;
eighth, centrifuging the solid obtained in the seventh step again;
and a ninth step of adding Buffer AE or sterilized water to the solid obtained in the eighth step, standing at room temperature for 1min, and centrifuging again to obtain DNA.
3. The PCR detection method of Babesia gibsoni as claimed in claim 2, wherein: the incubation temperature in the third step is 56 ℃ for 10 min.
4. The PCR detection method of Babesia gibsoni as claimed in claim 2, wherein: the rotation speed of centrifugation in the fifth step and the sixth step is 8000rpm, and the duration is 1 min.
5. The PCR detection method of Babesia gibsoni as claimed in claim 2, wherein: and in the seventh step, the rotating speed of the centrifugation is 12000rpm, and the time duration is 3 min.
6. The PCR detection method of Babesia gibsoni as claimed in claim 2, wherein: and in the eighth step, the rotating speed of the centrifuge is 12000rpm, and the time duration is 1 min.
7. The PCR detection method of Babesia gibsoni as claimed in claim 2, wherein: and in the ninth step, the rotating speed of the centrifugation is 8000rpm, and the time duration is 1 min.
8. The method for PCR detection of Babesia gibsoni according to claim 1, wherein: the amplification system of the second step is as follows:
the total volume of the reaction system is 25 mul, and the reaction components are as follows: 8. mu.l of sterilized water, 12.5. mu.l of rtaqMix, 1. mu.l of gi.BaB-1, 1. mu.l of gi.BaB, 2.5. mu.l of sample DNA extract;
gi.BaB-1:5’-CTCGRCTACTTGCCTTGT-3’；
gi.BaB:5’-CGAARCTGAAAYAACGG-3’。
9. the PCR detection method of Babesia gibsoni as claimed in claim 8, wherein: the second step of amplification reaction program is:
the first step, pre-amplification is carried out at 94 ℃ for 5 min;
secondly, amplifying at 94 ℃ for 30 s;
step three, amplifying at 60 ℃ for 30 s;
fourthly, amplifying at 72 ℃ for 42 s;
the first to fourth steps were performed for 35 cycles before amplification at 72 ℃ for 5 min.
10. The method for PCR detection of Babesia gibsoni according to claim 1, wherein: the method for detecting the electrophoresis of the amplification product in the second step comprises the following steps: and (3) adopting 8 mu l of the three-step PCR amplification product, adopting a 1.5% agarose platform, carrying out electrophoresis at 5V/cm for 30min, stopping when bromophenol blue reaches the bottom, and observing a 671bp nucleic acid fragment in an ultraviolet detector, thus determining that the detected sample is positive to the Babesia gibsonii nucleic acid.
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| WO2016181297A1 (en) * | 2015-05-12 | 2016-11-17 | Narodowy Instytut Zdrowia Publicznego - Państwowy Zakład Higieny | Method of detection and/or identification of protozoa of genus babesia, primer for use in this method and kit for use in methods of diagnosis of symptomatic and asymptomatic babesiosis |
| CN206512204U (en) * | 2016-12-30 | 2017-09-22 | 中瑞动检（北京）生物技术有限公司 | A kind of PCR detection kit for detecting this burnt worm of dog BABEI |
| CN108486223A (en) * | 2018-04-18 | 2018-09-04 | 华中农业大学 | A kind of Ji Shi Babesias RPA molecular detecting methods |
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