CN104561343A - Primer and kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi - Google Patents
Primer and kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi Download PDFInfo
- Publication number
- CN104561343A CN104561343A CN201510035284.2A CN201510035284A CN104561343A CN 104561343 A CN104561343 A CN 104561343A CN 201510035284 A CN201510035284 A CN 201510035284A CN 104561343 A CN104561343 A CN 104561343A
- Authority
- CN
- China
- Prior art keywords
- babesia
- seq
- primer
- strain
- mohs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a primer and a detecting kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi. The primers in the primer sequence and the kit disclosed by the invention are respectively primers in SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, and probe primers SEQ ID No.3 and SEQ ID No.6. Through using the primer and the kit disclosed by the invention for detection, the advantages of high sensitivity, high speed, high specificity and the like are achieved, and the primer and the kit disclosed by the invention can be widely applied to the qualitative and quantitative detection of pathogens.
Description
Technical field
The present invention relates to a kind of primer pair and the test kit that detect zooparasite.Exactly, the present invention relates to a kind of primer and the detection kit of distinguishing detection low pathogenicity and highly pathogenic babesia motasi.
Background technology
The Babesia now reported has more than 100 plants, wherein name and be recognized the Babesia infecting sheep have sheep Babesia (
b. ovis), Mohs Babesia (
b. motasi), coarse Babesia (
b. crassi) and some Babesia U sps.Sheep Babesia by Rh (
rhipicephalus) hard tick propagate, be small-sized Babesia pathogenic by force, its biological characteristics and ox Babesia basically identical; Coarse Babesia is the large-scale Babesia that a kind of virulence is low, breeds in the mode of quaternary fission or binary fission, and its communication media tick and circulation way it be unclear that, and only finds in Iran and Turkey at present; And Mohs Babesia is the polypide the most extensive, characteristic of division is the most complicated that distributes in the Babesia of infection sheep, they are pathogenic different because of its difference distributed, as stronger in having of being separated with Mediterranean in southern Europe is pathogenic, and Northern Europe be separated pathogenic more weak, and these two kinds of Mohs Babesias be all by Haemaphysalis punctata (
haemaphysalis punctata) the large-scale Babesia propagated, at the Mohs Babesia that Sahara is separated, in form between known Mohs Babesia and sheep Babesia, but its communication media is not the tick of Haemaphysalis.According to pathogenic, difference, morphology, serology and the molecular classification Evolution analysis result to sheep and goat infectivity of Babesia, Uilenberg (2006) thinks in Mohs Babesia at least containing two or more kinds or subspecies.
China, before 1996, only has the report of 2-3 example sheep Babesia Gibsoni, then respectively in Gansu, there is the report of morbidity in Hebei, Liaoning, Yunnan, Shanxi, the ground such as Henan and Xinjiang.The Epidemic Scope of this disease in expanding trend year by year, may with Tick victor distribution range extensively have certain relation.This research team in last century Mo, the reconnaissance investigation of carrying out to China's part provinces and regions sheep Babesia Gibsoni.By blood inoculation and tick propagation experimentation, successively ground separation obtains the Babesia strains of 9 strain sheep in China Liaoning, Hebei, Gansu, Hubei, Xinjiang etc.Epidemiology survey finds that this disease is extensively popular in China, and some local harm are serious, positive rate more than 30%, the breeding sheep that cause of disease is introduced other places and lamb pathogenic comparatively strong, seriously restrict the breed improvement of sheep and the development of sheep husbandry.Carry out molecular classification research with the rrna 18S rRNA gene of pyriform worm (comprising Babesia and Taylor worm) to find; all there is nearer sibship Lintan, Gansu, the strain isolated of fiber crops when, Zhuhe, sky Ning County and Hebei, Hubei and Liaoning with Mohs Babesia; so name as Mohs Babesia (Liu et al., 2007; Guan et al., 2009), and carry out finding in the research of molecular classification with rrna 28S rRNA and transcribed spacer ITS gene, plastosome CoI gene etc.: the Mohs Babesia of China can be divided into two Asias to prop up; One is the Lintan strain of Mohs Babesia and the strain of Mohs Babesia God blessings of low pathogenicity, and two is highly pathogenic Mohs Babesia Ning County's strain and Mohs Babesia Hebei strain (Niu et al., 2009a).2010, (the Guan et al. such as Guan, 2010e) confirm that the positive serum that sheep is infected in Mohs Babesia Lintan strain whole worm antigen and the strain of Mohs Babesia God blessings has strong cross reaction, and there is no cross reaction with the positive serum of the Ning County's strain of Mohs Babesia and Hebei strain.These results conform to from the conclusion of Uilenberg: Chinese Mohs Babesia also at least may exist two different subspecies.Confirm through propagation experimentation, the communication media between the different subspecies of Mohs Babesia is not identical yet, and the communication media of some subspecies is not yet studied clear at present.
