CN108486224B - Method for detecting Babesia corpuscula RPA molecules of field mice - Google Patents
Method for detecting Babesia corpuscula RPA molecules of field mice Download PDFInfo
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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
Abstract
The invention discloses a method for detecting rice field babesia RPA, which comprises the steps of firstly designing a pair of specific primers and a probe according to the mitochondrial COX I gene sequence of the rice field babesia, wherein the nucleotide sequences of the primer pair are respectively SEQ ID NO:2 to SEQ ID NO: and 4, extracting the total DNA in the mouse blood sample, carrying out recombinase combined enzyme amplification reaction, and finally detecting the amplification product by adopting a lateral flow assay test strip. The invention has high sensitivity and strong specificity, and can quickly identify the Babesia microti.
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 tenuis.
Technical Field
The babesiosis disease of the hamsters is infected and transmitted through hard tick bites, blood transfusions and other ways, has no obvious seasonality and wide regional distribution, infected hosts can have serious clinical symptoms and even die, and common hosts are mostly recessive infections and become transmission sources. Since the first human case was reported in south slav in 1957, the infection of babesia microti in our country has been reported, but the pathogenesis of this zoonosis is still unclear.
The vertical transmission of babesia microti can be achieved in mice, which means that mice infected with the babesia microti can enhance the transmission of babesia microti by virtue of their strong reproductive capacity. But also survive long term in the host by evading the host's immune response to it via immune evasion mechanisms, and although the patient is cured by treatment, it is likely to relapse upon discontinuation of drug therapy.
At present, microscopic examination of blood smears is the gold standard for detection of Babesia. Since babesia is similar in morphology to plasmodium, this approach has limitations requiring trained personnel to operate. Patients with very low parasitemia have difficulty in microscopic diagnosis in the early or chronic stages of infection. Recently developed PCR-based detection methods, including real-time PCR, have higher specificity and sensitivity and are faster and simpler to operate than serological methods and conventional PCR methods. Indirect immunofluorescence assay (IFA) is also a more commonly used method in the laboratory, with higher specificity but requiring fluorescent microscopy for observation. Enzyme-linked immunosorbent assay (ELISA) has the advantages of sensitivity, simplicity, easy standardization and the like and is used for clinically detecting Babesia, but the specificity is not high enough and the time consumption is long. Thus, there is still a lack of molecular detection methods to detect and distinguish various babesia infections.
The gene of the rice field babesia is specifically amplified based on the molecular level, but common PCR, nested PCR, fluorescent quantitative PCR and the like all have to rely on expensive instruments, the detection cost is high, the technology which has higher requirements on detection personnel and the like cannot be carried out in places 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 one 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 Babesia disease and laboratory screening identification for non-clinical diagnosis.
Disclosure of Invention
The invention aims to combine recombinase polymerase isothermal amplification technology (RPA) with lateral flow analysis technology (LF), so that the detection of the Babesia microti is easier to judge, and the invention has wide application prospect in primary clinical rapid detection.
The specific scheme of the invention comprises the following steps:
1) designing a primer: according to the reported full-length sequence (11109bp) of the mitochondrial gene of the Babesia microti, compared with other genes, such as COX III, cob and the like, the COX I gene (1422bp) has higher specificity. Therefore, a pair of specific primers and probes are designed by selecting COX I gene (shown in SEQ ID NO: 1 of the sequence table), and the nucleotide sequences of the primers and the probes are shown as follows:
the upstream primer F1: 5'-attatagcaatggcatctatagcgatcattggatg-3', respectively;
the downstream primer R1: 5'-atttgtaggatcaaatagcatagtgttatagtgcc-3', respectively;
5'-ccaactggtaacaagatattcaattggatatgtactttgcaaagtact-3' Probe
The above nucleotide sequences are designated as SEQ ID NO 2 to SEQ ID NO 4 in sequence in the sequence listing.
2) Extraction of total DNA from mouse blood samples: the total DNA of the blood sample was extracted using a blood/cell/tissue genomic DNA extraction kit (centrifugal column type, available from QIAGEN, Catalogue No.51304) according to the instructions of the kit.
3) Recombinase polymerase isothermal amplification: a recombinase-combined enzyme amplification (RPA) reaction system is established, 29.5ul of the Buffer solution of the regeneration Buffer and 11.2ul of ddH2O, 2.1ul of upstream primer, 2.1ul of downstream primer, 2ul of DNA template, 0.6ul of probe, and 2.5ul of 280mM magnesium acetate. Placing the 50ul total reaction solution in an incubator at 37 deg.C (keeping out of the sun), and reacting for 10 min;
4) and (3) analyzing an amplification product: and detecting the amplification product by using a lateral flow assay test strip.
