CN108486223B - Babesia gibsoni RPA molecular detection method - Google Patents
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Abstract
The invention discloses a method for detecting Babesia gibsoni RPA molecules, which comprises the steps of firstly designing a pair of specific primers and corresponding probes according to the mitochondrial COX I gene sequence of Babesia gibsoni, wherein the nucleotide sequences of the primer pair are respectively SEQ ID NO: 2 to SEQ ID NO: and 4, extracting total DNA in the dog blood sample, carrying out recombinase combined enzyme amplification reaction, and finally detecting an amplification product by adopting a lateral flow assay test strip. The invention has the advantages of rapidness, simplicity, practicability, high sensitivity, good specificity and the like.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a Recombinase polymerase isothermal amplification (RPA) molecular detection method for Babesia gibsoni.
Technical Field
The babesiosis gibsoni disease is a blood protozoa disease taking ticks as a transmission medium, is frequently generated in summer and autumn where ticks propagate, and mainly causes dog high fever, anemia, jaundice and hemoglobinuria. Since Bauer F detected Babesia in dogs in 1967, scientists of Birkenhueer et al discovered this species of Babesia in different regional dogs in succession. And is therefore considered a novel canine epidemic.
The main pathogenic stage of the disease is parasitizing red blood cells of vertebrates with protozoa of the genus Babesiae (Babesiae) of the family Babesiae, in which the bodies divide, multiply, cause lysis of the red blood cells and produce toxins. The current detection methods are divided into direct methods and indirect methods. The direct method is used for smear microscopy of peripheral blood of sick animals, is simple and convenient, has high specificity but poorer sensitivity, is closely related to the insect staining rate of the animals, and is relatively greatly interfered by external factors such as environment, personnel and the like. The indirect method is an immunological diagnostic method, and mainly includes complement binding reaction, indirect immunofluorescence assay, ELISA, radioimmunoassay and the like. Of these, indirect immunofluorescence assays and complement fixation reactions are the most sensitive. The methods are complicated, and are only suitable for laboratory examination but not clinical diagnosis.
The pathogen identification can be timely and accurately carried out by carrying out PCR amplification on the Cobemyces gibsonii COX I gene based on the molecular level, but the common PCR, the nested PCR with higher sensitivity and the fluorescent quantitative PCR all have to depend on expensive precise instruments, have high detection cost, long time consumption, have higher requirements on detection personnel and the like, and cannot be carried out in basic laboratories with poorer conditions. The recombinase polymerase isothermal amplification can amplify target sequences efficiently, quickly, specifically and sensitively under isothermal conditions. The method does not need special equipment, only needs an instrument (such as a water bath or a portable constant temperature electric heater) capable of providing a constant temperature environment, thereby greatly reducing the detection cost, and can be more widely applied to clinical diagnosis of canine Babesia gibsonii disease and laboratory screening and identification of Babesia gibsonii for non-clinical diagnosis.
Disclosure of Invention
The invention aims to provide a rapid, sensitive, specific, simple and practical RPA molecular detection method for Babesia gibsoni.
The specific scheme of the invention comprises the following steps:
1) designing a primer: a pair of specific primers and corresponding probes are designed according to a mitochondrial COX I gene sequence (shown in a sequence table SEQ ID NO: 1) of Babesia gibsoni with an accession number of AB499087.1, which has been reported in 2016, and the nucleotide sequences of the primer pairs are shown as follows:
the upstream primer F1: 5'-ATAGTTTATTGCTTCAGCCAATAGCTTTCTGTTTGG-3', respectively;
the downstream primer R1: 5'-TATCTACAGTTTGACCAATTGATTTTAAAGCGCC-3', respectively;
and (3) probe: 5' -ATAATATTTGGTTTACTTGCCTCAGGTATAGCTAGTGCTATGAGTG-3
The nucleotide sequences of the above primer pairs, which have been processed into computer-readable copies of the nucleotide sequences of the patent programs using dedicated software, are designated in sequence as SEQ ID NOs: 2 to SEQ ID NO: 4.