The Babesia Gibsoni of people is reported in nineteen sixty the earliest, primarily of two kinds infect people Babesia disease pathogen-vole Babesia (
b. microti) and difference Babesia (
b. divergens) cause, patient often with concurrent various syndromess such as severe anemia, interval high heat, sore muscle, tics, can cause death during severe infections.The Babesia of people is not infected before research in recent years shows, due to entomophila distribution range constantly extensively, the host range of the pathogenic cause of disease of carrier's Babesia is also in expansion, constantly there is (Schnittger L in the new pathogenic cause of disease of people's Babesia, et al., 2012), this disease progressively becomes a kind of global mankind and newly sends out parasitosis and be subject to public attention.Record according to current document, now mainly contain the ability that four large class Babesias have Zoonosis, the first kind is vole Babesia, and it is a kind of and the closely-related Zoonosis parasite of rodent, and U.S. most people infect Babesia case and caused by it; Equations of The Second Kind is rebaptism
b. duncani, the similar vole Babesia of form, but the two sibship is far away; 3rd class comprise difference Babesia (
b. divergence) and class difference Babesia,
b. venatorum(being also referred to as EU1); 4th class is Korea S newfound Babesia KO1 type, and very similar to the Mohs Babesia Hebei pathogen strain form of China, molecular classification analysis shows that both homologys are up to 98%, and nucleotide sequence only differs from 13 bases.
Present stage, mainly there is sheep Babesia-Mohs Babesia and the sheep Babesia U sp Xinjiang Strain of two kinds in China.But between the different Local Isolates of Mohs Babesia, because it is pathogenic, there is some difference for communication media and serology, binding molecule taxonomy shows that Mohs Babesia exists two different subspecies, i.e. the Mohs Babesia subspecies of low pathogenicity, as Lintan strain and God blessings strain; Highly pathogenic Mohs Babesia subspecies, as Ning County's strain and Hebei strain.
The gold standard that Babesia detects is the mode adopting blood smear dyeing microscopic examination, but blood smear dyeing microscopic examination result is subject to the impact of several factors, and recall rate is low, and accuracy is poor.Though Standard PCR, sleeve type PCR and the nuclei aoid methods such as real-time fluorescence PCR based on SYBR Green dyestuff have application, the shortcomings such as it is low that Standard PCR exists sensitivity, poor specificity, and sense cycle is long; Sleeve type PCR is highly sensitive, but the PCR of two steps reacts loaded down with trivial details, easily pollutes, and is not suitable for large-scale epidemiology survey.
Summary of the invention
The invention provides one and can overcome prior art deficiency, the primer and test kit that detect low pathogenicity and highly pathogenic babesia motasi can be distinguished.
The primer pair gene order detecting low pathogenicity babesia motasi of distinguishing of the present invention is: SEQ ID No.1 and SEQ ID No.2; Distinguishing the primer pair gene order detecting highly pathogenic babesia motasi is: SEQ ID No.4 and SEQ ID No.5.