Lateral flow-out test strip: taking 2ul of the final reaction product amplified by 50ul of recombinase polymerase, adding the final reaction product into a 1.5mL centrifuge tube containing 98ul 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.
The invention successfully establishes a rapid, sensitive, specific, simple and practical molecular detection method for the Babesia microti, and compared with other traditional PCR methods, the invention has the following advantages:
1. the method has high sensitivity: the results show that the RPA method of the present invention has a copy number of 1.45X 107Positive gDNA of copies/ml was diluted to 0.25 worm/ul and was still detectableAnd (4) positive. I.e. the minimum detection limit is 0.25 worm bodies/ul.
2. The method has strong specificity: 1 pair of primers and 3 specific target sequences of the probe are identified, so that the high specificity of the amplification of the RPA method is ensured.
3. The method is quick and time-saving: the time is short, and the reaction is usually completed within 15 min.
4. The method has wide reaction temperature range: the amplification temperature is 25-50 deg.C. I.e., amplification can be accomplished at room temperature. This low temperature operation also effectively prevents background noise, making the results more accurate.
5. The method has flexible volume: the method can be used for micro-volume reaction and large-volume reaction systems. The smaller reaction volume can save consumables.
6. The method has simple detection result: the test strip color development condition is directly observed by naked eyes, and the test line quality control line is both colored, so that the test strip can be qualitatively judged to be positive by the babesia volvacea.
Drawings
FIG. 1: screening for RPA amplification temperature. In the figure: the amplification temperatures of test strips No. 1-7 were 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: screening for RPA amplification time. In the figure: the amplification time corresponding to the test paper strip No. 1-6 is 5min, 10min, 15min, 20min, 25min and 30min respectively; test strip No. 7 is negative control water.
FIG. 3: and (3) carrying out electrophoresis result on the specific detection of the rat babesia COX I gene by using the RPA primer. In the figure: lane M: DNA Marker I, 1 is the babesia microti, 2 is the babesia canis, 3 is the babesia orientalis, 4 is the theileria buffalo, 5 is the plasmodium falciparum, 6 is the toxoplasma gondii, 7 is the healthy BALB/c mouse, 8 is the healthy person, 9 is the haemaphysalis longicornus, 10 is water.
FIG. 4: and (3) detecting the result of the lateral flow analysis test strip of the COX I gene RPA amplification product. In the figure: 1 is healthy BALB/c mouse, 2 is healthy human, 3 is healthy cow, 4 is haemaphysalis longicornus, 5 is canine Babesia gibsoni, 6 is Babesia orientalis, 7 is Plasmodium falciparum, 8 is Toxoplasma gondii, 9 is Theileria buffalo, 10 is Babesia microti, and 11 is water.
FIG. 5: the results were electrophoresed on a normal PCR agarose gel using 18S rRNA primers. In the figure: lane M: DNA Marker2K, 1 is water, 2 is the sample to be detected, and 3 is the positive control.
FIG. 6: and (3) experimental results of sensitivity of the detection method for the rice field babesia RPA. 1-8 templates are respectively 106Individual worm body/ul, 103Individual worm body/ul, 102DNA of individual worm/ul, 10 worm/ul, 1 worm/ul, 0.5 worm/ul, 0.25 worm/ul, 0.125 worm/ul; 9 is a negative control.
Detailed Description
Example 1: detection of Babesia microti on blood samples
Extraction of total DNA from BALB/c mouse blood samples: total DNA was extracted from blood samples using the QIAamp DNA Mini Kit (Qiagen, Germany) following the Kit instructions.
2. Selection of genes
Analysis of more than 10 papers on the detection of Babesia microti revealed that most 18S rRNA was used as the target sequence for detection, but the specific region was short, and it was not easy to synthesize the primer for RPA. We select the mitochondrial gene and compare the mitochondrial full length (11109bp) of different insect strains published in Gen Bank with Bioedit software to find that the Bactrois babesi COX I gene (1422bp) shows higher specificity to protozoa and various hosts than other genes (COX III, cob and the like). Therefore, a region of the rice babesia with high COX I gene specificity was selected as a candidate template for primer design.
3. Determination of target sequences
By utilizing Genbank to consult mitochondrial genome sequences of different geographical strains of the Babesia microti, related documents are analyzed, a Babesia conservative sequence is determined, and a conservative sequence (about 200 bp) with higher homology in COX I genes is selected as a specific target sequence for detection. Therefore, the target sequence is highly conserved in the Babesia microti strain, and is highly specific compared with other species genes, and the design requirement of the primer is further met.