2) extracting total DNA in a dog blood sample, and carrying out recombinase combined enzyme amplification reaction;
3) and detecting the amplification product by using a lateral flow assay test strip.
Preferably, the recombinase is combined with an enzyme amplification reaction system comprising: 29.5. mu.L of the hydration Buffer, 11.2. mu.L of ddH2O, 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 2. mu.L of the DNA template, 0.6. mu.L of the probe, 2.5. mu.L of 280mM magnesium acetate.
Preferably, the conditions of the recombinase-combined enzyme amplification reaction are as follows: reacting for 20min at 30-40 ℃ in the dark.
Preferably, the lateral flow assay test strip detection method is as follows: and (3) adding 2 mu L of a final reaction product amplified by 50 mu L of recombinase polymerase into a 1.5mL centrifuge tube containing 98 mu L of MGCB running buffer, mixing uniformly, vertically putting the test strip into the centrifuge tube, reacting for 3min, taking out the test strip and taking a picture.
The results show that: only the quality control line is developed into a negative sample; the detection line and the quality control line are both developed, and the corresponding negative sample is normally developed to be a positive sample.
The invention successfully establishes the RPA molecular detection method for the Babesia gibsoni, and has the following advantages compared with other traditional methods:
1. the invention is economical and practical: the reaction is carried out under the condition of constant temperature, an expensive PCR instrument is not needed, and only equipment capable of providing constant temperature, such as a water bath kettle, is needed to complete the reaction.
2. The method has high sensitivity: the results show that the RPA method of the present invention has a copy number of 1.58X 106Positive gDNA of copies/ml can still detect positive amplification bands when diluted to 0.5 worm bodies/ul, and the detection limit is 0.5 worm bodies/ul. The sensitivity is 10-100 times higher than that of common PCR.
3. The method of the invention has strong specificity: 1 pair of primers identifies 3 specific sequence regions of the target sequence, thereby ensuring the high specificity of the amplification of the RPA method.
4. The invention is quick and time-saving: the amplification reaction is carried out for 20min, the test strip detects the amplification product for 3min, and the total time consumption is less than 1 h.
5. The invention has simple detection: the reaction product is directly observed by naked eyes and is subjected to color development by a test paper strip to qualitatively judge whether the COX I target sequence of the Babesia gibsoni is amplified or not.
Drawings
FIG. 1 shows the results of screening for temperature conditions for RPA amplification. In the figure: the test paper strips No. 1-7 have temperatures of 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C, respectively.
FIG. 2 shows the results of screening for RPA amplification time. In the figure: the test strip No. 1 is a negative control; the reaction time of the test paper strip No. 2-7 is 5min, 10min, 15min, 20min, 25min and 30min respectively.
FIG. 3 shows the result of electrophoresis of the specific detection of the COX I gene of Babesia gibsoni. In the figure: lane M: DNA Marker; 1: babesia dunnii; 2: babesia gemmifera; 3: babesia microti; 4: babesia orientalis; 5: plasmodium falciparum; 6: theileria annulata; 7: toxoplasma gondii; 8: haemaphysalis longicornis; 9: a healthy dog; 10: water; 11: and (4) positive control.
FIG. 4 shows the test results of lateral flow assay test strips for the COX I gene of Babesia gibsoni. In the figure: 1: a positive control; 2: babesia gemmifera; 3: babesia microti; 4: babesia orientalis; 5: plasmodium falciparum; 6: theileria annulata; 7: toxoplasma gondii; 8: haemaphysalis longicornis; 9: a healthy dog.
FIG. 5 shows the result of electrophoresis of Babesia gibsoni in total DNA of canine blood using 18S rRNA universal primers. In the figure: lane M: DNA Marker 2KII, lanes 1 and 2 are samples to be detected, 3 is a positive control, 4 is a negative control, and the result shows that the two samples are positive.