Comprising sequence in the test kit distinguishing detection low pathogenicity and highly pathogenic babesia motasi of the present invention is: the primer of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5 and probe primer SEQ ID No.3 and SEQ ID No.6.
For convenience of using, also have in the test kit distinguishing detection low pathogenicity and highly pathogenic babesia motasi of the present invention: standard positive plasmid template pGEM-Teasy-18S rRNABLT and pGEM-Teasy-18S rRNABNX.
5. the condition that the differentiation detection low pathogenicity described in claim 3 and 4 and the test kit of highly pathogenic babesia motasi carry out pcr amplification is:
By the condition that primer of the present invention and test kit carry out increasing be:
The condition of the Mohs Babesia pcr amplification of low pathogenicity is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 40 circulations;
The condition of highly pathogenic Mohs Babesia pcr amplification is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 60 DEG C of 10s, 72 DEG C of 30s, 40 circulations.
The present invention advises having following three parts compositions when embody rule: 1) standard positive plasmid template pGEM-Teasy-18S rRNABLT, the forward primer SEQ ID No.1 of the Mohs Babesia subspecies of specific detection low pathogenicity and reverse primer SEQ ID No.2(10 μ Μ) each 0.5 μ L, fluorescent probe SEQ ID No.3(5 μM) 1.0 μ L; 2) standard positive plasmid template pGEM-Teasy-18S rRNABNX, the forward primer SEQ ID No.4 of the Mohs Babesia subspecies that specific detection is highly pathogenic and reverse primer SEQ ID No.5(10 μ Μ) each 0.5 μ L, fluorescent probe SEQ ID No.6(5 μM) 1.0 μ L; 3) the public reagent fluorescence quantitative PCR reaction solution of two kinds of detection methods, negative quality control standard product are applicable to.Fluorescence quantitative PCR reaction solution is wherein by Premix Ex TaqTM(2 ×) 12.5 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, sterile purified water 8.0 μ L.
The present invention is a kind of TaqMan Real-Time Fluorescent Quantitative PCR Technique.Fluorescence PCR assay susceptibility is high, high specificity, detection speed are fast.TaqMan probe fluorescent quantitative PCR technique is pcr amplification adds pair of primers while, add a specific fluorescent probe, and this probe is an oligonucleotide, and two ends mark a reporter fluorescence group and a quenching fluorescence group respectively.When probe is complete, the fluorescent signal that reporter group is launched is quenched group absorptions; When just starting, probe is combined on any strand of DNA; During PCR amplification, probe enzyme is cut degraded by 5 ' to 3 ' 5 prime excision enzyme activity of Taq enzyme, reporter fluorescence group is separated with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescent signal, namely often increase a DNA chain, just have a fluorescence molecule to be formed, the accumulation and the PCR primer that achieve fluorescent signal form Complete Synchronization.The method susceptibility is high, high specificity, sense cycle are short, compared with Standard PCR, there is not crossed contamination and nucleic acid dye to shortcomings such as the harm of human body, is applied to the detection of various pathogenic agent in recent years gradually.
The present invention is for target with pyriform worm (comprising Taylor worm and Babesia) rrna 18S rRNA, excavate the target gene 18S rRNA being suitable as pathogeny differential diagnosis, set up and simultaneously can distinguish the existing different subspecies of Mohs Babesia of China in animal host with natural medium tick body, be i.e. the TaqMan real-time fluorescence quantitative PCR diagnostic techniques of the Mohs Babesia Lintan of low pathogenicity, God blessings strain and highly pathogenic Mohs Babesia Ning County, Hebei strain.The advantages such as this technology is highly sensitive with it, speed fast, high specificity, can be widely applied in the quantitative and qualitative analysis context of detection of pathogenic agent.And the method is carried out amplification and detect in real time in closed system, avoids environmental pollution, shorten detection time, be particularly useful for the Detection and diagnosis of the batch samples of acute and subclinical infection.