4. Primer design
According to the design principle of related primers and probes and with reference to the design requirements of primers and probes of a Twist Amp kit, a COX I gene part conserved fragment (244bp) is selected as a target fragment, and the following primers and probes are synthesized by Shanghai Biotechnology company.
The upstream primer F1: 5'-ATTATAGCAATGGCATCTATAGCGATCATTGGATG-3', respectively;
the downstream primer R1: 5 '-biotin-ATTTGTAGGATCAAATAGCATAGTGTTATAGTGCC-3'; (wherein biotin is biotin, and the primer marked with biotin is equivalent to further marking the amplification product and then being captured by a biotin ligand on the test strip detection line.)
And (3) probe: 5'-FAM-CCAACTGGTAACAAGATATTCAATTGGATA- (THF) -TGTACTTTGCAA AGTACT-SPC3-3' (wherein FAM, THE and SPC3 are carboxyfluorescein, tetrahydrofuran and a blocking group C3 Spacer. FAM carboxyfluorescein labeled specific probe is specifically combined with a Biotin labeled nucleic acid amplification product, and is dripped on a test strip to be combined with a colloidal gold labeled anti-FAM antibody to form a ternary complex, and when THE ternary complex is diffused to a detection line, THE ternary complex is captured by a Biotin ligand to form THE detection line, and an unhybridized FAM labeled probe is combined with THE colloidal gold labeled anti-FAM antibody to form a Biotin-free binary complex which is combined on a quality control line.)
5. Recombinase polymerase amplification: establishing a Recombinase Polymerase Amplification (RPA) reaction system: first, 29.5ul of Rehydration Buffer, 11.2ul of ddH was added2O, 2.1ul of upstream primer, 2.1ul of downstream primer, 2ul of DNA template and 0.6ul of probe are mixed and gently shaken, centrifuged for several seconds; adding the mixed solution into a 0.2mL Twist Amp Basic RT reaction tube containing the freeze-dried enzyme powder, and repeatedly allowing for several times; ③ adding 2.5 mu l of magnesium acetate of 280m M, and repeatedly sucking for a plurality of times; fourthly, 50 mul of the total reaction solution is respectively placed at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ for 20 min. Selecting the optimal temperature, optimizing the time, and reacting for 5min, 10min, 15min, 20min, 25min and 30min in sequence.
6. Recombinase polymerase reaction amplification product analysis:
lateral flow-out test strip: taking 2ul of the final reaction product of recombinase polymerase amplification, adding the final reaction product into a 1.5mL centrifuge tube containing 98 mul of MGCB running buffer, mixing uniformly, vertically placing 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 of the negative sample is developed; the detection line and the quality control line of the positive sample are developed.
The temperature gradient results are shown in figure 1, and it can be seen that the bands are clearer when the reaction temperature is 30-45 ℃; the time gradient results are shown in FIG. 2, and it can be seen that the RPA amplification time of 10min is the best.
Example 2: specificity test of detection method of babesia microti RPA
As shown in Table 1, the strains of Theileria buffalo, Babesia gibsoni, Babesia orientalis and Toxoplasma gondii used in the specificity test were insect strains isolated and preserved by the animal parasite laboratory in which the present applicant was 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. Furthermore, gDNA from human, canine, buffalo and BALB/c mice was provided by the animal parasite laboratory in which the applicant was located.
TABLE 1 specificity test for the detection of Babesia microti 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.
Extraction of total DNA: total DNA was extracted from the above ten samples according to the method described in the blood/cell/tissue genomic DNA extraction kit (centrifugal column type) (purchased from QIAGEN, Cat. No. 51304).
Recombinase Polymerase Amplification (RPA) reaction: 1) establishing a recombinase combined enzyme amplification (RPA) reaction system: mixing 29.5. mu.l of the Buffer solution of the regeneration Buffer, 11.2. mu.l of ddH2O, 2.1. mu.l of the upstream primer, 2.1. mu.l of the downstream primer, 2. mu.l of the DNA template and 0.6. mu.l of the probe, slightly shaking, and centrifuging for several seconds; adding the mixed solution into a 0.2mL Twist Amp Basic RT reaction tube containing the freeze-dried enzyme powder, and repeatedly allowing for several times; ③ adding 2.5 mul of 280mM magnesium acetate, and repeatedly sucking for several times; fourthly, respectively placing the 50 mul total reaction solution into an incubator at 30 ℃ (keeping out of the sun) and reacting for 10 min;
recombinase polymerase reaction amplification product analysis:
agarose gel electrophoresis: mu.l of the final reaction product amplified by the recombinase polymerase is added into 5% agarose gel containing 0.5ug/mL Ethidium Bromide (EB) dye, electrophoresis is carried out for 20min under 100V voltage, and the pattern of the amplified product is observed by imaging on a gel phase system. A single band of an amplification product at 244bp is positive to the hamster babesia. The results are shown in FIG. 3.