FIG. 6: is the result of the sensitivity experiment of the canine Babesia gibsoni RPA detection method. In the figure: 1 to 8 are each 106Individual worm body/ul blood, 10 worm bodies/ul, 5 worm bodies/ul, 2.5 worm bodies/ul, 1.25 worm bodies/ul, 1 worm body/ul, 0.5 worm bodies/ul and 0.25 worm body/ul blood, wherein 1 is a positive standard, 2-8 templates are DNA diluted by the positive standard, and 9 templates are negative control ddH2O。
Detailed Description
Example 1: detection of Babesia gibsoni on canine blood samples
1. Extraction of total DNA from dog blood sample
Total DNA was extracted from a canine blood sample using a blood/cell/tissue genomic DNA extraction kit (spin column type) (purchased from QIAGEN, Catalogue No.51304, according to the instructions of the kit).
2. Primer design
The upstream primer F1: 5'-ATAGTTTATTGCTTCAGCCAATAGCTTTCTGTTTGG-3', respectively;
the downstream primer R1: 5 '-biotin-TATCTACAGTTTGACCAATTGATTTTAAAGCGCC-3'; (wherein biotin is a biotin label, and the primer marked with biotin is equivalent to further marking an amplification product and then captured by a biotin ligand on a test strip detection line.)
And (3) probe: 5 '-FAM-ATAATATTTGGTTTACTTGCCTCAGGTATA-THE-GCTAGTGCTATGAGTG-SPC 3-3' (wherein FAM, THE and SPC3 are respectively carboxyl fluorescein, tetrahydrofuran and a blocking group C3 Spacer. FAM carboxyl fluorescein labeled specific probe is specifically combined with a Biotin labeled nucleic acid amplification product, THE specific probe is dripped on a test strip to be combined with a colloidal gold labeled anti-FAM antibody to form a ternary complex, when THE ternary complex is diffused to a detection line, THE ternary complex is captured by a Biotin ligand to form THE detection line, an unhybridized FAM labeled probe is combined with THE colloidal gold labeled anti-FAM antibody to form a Biotin-free binary complex, and THE complex is combined on a quality control line.)
3. Recombinase polymerase amplification
Establishing a Recombinase Polymerase Amplification (RPA) reaction system which comprises the following steps: 29.5 μ LRehydration Buffer, 11.2 μ L ddH2O, 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 2. mu.L of the DNA template, 0.6. mu.L of the probe, 2.5. mu.L of 280mM magnesium acetate. Wherein the positive control is gDNA of the dog with the positive identification of the Babesia gibsoni, and the negative control is sterilized double distilled water. The 50 μ L total reaction solution is respectively placed at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, and acted for 30 min. Selecting the optimal temperature, optimizing the time, and reacting for 5min, 10min, 15min, 20min, 25min and 30min in sequence.
The result of the temperature gradient detection is shown in FIG. 1, and it can be seen from the graph that the band is clearer when the reaction temperature is 30-40 ℃; the time gradient test results are shown in FIG. 2, and it can be seen that the RPA amplification time of 20min is the best.
4. Recombinase polymerase reaction amplification product analysis
Lateral flow-out test strip: and adding 2 mu L of final reaction product amplified by 50 mu L of recombinase polymerase into a 1.5mL centrifuge tube containing 98 mu L of MGCB running buffer, mixing uniformly, vertically putting the test strip into the centrifuge tube, and reacting for 3 min. The test strip was removed and photographed. The results show that: only the quality control line is developed into a negative sample; the detection line and the quality control line are both developed, and the corresponding negative sample is normally developed to be a positive sample.
Example 2: specificity test of canine Babesia gibsoni RPA detection method
As shown in Table 1, the Babesia gibsoni, Babesia gemmifera, Babesia microti, Babesia orientalis, Theileria annulata and Toxoplasma gondii strains used in the specificity test were insect strains isolated and stored by the animal parasite laboratory in which the present applicant is present. Plasmodium falciparum DNA is a great gift of medicine from the third army of Chongqing. All insect strains were stored as frozen anticoagulated blood. In addition, Haematococcus longipes and canine gDNA were provided by the animal parasite laboratory in which the Applicant was located.