Accompanying drawing explanation
The Mohs Babesia subspecies real-time fluorescence quantitative PCR specific amplification curve of Fig. 1 low pathogenicity.
The Mohs Babesia subspecies real-time fluorescence quantitative PCR specific amplification curve that Fig. 2 is highly pathogenic.
Embodiment
1. construction recombination plasmid standard substance.
(1) according to the Mohs Babesia Lintan strain rrna 18S rRNA gene order infecting sheep, Primer premier 5.0 and Oligo 7 is used to design primer, the object clip size of pre-acquired is Mohs Babesia subspecies Lintan strain 661bp and Mohs Babesia subspecies Ning County strain 666bp respectively, is synthesized by precious biotinylated biomolecule engineering (Dalian) company limited.Primer pair sequence is: BLTC-S(forward primer): 5 '-AGAAACGGCTACCACATC-3 ' SEQ ID No.7, BLTC-AS(reverse primer): 5-CTTGCGACCATACTCCC-3 ' SEQ ID No.8.
(2) the Mohs Babesia subspecies Lintan strain of preserving with laboratory and the DNA of Mohs Babesia subspecies Ning County strain increase for template, adopt the PCR reaction system of 50 μ L, reaction conditions is 94 DEG C of denaturation 4min, then 94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations, 72 DEG C extend 7min.Get 5 μ L amplified production agarose gel electrophoresis to identify.Be accredited as positive PCR primer sepharose and reclaim purification kit (TaKaRa, Dalian) carry out purifying recovery, recovery product is connected to pGEM-Teasy carrier (Promega, America) competent escherichia coli cell JM-109(TaKaRa is transformed into after, Dalian) in, carry out blue hickie screening, picking positive colony (day shift) carries out PCR and order-checking qualification.
(3) extracting plasmid to being accredited as positive clone, using NanoDrop 2000/2000C(Thermo Scientific, America) ultramicrospectrophotometer mensuration plasmid concentration, and be converted into copy/μ L, in this, as plasmid standard.By the plasmid standard built, serial dilution is final concentration is 1.0 × 10
9-1.0 × 10
0copy/μ L, to carry out real-time fluorescence quantitative PCR amplification.
2. real time fluorescence quantifying PCR method
(1) Responsiveness
1) the Mohs Babesia subspecies Lintan of low pathogenicity, God blessings strain: quantitative PCR reaction system is 25 μ L:Premix Ex Taq
tM(2 ×) 12.5 μ L, forward primer SEQ ID No.1 and reverse primer SEQ ID No.2(10 μ Μ) each 0.5 μ L, fluorescent probe SEQ ID No.3(5 μM) 1.0 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, genomic DNA template 2 μ L, sterile purified water 8.0 μ L.Reaction conditions is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 45 circulations.
2) highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain: quantitative PCR reaction system is 25 μ L:Premix Ex Taq
tM(2 ×) 12.5 μ L, forward primer SEQ ID No.4 and reverse primer SEQ ID No.5(10 μ Μ) each 0.5 μ L, fluorescent probe SEQ ID No.6(5 μM) 1.0 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, genomic DNA template 2 μ L, sterile purified water 8.0 μ L.Reaction conditions is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 60 DEG C of 10s, 72 DEG C of 30s, 45 circulations.1) with 2) reaction system in the middle of, at the end of the elongating temperature of each circulation, set fluorescence signal acquisition point detect in real time, each template concentrations does 3 repetition, the calculating variation coefficient of averaging, and tests and all establishes negative control.Its result shows: template amount is linearly relevant to corresponding Ct value, expands why curve is S-type.