Lateral flow-out test strip: and (3) adding 2 mu l of the final reaction product amplified by the recombinase polymerase into a 1.5mL centrifuge tube containing 98 mu l of MGCB running buffer, mixing uniformly, vertically placing 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. The results are shown in FIG. 4.
The test results show that the specificity and the accuracy of the method for detecting the Babesia microti are high.
Example 3: sensitivity test of detection method of babesia microti RPA
The sensitivity of the method for detecting the rice field babesia RPA is detected by using COX I gene positive gDNA as a template. The method comprises the following steps:
1. the method of example 1 extracts total DNA template from blood samples of BALB/c mice.
2. The target fragment was amplified by the usual PCR method: the 18S rRNA gene of the rice mouse babesia was amplified by using the total DNA of the blood of the mouse stained with BALB/c, and the nucleotide sequences of the primer pairs are shown below.
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) 0.3ul, 10 XPCR Buffer (Mg)2+Plus)2.5ul, dNTP mix (2.5 mM each) ul, template DNA 2ul, ddH2O16.2 ul, 1ul each of the upstream and downstream primers. The total reaction was 25 ul.
PCR product analysis: mu.l of the PCR final product was added to 2% agarose gel containing 0.5ug/mL Ethidium Bromide (EB) dye, electrophoresed at 100V for 20min, and the amplified product was visualized by imaging on a gel imaging system. A single band of the amplification product at 400bp is positive to the hamster 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 rat babesia.
5. Amplifying positive gDNA by fluorescent quantitative PCR and calculating copy number
The number of copies of the positive gDNA of the hamster babesia was calculated as: 1.45X 107copies/ml
By ddH2O10-fold dilution of the gDNA, i.e., 10 dilution of the gDNA5、104、103、10210, 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: taking 10ul of standard positive gDNA and adding 90ul of ddH2O, mixing well, taking 10ul, adding into another tube with 90ul ddH2In the EP tube of O, 10 mul of the mixture is taken and added into the existing 90ul ddH of the next tube after being mixed evenly again2And O, and the like in the EP pipe. The 2-fold dilution was performed by adding 20. mu. LddH to 20ul of standard positive gDNA2O, mixing well, adding 20ul of the mixture into another tube with 20ul of ddH2In the EP tube of O, 20 mul of the mixture is added into the existing 20ul ddH of the next tube after being mixed uniformly again2And O, and the like in the EP pipe.
RPA sensitivity assay: mixing 29.5ul of the Buffer solution of the regeneration Buffer, 11.2ul of ddH2O, 2.1ul of the upstream primer, 2.1ul of the downstream primer, 2ul of the DNA template and 0.6ul of the probe, slightly oscillating, and centrifuging for several seconds; adding the mixed solution into a Twist Amp Basic RT reaction tube of 0.2m L containing freeze-dried enzyme powder, and repeating the suction for several times; ③ adding 2.5ul of 280mM magnesium acetate, and repeatedly sucking ul for a plurality of times; and fourthly, respectively placing the 50 mu L of total reaction solution into an incubator at 30 ℃ (keeping out of the sun) and reacting for 10 min.
7. And (3) analyzing an amplification product: taking 2ul of the final reaction product of recombinase polymerase amplification, adding the final reaction product into a 1.5mL centrifuge tube containing 98ul 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. The results are shown in FIG. 6.
The results show that the RPA method has a copy number of 1.45X 107Faint bands were still detectable when the positive plasmids of copies/ml were diluted to 0.25 worm/ul blood.