TABLE 1 specificity assay for the detection of Babesia gibsoni RPA
Description of table 1: and the + represents the color development of the test strip detection line, and the-represents the non-color development of the test strip detection line.
Recombinase polymerase reaction amplification product analysis:
agarose gel electrophoresis: mu.L of the final reaction product of 50. mu.L of RPA amplification was added to a 2% agarose gel containing 0.5. mu.g/mL Ethidium Bromide (EB) dye, electrophoresed at 100V for 20min, and the amplified product was visualized on a gel-phase system. As shown in FIG. 3, only 11 lanes showed a single band at 233bp, which was detected as positive for Babesia gibsoni, and none of the other lanes detected the target band, indicating that the primer specificity is better.
Lateral flow-out test strip: taking 2 mu L of final reaction product of 50 mu L of RPA amplification, adding the final reaction product into a 1.5mL centrifuge tube containing 98 mu L of MGCB running buffer, mixing uniformly, vertically putting the test strip into the centrifuge tube, and reacting for 3 min. The test strip was removed and photographed. The results are shown in FIG. 4, and the test paper strips No. 2-9 only have the quality control line for color development, which indicates that the template DNA No. 2-9 is a negative sample; the test strip No. 1 and the quality control line are both colored, which indicates that the template DNA No. 1 is a positive sample.
The test results show that the specificity and the accuracy of the method for detecting the Babesia gibsoni are high.
Example 3: method for detecting sensitivity test of canine Babesia gibsoni RPA
The sensitivity test of the Babesia gibsoni RPA method is carried out by taking the Babesia gibsoni positive gDNA as a template. The method comprises the following steps:
1. blood total DNA template was extracted from canine blood samples.
2. Ordinary PCR identification of insect strains: 18S rRNA gene part of Babesia gibsoni in total DNA of canine blood is amplified by using 18S short universal primers, and the nucleotide sequences of the primer pairs are shown as follows.
The upstream primer F2: 5'-AACCTGGTTGATCCTGCCAGTAGTCAT-3', respectively;
the downstream primer R2: 5'-GATCCTTCTGCAGGTTCACCTAC-3', respectively;
the reaction system is as follows: TaKaRa Taq enzyme (5U/. mu.l) 0.3. mu.L, 10 XPCR Buffer (Mg)2+Plus) 2.5. mu.L, dNTP mix (2.5mM) 4. mu.L, template DNA 2. mu.L, ddH2O16.2. mu.L, 1. mu.L each of the upstream and downstream primers. The total reaction volume was 25. mu.L.
PCR product analysis: mu.L of the PCR final product was added to 2% agarose gel containing 0.5. mu.g/mL Ethidium Bromide (EB) dye, electrophoresed at 100V for 20min, and the amplified product was visualized on a gel imaging system. A single band of the amplification product at 400bp is positive to Babesia. The results are shown in FIG. 5.
4. Identification of positive gDNA:
the amplified target gene was excised from the agarose gel, and the DNA was recovered using an agarose gel recovery kit (available from TiangGen Co., Ltd.) and sent to the assay.
And (3) introducing the sequencing result into an NCBI database for sequence comparison, and confirming that the gDNA is a positive template of the canine Babesia gibsoni.
5. Amplifying positive gDNA by fluorescent quantitative PCR and calculating copy number
The copy number of the canine Babesia gibsoni positive gDNA is calculated as: 1.58X 106copies/ml。
By ddH2O10-fold dilution of the gDNA, i.e., 10 dilution of the gDNA5、104、10310210, 1; then diluting 10 polypide/ul 2 times, namely diluting to 5, 2.5 and 1.25 polypide/ul respectively; 1 worm body/ul is diluted 2 times to 0.5 and 0.25 worm bodies/ul. The specific dilution method is as follows: 10-fold dilution: add 90. mu.L ddH to 10. mu.L of standard positive gDNA2O, mixing well, taking 10 mu L, adding into another tube with 90 mu L ddH2In the EP tube of O, 10 mul is taken and added into the 90 mul ddH existing in the next tube after being mixed evenly again2And O, and the like in the EP pipe. In the 2-fold dilution, 20. mu.L of standard positive gDNA was added to 20. mu.L of ddH2O, mixing well, taking 20 mu L, adding into another tube with 20 mu L ddH2In the EP tube of O, 20. mu.L of the mixture is added into the existing 20. mu.L ddH of the next tube after being mixed uniformly again2And O, and the like in the EP pipe.