(2) specificity analyses
1) the Mohs Babesia subspecies Lintan of low pathogenicity, God blessings strain: utilize above-mentioned set up real time fluorescence quantifying PCR method, each sample detects Mohs Babesia subspecies Lintan strain and God blessings strain in triplicate respectively; Set the Ning County's strain of Mohs Babesia subspecies and Hebei strain, sheep Babesia U sp Xinjiang Strain, the Theileria luwenshuni of sheep, T.uilenbergi, continuous Theileria SP, sheep without slurry, clean sheep genome and aqua sterilisa as negative control simultaneously.Result shows: only have the Lintan strain of 1-Mohs Babesia subspecies, the strain of 2-Mohs Babesia subspecies God blessings is positive, the Ning County's strain of Mohs Babesia subspecies and Hebei strain, other relevant cause of disease and the water of sheep Babesia U sp Xinjiang Strain, sheep are all negative (Fig. 1).
2) highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain: utilize above-mentioned set up real time fluorescence quantifying PCR method, each sample detects the Ning County's strain of Mohs Babesia subspecies and Hebei strain in triplicate, establish the Lintan strain of Mohs Babesia subspecies and God blessings strain, sheep Babesia U sp Xinjiang Strain simultaneously; The Theileria luwenshuni of sheep, T.uilenbergi, continuous Theileria SP, sheep are negative control without slurry, clean sheep genome and aqua sterilisa.Result shows: only have the subspecies Ning County's strain of 1-Mohs Babesia and the Hebei strain of 2-Mohs Babesia subspecies to be positive, and the Lintan strain of Mohs Babesia subspecies, God blessings strain, other relevant cause of disease of sheep Babesia U sp Xinjiang Strain and sheep and water are all negative (Fig. 2).
(3) stability is analyzed with repeatability
With regard to different detection methods, when analyzing, selected concentration is also different; To Mohs Babesia subspecies Lintan, the God blessings strain of low pathogenicity, getting concentration is 1.0 × 10
9-1.0 × 10
7the standard substance of 3 concentration of copy/μ L carry out stability analysis; To highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain, getting concentration is 1.0 × 10
9-1.0 × 10
8the standard substance of 2 concentration of copy/μ L carry out stability analysis.Two kinds of methods all first carry out batch interior replica test, and to the standard substance of same batch of dilution, each concentration duplicate detection four times, analyzes the variation coefficient of Ct value.And then replica test between carrying out batch, detect the standard substance of the dilution of 3 different batches, the Ct value variation coefficient judges its stability by analysis.Repeatedly repeat these two experiments three times, the data difference nonsignificance of acquisition, illustrates that the detected result between its different batches has its comparability, has good repeatability.
<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120> distinguishes the primer and test kit that detect low pathogenicity and highly pathogenic babesia motasi
<160> 8
<210> 1
<211> 18
<212> DNA
<213> artificial sequence (forward primer OBLMT-S)
<400>
tgccggttca tgaatttg 18
<210> 2
<211> 19
<212> DNA
<213> artificial sequence (reverse primer OBLMT-AS)
<400>
gcaagtcata ctgcgtatc 19
<210> 3
<211> 21
<212> DNA
<213> artificial sequence (probe primer OBLMT-Prob)
<400>
ctgcgtcctt catcgttgtg t 21
<210> 4
<211> 19
<212> DNA
<213> artificial sequence (forward primer OBHN-S)
<400>
aggcagtaat tgctaagtg 19
<210> 5
<211> 19
<212> DNA
<213> artificial sequence (reverse primer OBLMT-AS)
<400>
gcgaaaccaa atggtaatc 19
<210> 6
<211> 20
<212> DNA
<213> artificial sequence (probe primer OBHN-Prob)
<400>
cgctcactaa tggagaccgc 20
<210> 7
<211> 18
<212> DNA
<213> artificial sequence (forward primer BLTC-S)
<400>
agaaacggct accacatc 18
<210> 8
<211> 17
<212> DNA
<213> artificial sequence (reverse primer BLTC-AS)
<400>
cttgcgacca tactccc 17
Claims (5)
1. distinguish the primer pair detecting low pathogenicity babesia motasi, it is characterized in that primer pair gene order is: SEQ ID No.1 and SEQ ID No.2.