Sequence listing
<110> university of agriculture in Huazhong
<120> detection method of Babesia corpuscula RPA molecules of field mouse
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1422
<212> DNA
<213> Peterus babesi (Babesia microti)
<400> 1
ctatagcgtt aagatgtata gtaatacaac cttaatgata aatagcattg ttgaaccaat 60
actacataat gcattccatg accagacttc gtctccatag tcagggattc tccttggcag 120
cgtagcgaag cctaagaagt gcattggagt aaatgtcaac agtattgcta ataccattga 180
tggtaatact actattagac atctgttagt gataactact tcaagcatca atctctgagt 240
agaggcaata aaggctaata aagcaagtat tgctccaata ctcaagacga agtggaagtg 300
agctaccaca tacgctgtat cgtgtaaaga cacgtcaaca ccagcattgc ctaggactac 360
tccagtagta ccacctaagg tgaaggtaat gatgaaggct atgactatct gtagtatacc 420
aagatacttg tttacaacag taccttgtaa cgtacatatc caattgaaga tcttattacc 480
agttggtaat gcgatcagga ttgtagtagt ggtgaagtaa cttctagtat ctacttctac 540
acctgatgta tacatatggt gtacccatac taagcatcct ataatcgcta tagatgccat 600
tgcaataatc atagtctgat taccaaacac ctctttggat gtagtacagg ctaggatcaa 660
gctaatgata ccaaatgcag gaagtattaa cacatacact tctggatgtc caaagaccca 720
gaataaatgt tgatacaata ttggatcacc tgcatttgta ggatcaaata gcatagtgtt 780
ataatgccta tcagctaata acatgcagag agctgctgtt aacactggaa gtgtaatcaa 840
cagtagaact gatgttatat agatagacca tgccgttggt aattgtctat ctaagagcat 900
tccaatgcac tttaggctat taaatgtgca gataaagtta atgctgctca ttacactagc 960
tatacctgat ataatcaacc cgatgattat caaatccaca cttaatgtag taatggaagt 1020
acttagaggt ggatataaag tccatcctgt acctccactt acctcacagt aaagtgatga 1080
aaccacaaga gcaaatgcta atggttgcat tagtaaacta tacaagttag ctcttgggaa 1140
cacaacctcg ggagctccga ccagtattgg tagaaggtag ttaccgatac caccaaacag 1200
gccaggcatg atgttgaaga atatcatgat cacgccatgt aaagtgaaag ctaggttata 1260
gatatctact atgtctatag ctataaacct atctccacta ctacctagct ctcctctaat 1320
gattaggcta aggtaaaaac ctactatgcc aaatacatag gatagccaaa ggtagctaat 1380
gcctacgact ttatggttag cacatataga tgtgaataac at 1422
<210> 2
<211> 35
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
attatagcaa tggcatctat agcgatcatt ggatg 35
<210> 3
<211> 35
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
atttgtagga tcaaatagca tagtgttata gtgcc 35
<210> 4
<211> 48
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
ccaactggta acaagatatt caattggata tgtactttgc aaagtact 48
Claims (4)
1. A non-diagnosis purpose detection method for the rice field mouse babesia RPA is characterized by comprising the following steps:
1) designing a pair of specific primers and a probe according to the mitochondrial COX I gene sequence of the Babesia microti, wherein the sequences of the primers and the probe are as follows:
the upstream primer F1: 5'-ATTATAGCAATGGCATCTATAGCGATCATTGGATG-3', respectively;
the downstream primer R1: 5'-ATTTGTAGGATCAAATAGCATAGTGTTATAGTGCC-3', respectively;
and (3) probe: 5'-CCAACTGGTAACAAGATATTCAATTGGATATGTACTTTGCAAAGTACT-3'
2) Extracting total DNA in a mouse blood sample, and carrying out recombinase combined enzyme amplification reaction;
3) and detecting the amplification product by adopting a lateral flow assay test strip method.
2. The method for detecting babesia microti RPA as claimed in claim 1, wherein said recombinase-enzyme amplification reaction system comprises: 29.5ul of Rehydration Buffer, 11.2ul of ddH2O, 2.1ul of upstream primer, 2.1ul of downstream primer, 2ul of DNA template, 0.6ul of probe, and 2.5ul of 280mM magnesium acetate.
3. The method for detecting babesia microti RPA as claimed in claim 1, wherein said conditions for recombinase-combined enzyme amplification reaction are: and reacting for 10min at 30-40 ℃ in the dark.
4. The method for detecting babesia microti RPA of claim 1, wherein the lateral flow assay strip is a strip that: taking 50ul of the final reaction product of recombinase polymerase amplification, adding the final reaction product 2ul into a 1.5mL centrifuge tube containing 98ul 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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| AU4001199A (en) * | 1998-05-21 | 1999-12-06 | Igenex, Inc. | Improved methods for detecting a target nucleic acid fragment |
| US9970065B2 (en) * | 2014-02-04 | 2018-05-15 | Westchester Medical Center Advanced Physician Services, Pc | Real-time PCR assay for detection of Babesia microti and clinical utilization in diagnosis and treatment of babesiosis |
| EP2966177A1 (en) * | 2014-07-09 | 2016-01-13 | Vetgenomics, S.L. | Methods for detecting target DNA sequences |
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2018
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