RPA sensitivity assay: f1 and R1 are used as primers, and a probe is added to amplify the diluted positive gDNA. mu.L of diluted positive gDNA was collected, and the other reagents in the system were 29.5. mu.L of the Buffer solution for regeneration Buffer, 11.2. mu.L of ddH2O, 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 2. mu.L of the DNA template, 0.6. mu.L of the probe, and 2.5. mu.L of 280mM magnesium acetate. Wherein the positive control is gDNA of the dog with the positive identification of the Babesia gibsoni, and the negative control is sterilized double distilled water. The total reaction solution (50. mu.L) was incubated at 37 ℃ for 20 min.
7. And (3) analyzing an amplification product: taking 2 mul of the final reaction product amplified by 50 mul of recombinase polymerase,adding into a 1.5mL centrifuge tube containing 98 μ L MGCB running buffer, mixing, vertically putting the test strip into the centrifuge tube, and reacting for 3 min. The test strip was removed and photographed. The results show that: only the quality control line is developed into a negative sample; the detection line and the quality control line are both developed, and the corresponding negative sample is normally developed to be a positive sample. As a result, as shown in FIG. 6, the 7 th test strip test line still developed color. The results show that the RPA method has a copy number of 1.58X 106Faint bands were still detectable when the positive plasmids of copies/ml were diluted to 0.5 worm/ul blood.
Sequence listing
<110> university of agriculture in Huazhong
<120> method for detecting Babesia gibsoni RPA molecules
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1575
<212> DNA
<213> Babesia gibsoni mitochondrial COX I Gene ()
<400> 1
atggtgcccc aggccctgct cttcgtgcct ctgctcgtct ttccactgtg cttcggcaaa 60
tttcccatct acactattcc tgacaagctg ggaccctgga gtcctatcga tattcaccat 120
ctgtcatgcc ctaacaatct cgtggtcgag gatgaagggt gtaccaacct gtcaggtttc 180
agctacatgg agctgaaagt ggggtatatc ctcgctatta aggtcaacgg cttcacatgc 240
actggagtgg tcaccgaggc agaaacctac acaaattttg tgggctatgt caccacaact 300
ttcaagagga aacactttag accaacaccc gacgcctgtc gcgccgctta caactggaag 360
atggctggcg atccacgata tgaggaatct ctgcacaatc cttacccaga ctatagatgg 420
ctgcggacag tgaagaccac aaaagagagc ctggtcatca ttagcccatc cgtcgcagac 480
ctggatccct acgatagatc cctgcactct cgggtgtttc cctctggcaa gtgcagtgga 540
gtggccgtca gctccactta ctgtagcacc aaccatgatt atactatctg gatgccagag 600
aatccccggc tgggaatgtc ctgcgacatt ttcacatcta gtcgcgggaa gcgagccagt 660
aaagggtcag agacttgtgg ttttgtggac gaaaggggcc tgtataagag cctcaaagga 720
gcttgcaagc tgaaactctg tggcgtgctg ggactcagac tgatggatgg aacctgggtc 780
tcaatgcaga caagcaacga gactaagtgg tgcccccctg acaaactcgt gaatctgcac 840
gacttcaggt ccgatgagat cgaacatctg gtggtcgagg aactcgtgcg aaaaagggag 900
gaatgtctcg atgctctgga gtctatcatg actaccaagt ctgtgagttt taggagactc 960
agtcacctga gaaagctcgt ccctggcttc ggaaaagcat acaccatctt taacaagaca 1020
ctgatggaag cagacgccca ttataaaagc gtggagacct ggaatgaaat cctgccatcc 1080
aagggatgcc tccgagtcgg aggacgctgt caccctcatg tgaacggcgt cttctttaat 1140
ggaatcattc tggggcctga cggtaacgtg ctgatcccag agatgcagtc aagcctgctc 1200
cagcagcaca tggagctgct cgaatcctct gtgattcctc tggtccatcc actcgcagat 1260
ccctctacag tgttcaagga cggggatgag gccgaagact ttgtggaggt ccacctgcca 1320
gatgtgcata accaggtgtc tggggtcgac ctcggtctgc ccaattgggg gaagtacgtg 1380
ctgctcagcg ccggtgcact gactgctctc atgctgatca ttttcctgat gacctgctgt 1440
cgacgagtga accggtccga gcctactcag cacaatctgc gagggaccgg tagagaagtg 1500
tccgtcacac cacagtctgg caaaatcatt agttcatggg agagccataa gtccgggggt 1560
gaaacacgcc tgtga 1575
<210> 2
<211> 36
<212> DNA
<213> Artificial sequence ()
<400> 2
atagtttatt gcttcagcca atagctttct gtttgg 36
<210> 3
<211> 34
<212> DNA
<213> Artificial sequence ()
<400> 3
tatctacagt ttgaccaatt gattttaaag cgcc 34
<210> 4
<211> 46
<212> DNA
<213> Artificial sequence ()
<400> 4
ataatatttg gtttacttgc ctcaggtata gctagtgcta tgagtg 46
Claims (5)
1. A method for detecting the RPA molecule of the Babesia gibsoni with non-diagnosis purpose is characterized by comprising the following steps:
1) designing a pair of specific primers and corresponding probes according to the mitochondrial COX I gene sequence of Babesia gibsoni, wherein the nucleotide sequences of the primer pairs are as follows:
the upstream primer F1: 5'-ATAGTTTATTGCTTCAGCCAATAGCTTTCTGTTTGG-3', respectively;
the downstream primer R1: 5'-TATCTACAGTTTGACCAATTGATTTTAAAGCGCC-3', respectively;
and (3) probe: 5'-ATAATATTTGGTTTACTTGCCTCAGGTATAGCTAGTGCTATGAGTG-3'
2) Extracting total DNA in a dog blood sample, and carrying out recombinase combined enzyme amplification reaction;
3) and detecting the amplification product by using a lateral flow assay test strip.
2. The method for detecting babesia gibsoni RPA molecules according to claim 1, wherein: a biotin label is connected to THE 5' end of THE downstream primer R1 sequence, a FAM carboxyfluorescein label is connected to THE 5' end of THE probe sequence, a THE tetrahydrofuran label is inserted between THE 30 th site and THE 31 th site of THE probe sequence, and a SpC3 blocking group is connected to THE 3' end of THE probe sequence.
3. The babesia gibsoni RPA fraction of claim 1The detection method is characterized in that the recombinase combined enzyme amplification reaction system comprises: 29.5. mu.L of the hydration Buffer, 11.2. mu.L of ddH2O, 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 2. mu.L of the DNA template, 0.6. mu.L of the probe, 2.5. mu.L of 280mM magnesium acetate.
4. The method for detecting the RPA molecule of Babesia gibsoni as claimed in claim 1, wherein the conditions of the recombinase-combined enzyme amplification reaction are as follows: reacting for 20min at 30-40 ℃ in the dark.
5. The method for detecting the babesia gibsoni RPA molecule according to claim 1, wherein the lateral flow assay test strip comprises: and (3) adding 2 mu L of a final reaction product amplified by 50 mu L of recombinase polymerase into a 1.5mL centrifuge tube containing 98 mu L of MGCB running buffer, mixing uniformly, vertically putting the test strip into the centrifuge tube, reacting for 3min, taking out the test strip and taking a picture.
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| CN110229827B (en) * | 2019-05-22 | 2020-12-18 | 华中农业大学 | Babesia gibsoni surface protein and application thereof |
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| CN105734158A (en) * | 2016-04-28 | 2016-07-06 | 新疆农业大学 | Fluorescent PCR (polymerase chain reaction) detection kit for babesia caballi disease |
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