2. distinguish the primer pair detecting highly pathogenic babesia motasi, it is characterized in that primer pair gene order is: SEQ ID No.4 and SEQ ID No.5.
3. distinguish the test kit detecting low pathogenicity and highly pathogenic babesia motasi, it is characterized in that comprising sequence in test kit is: the primer of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5 and probe primer SEQ ID No.3 and SEQ ID No.6.
4. the test kit distinguishing detection low pathogenicity and highly pathogenic babesia motasi according to claim 3, is characterized in that also having in test kit: standard positive plasmid template pGEM-Teasy-18S rRNABLT and pGEM-Teasy-18S rRNABNX.
5. the condition that the differentiation detection low pathogenicity described in claim 3 and 4 and the test kit of highly pathogenic babesia motasi carry out pcr amplification is: the condition of the Mohs Babesia pcr amplification of low pathogenicity is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 45 circulations; The condition of highly pathogenic Mohs Babesia pcr amplification is: 95 DEG C of denaturation 30s, then 95 DEG C of 5s, 60 DEG C of 10s, 72 DEG C of 30s, 45 circulations.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510035284.2A CN104561343B (en) | 2015-01-24 | 2015-01-24 | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi |
| CN201610338651.0A CN106434861A (en) | 2015-01-24 | 2015-01-24 | Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit |
| CN201610338637.0A CN106434860A (en) | 2015-01-24 | 2015-01-24 | Primer and kit for distinguished detection of high-pathogenicity babesia motasi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510035284.2A CN104561343B (en) | 2015-01-24 | 2015-01-24 | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610338651.0A Division CN106434861A (en) | 2015-01-24 | 2015-01-24 | Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit |
| CN201610338637.0A Division CN106434860A (en) | 2015-01-24 | 2015-01-24 | Primer and kit for distinguished detection of high-pathogenicity babesia motasi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104561343A true CN104561343A (en) | 2015-04-29 |
| CN104561343B CN104561343B (en) | 2017-06-20 |
Family
ID=53078417
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610338637.0A Pending CN106434860A (en) | 2015-01-24 | 2015-01-24 | Primer and kit for distinguished detection of high-pathogenicity babesia motasi |
| CN201510035284.2A Active CN104561343B (en) | 2015-01-24 | 2015-01-24 | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi |
| CN201610338651.0A Pending CN106434861A (en) | 2015-01-24 | 2015-01-24 | Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610338637.0A Pending CN106434860A (en) | 2015-01-24 | 2015-01-24 | Primer and kit for distinguished detection of high-pathogenicity babesia motasi |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610338651.0A Pending CN106434861A (en) | 2015-01-24 | 2015-01-24 | Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN106434860A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593826A (en) * | 2019-01-14 | 2019-04-09 | 中国农业科学院兰州兽医研究所 | A kind of Mohs Babesia detection kit |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10760137B2 (en) * | 2017-07-18 | 2020-09-01 | Roche Molecular Systems, Inc. | Compositions and methods for detection of Babesia |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597650A (en) * | 2009-06-01 | 2009-12-09 | 中国农业科学院兰州兽医研究所 | Detect detection film and the method for cattle and sheep pyriform worm in the blood tick body of Qinghai |
| CN103103286B (en) * | 2013-03-05 | 2014-10-15 | 扬州大学 | Primer, probe and kit for fluorescence quantitative PCR (Polymerase Chain Reaction) detecting and typing of babesia |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550446A (en) * | 2009-01-07 | 2009-10-07 | 中国农业科学院兰州兽医研究所 | Detecting membrane used for detecting Theileria luwenshuni, Theileria uilenbergi and ovine Piroplasma |
| CN103710433B (en) * | 2013-11-14 | 2016-02-03 | 中国检验检疫科学研究院 | For detecting primer and the real-time fluorescence quantitative PCR test kit of Babesia |
-
2015
- 2015-01-24 CN CN201610338637.0A patent/CN106434860A/en active Pending
- 2015-01-24 CN CN201510035284.2A patent/CN104561343B/en active Active
- 2015-01-24 CN CN201610338651.0A patent/CN106434861A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597650A (en) * | 2009-06-01 | 2009-12-09 | 中国农业科学院兰州兽医研究所 | Detect detection film and the method for cattle and sheep pyriform worm in the blood tick body of Qinghai |
| CN103103286B (en) * | 2013-03-05 | 2014-10-15 | 扬州大学 | Primer, probe and kit for fluorescence quantitative PCR (Polymerase Chain Reaction) detecting and typing of babesia |
Non-Patent Citations (3)
| Title |
|---|
| ALLEN E. TEAL,ET AL: "A New Real-Time PCR Assay for Improved Detection of the Parasite Babesia microti", 《J. CLIN. MICROBIOL》 * |
| ROS-GARCÍA ET AL: "Assessment of exposure to piroplasms in sheep grazing in communal mountain pastures by using a multiplex DNA bead-based suspension array", 《PARASITES & VECTORS》 * |
| 罗金等: "驽巴贝斯虫病PCR检测方法的建立和应用", 《中国畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593826A (en) * | 2019-01-14 | 2019-04-09 | 中国农业科学院兰州兽医研究所 | A kind of Mohs Babesia detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106434861A (en) | 2017-02-22 |
| CN106434860A (en) | 2017-02-22 |
| CN104561343B (en) | 2017-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Garrido et al. | Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis | |
| CN104862406B (en) | A kind of primer for field quick detection mycobacterium tuberculosis complex and probe and its kit | |
| CN105567871A (en) | RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof | |
| CN104561344B (en) | Detectable primer pair and kit with differentiation sheep Babesia not of the same race | |
| Luigi et al. | Development of quantitative real-time RT-PCR for the detection and quantification of Peach latent mosaic viroid | |
| CN103627812B (en) | Main pathogen of bovine mastitis Quadruple-PCR quick detection kit | |
| CN108504778A (en) | Kit that is a kind of while detecting porcine circovirus 2 type and porcine pseudorabies virus and application | |
| JP5522820B2 (en) | Method for detecting pathogens of strawberry important diseases and primers for detection | |
| CN101481742B (en) | Detection kit for Mycoplasma hyopneumoniae and use thereof | |
| Delić | Polymerase chain reaction for phytoplasmas detection | |
| CN106434935A (en) | Composition and method for identifying pasteurella multocida and/or haemophilus parasuis | |
| CN104561343A (en) | Primer and kit for distinguishing and detecting low-pathogenicity and high-pathogenicity babesia motasi | |
| CN111394483B (en) | Kit and detection method for simultaneously detecting three fish-source streptococci | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN106435007A (en) | Primer, probe, kit and method for detecting bacillus erysipelatos-suis by fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN110184266A (en) | Citrus leaf DNA rapid extracting method and its application in Citrus Huanglongbing pathogen detection | |
| CN115747361A (en) | Real-time fluorescent MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method | |
| CN110564882A (en) | Dual TaqMAN probe fluorescent quantitative PCR detection method for equine piroplasmosis | |
| CN104593493B (en) | A kind of real-time fluorescence quantitative PCR kit for detecting sheep Babesia | |
| del Campo et al. | A rapid and cost–efficient DMSO–based method for isolating DNA from cultured lichen photobionts | |
| AU2019100071A4 (en) | Pcr detection kit for rapidly identifying salmonella of specific serotypes | |
| CN103740822B (en) | Fluorescence quantitative PCR (polymerase chain reaction) combined rapid detection kit and method for SPF (specific pathogen free) mice pathogenic bacteria nucleic acid | |
| CN103740839A (en) | Universal type kit and method for detecting clostridium botulinum by fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN113493846B (en) | Citrus flavedo virus molecular fluorescence RAA detection primer probe set, kit and method | |
| CN110358851B (en) | Nucleic acid sequence, primer, method and kit for detecting bacillus cereus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |