CN110551839A - Duncan babesia dongchensis identification and detection kit and detection method - Google Patents
Duncan babesia dongchensis identification and detection kit and detection method Download PDFInfo
- Publication number
- CN110551839A CN110551839A CN201910775862.4A CN201910775862A CN110551839A CN 110551839 A CN110551839 A CN 110551839A CN 201910775862 A CN201910775862 A CN 201910775862A CN 110551839 A CN110551839 A CN 110551839A
- Authority
- CN
- China
- Prior art keywords
- babesia
- detection
- dunnii
- duncan
- kit
- 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.)
- Pending
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
- C12Q1/6844—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
- 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
Abstract
The invention discloses a TaqMan fluorescent quantitative PCR method and a kit for identification and detection of Babesia dunnii, and relates to a detection technology of a Babesia dunnii protogene in blood. Can be used for qualitative and quantitative detection of the Duncan Babesia. Primers and probes for identification and detection are designed by taking the Babesia dunnii 18S RNA as a target gene, and a TaqMan real-time fluorescent quantitative PCR method for detecting the Babesia dunnii is established. The method and the kit have the advantages of good specificity, high sensitivity and high detection speed. Is suitable for the rapid detection and large-scale epidemiological investigation of animal bodies, transmission medium ticks and clinical samples.
Description
Technical Field
The invention relates to a Duncan Babesia dunnii pathogeny detection kit and a detection method, in particular to a Duncan Babesia dunnii pathogeny detection kit and a non-disease diagnosis method caused by Duncan Babesia dunnii.
Background
Babesiosis (babesiosis) is a blood protozoal disease transmitted by vector ticks, which is caused by infecting humans or animals with protozoa belonging to the phylum apicomplexa, the family babesidae, the genus babesia, and is widely prevalent in tropical, subtropical and temperate zones, causing huge economic losses to the breeding industry and a huge threat to human health (Lobo et al.2013; Vannier and Krause 2012). Babesiosis was first discovered in romania in 1888, the etiology was babesia bovis (Babes V, 1888). Two years later, similar polypodies were also found in texas bovine red blood cells and were named gembobesia gemmifera. To date, more than 100 species of Babesia have been reported worldwide, with three of the more prevalent human and veterinary species, including Babesia divergens (Babesia divergens), Babesia microti, Duncan Denkenella (Babesia dunnii) (Levine, 1988; Jia et al, 2018, Kim et al, 2007; Saito-Ito et al, 2004).
With the rapid development of the tourism industry, the frequent progress of international trade and the cross-provincial animal trade, new release and recurrent infectious diseases are in an ascending trend. Babesiosis is also a growing concern, and its death may occur especially in immunocompromised persons due to the increase in the number of cases. The Duncan Babesia dunnii is a pathogen with strong pathogenicity, the conventional detection method of the pathogen is a classical blood smear staining method, the pathogen is difficult to detect under the condition of low insect staining rate, the condition of omission easily occurs, and the skill requirement of operators on the identification of the insect species which is mainly characterized by morphology is high. The molecular biological method established based on the polymerase chain reaction comprises a method for detecting the babesia by combining a PCR (polymerase chain reaction) method taking 18S as a target gene with a gene sequencing method, and can also be used for detecting the infection of the babesia dunken, but the method does not have the specificity of the species, is long in time consumption (about 3 to 5 days) and cannot meet the clinical rapid detection requirement. At present, no relevant report exists on a detection method for specificity of the Deng Babesia dunnii, so that a method which is rapid, high in sensitivity and good in specificity and can be used for clinical rapid detection is urgently needed to be developed.
Disclosure of Invention
The invention provides a Duncan Babesia dunnii identification and detection kit which can overcome the defects in the prior art, and also provides a Duncan Babesia dunnii identification and detection method.
The Duncan Babesia dunnii test kit comprises two amplification primers with sequences of SEQ ID No.1 and SEQ ID No.2, and a probe primer with a sequence of SEQ ID No.3, wherein the probe primer has a 5 '-end mark and a 3' -end mark HBQ 1.
Preferably, the Duncan Babesia dunnii detection kit also comprises a discriminating detection reagent Master Mix.
For convenient use, the Duncan Babesia dunnii test kit of the invention also comprises standard positive plasmids for fluorescent quantitative detection, standard negative controls and required conventional reagents.
The use method of the Duncan Babesia dunnii detection kit comprises the following steps:
(1) Extracting the genomic DNA of a sample to be detected; (2) and (2) taking the extracted genome DNA as a template, and amplifying by using an amplification reaction system and amplification conditions established by the primer and the probe, wherein the amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min for one cycle; 5s at 95 ℃, 30s at 55 ℃ and 40 cycles; (3) and (3) forming a Ct curve by using the amplification product, and judging whether the sample is positive to the Duncan Babesia dunnii according to the Ct value of the sample to be detected.
the Duncan's Babesia dunnii detection method for non-disease detection purpose of the invention is: extracting genome DNA of a sample to be detected, amplifying the obtained DNA to be detected by using an amplification primer in the kit, and judging a detection result according to the Ct value of the sample to be detected (the result is negative if the Ct value is more than 35, and the result is positive if the Ct value is less than 35).
The invention relates to a method and a kit for detecting Duncan Babesia dunnii by using TaqMan real-time fluorescent quantitative PCR technology.
TaqMan real-time fluorescent quantitative PCR is a method for introducing a fluorescent probe during PCR amplification, and a fluorescent reporter group and a fluorescent quencher group are respectively marked at two ends of the probe. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, the probe is combined on the target gene, and the Taq enzyme has 5-3 exonuclease activity during PCR amplification to degrade the probe, so that the reporter group and the quenching group are separated, and a fluorescent detection system can receive the fluorescent signal. And (3) fluorescent signals are formed when one DNA molecule is amplified, so that the synchronous accumulation of PCR amplification and the fluorescent signals is realized. The kit realizes efficient and rapid detection of target genes, is widely applied to pathogen detection of bacteria, viruses, parasites and the like, and is suitable for clinical detection and epidemiological investigation of a large number of samples.
Compared with the existing detection method, the method has the following advantages:
(1) The invention has fast detection speed: the whole detection process is completed within 90 minutes, the requirement on the skill of operators is low, and the operators with molecular biology operation skills can smoothly complete the operation. The subsequent treatment of the amplification product, such as agarose gel electrophoresis, enzyme digestion identification, gene sequencing and the like, is not needed, so that the pollution risk of the amplicon is reduced.
(2) The invention has high specificity: the marking probe BWA-P is specifically combined with a specific sequence of the Babesia dunkensii, so that the high specificity of the method is ensured, and the method has no cross reaction with 14 middle Piroporms of other infected animals (Babesia bovis, Babesia gemmifera, Babesia ova, Babesia gigas, Tabesia maxima, Tabesia circinata, Taylophila orientalis, Theileria sinensis, Babesia mowazei, Babesia incerta, Taylophila unibraziana, and Taylophila ovis) or human (Babesia divergens and Babesia microti).
(3) The application range is wide: laboratories with fluorescent quantitative PCR instruments can use the kit to detect the Duncan Babesia dunnii, 96 samples can be detected at one time, and the kit is suitable for detection of a large number of clinical samples and epidemiological investigation.
Drawings
FIG. 1. fluorescent quantitative PCR primer and probe specificity detection of Duncan Babesia dunnii. Wherein: 1 is the Duncken Babesia dunnii blood genome with the staining rate of 5%, 2 is the Moschs Babesia skatz strain, the Moschs Babesia ningham county strain, the Moschs Babesia Hebei strain, the Moschs Babesia lintan strain, Babesia unfixed Sinkiang strain, Babesia unfixed Dunhuang strain, Theileria luwenshuni, Theileria uilenbergi, Taylophila ovis, Babesia bovis, Babesia gemmifera, Babesia maxima, Cosbestus ovatus, Taylotheileria orientalis, Theileria sinensis, Babesia divergens (18S rRNA recombinant plasmid), and Babesia volvata (18S recombinant rRNA plasmid).
FIG. 2 shows the optimal concentration screening of primers and probes.
FIG. 3. fluorescent quantitative PCR sensitivity detection of Barbes dunken.
FIG. 4 clinical sample testing.
Detailed Description
The specific scheme of the invention comprises the following contents:
The experimental procedures in the following examples are conventional unless otherwise specified. The primer sequence synthesis is completed by Shanghai bioengineering GmbH.
(1) Primers and probes:
an upstream primer BWA-F: 5'-GCAGTTAAAAAGCTCGTA-3'
The downstream primer BWA-R: 5'-AAACACTCTAATTTTCTC-3'
probe BWA-P: 5'-FAM-CTCTGGCGGTGGTTCTCC-HBQ1-3'
(2) extracting whole blood genome DNA of sample to be detected
200. mu.L of whole blood to be examined was collected, and genomic DNA was extracted using a blood genomic DNA extraction kit (kit purchased from QIAGEN, cat. 51306), and the procedure was carried out according to the instruction manual.
(3) Amplification reaction system
(4) And (3) amplification reaction conditions: preferably, the condition of the fluorescent quantitative PCR amplification reaction is pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, annealing and extension at 55 ℃ for 30s, 40 cycles.
(5) Determination of detection result
The Ct value of the positive control is less than 35, and the Ct value of the negative control is more than 35;
When the Ct value of the sample to be detected is less than 35, judging that the Duncan Babesia dunnii is positive;
When the Ct value of the sample to be detected is more than 35, the sample is negative
Example 1: detection of Babesia dunken in blood samples
1. Blood genomic DNA extraction
The extraction of blood DNA was carried out using the QIAamp DNA Mini Kit (Qiagen, Germany), and the detailed procedures were carried out according to the instructions.
2. Primer design
According to GenBank published Duncan Babesia dunnii 18S rRNA gene as a target sequence, after sequence alignment analysis, an amplification primer and a probe are designed in a variation region:
an upstream primer BWA-F: 5'-GCAGTTAAAAAGCTCGTA-3'
The downstream primer BWA-R: 5'-AAACACTCTAATTTTCTC-3'
Probe BWA-P: 5'-FAM-CTCTGGCGGTGGTTCTCC-HBQ1-3'
TaqMan fluorescent quantitative PCR amplification
reaction system:
the reaction tube is placed in a fluorescent quantitative PCR instrument, and the reaction procedure is as follows: activating and pre-denaturing Taq enzyme at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, denaturation at 55 ℃ for 30s, and 40 cycles; fluorescence signals were collected at 55 ℃.
4. Determination of detection result
The Ct value of the positive control is less than 35, and the Ct value of the negative control is more than 35;
When the Ct value of the sample to be detected is less than 35, judging that the Duncan Babesia dunnii is positive;
And when the Ct value of the sample to be detected is more than 35, the sample to be detected is negative.
Example 2: specificity of TaqMan fluorescent quantitative PCR primer and probe
1. Blood genomic DNA extraction
The anticoagulation of Babesia equiva, Babesia cunningensis, Babesia equina Hebei, Babesia equina linza, Babesia equina Africa, Babesia equina Sinkiang, Babesia equina Dunhuang, Theileria luwenshuni, Taylor uilenbergi, sheep Theileria ovis, bovine Babesia equina, Babesia gemmifera, Babesia maxima, Babesia ovani, Theileria annulata, T.orientalis and T.mesochinensis was provided by Lanzhou veterinary institute tick and tick transmission research laboratory, and Babesia divergens (18SrRNA recombinant plasmid), Babesia microti (18S rRNA recombinant plasmid) were synthesized by Shanghai Biotech. Blood was collected at 200. mu.L and extracted with QIAamp DNA Mini Kit (Qiagen, Germany), and the detailed procedures were performed according to the instructions.
2. Isothermal amplification with crossed primers
Reaction system:
The reaction tube is placed in a fluorescent quantitative PCR instrument, and the reaction procedure is as follows: activating and pre-denaturing Taq enzyme at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, denaturation at 55 ℃ for 30s, and 40 cycles; fluorescence signals were collected at 55 ℃.
3. Determination of detection result
the Ct value of the positive control is less than 35, and the Ct value of the negative control is more than 35;
When the Ct value of the sample to be detected is less than 35, judging that the Duncan Babesia dunnii is positive;
And when the Ct value of the sample to be detected is more than 35, the sample to be detected is negative.
The experimental results are shown in figure 1, and the results show that the amplification primers and the detection probes used in the invention have good specificity and do not have cross reaction with other animal and human infected pyricularia.
Example 3: screening of TaqMan fluorescent quantitative PCR primer and probe concentration
1. Duncan Babesia dunnii genome DNA extraction
Golden hamster is intraperitoneally injected into the Duncan Babesia dunnii strain (ATCC PRA-302), the insect staining rate of a blood smear is 10% after 8 days, anticoagulation blood is collected from the heart after anesthesia, 200 mu L of blood is taken and extracted by a QIAamp DNA Mini Kit (Qiagen, Germany), and the specific operation steps are carried out according to the instruction.
2. Fluorescent quantitative PCR amplification
Reaction system:
The optimal primer concentration and probe concentration were selected with primer concentrations of 0.2, 0.4, 0.6, and 0.8. mu.L, and probe concentrations of 0.2, 0.4, 0.6, and 0.6. mu.L, respectively.
The reaction tube is placed in a fluorescent quantitative PCR instrument, and the reaction procedure is as follows: activating and pre-denaturing Taq enzyme at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, denaturation at 55 ℃ for 30s, and 40 cycles; fluorescence signals were collected at 55 ℃.
3. Determination of results
and taking the concentration of the primer and the probe with the amplification curve close to the S type and the minimum Ct value as the optimal primer and probe concentration of the fluorescent quantitative PCR.
The results are shown in FIG. 2, which shows that the optimal concentration of the primers and detection probes used in the present invention is 0.3. mu.M.
Example 3: sensitivity test for TaqMan primer amplification
1. DNA extraction of Babesia dunken
A200. mu.L sample of blood from a Dengken Babesia littoralis-infected golden hamster was extracted with the QIAamp DNA Mini Kit (Qiagen, Germany), and the specific procedures were performed according to the instructions.
2.18 preparation of S rRNA recombinant plasmid
(1) Amplification of 18S rRNA gene by using extracted Duncan Babesia dunnii genome as template
An amplification primer: PIRO-F140: 5'-CCATGGATAACCGTGCTAATTG-3'
PIRO-R1380：5’-CATCTAAGGGCATCACAGACC-3’
A50-mu-L PCR reaction system is adopted, the reaction condition is pre-denaturation at 94 ℃ for 3min, then extension is carried out for 5min at 94 ℃ for 30S, 55 ℃ for 30S and 72 ℃ for 1min in 35 cycles and 72 ℃. mu.L of the amplification product was identified by electrophoresis on a 1% agarose gel. The PCR product identified as positive is purified and recovered by an agarose gel recovery purification kit (zymo), and the recovered DNA fragment is connected with a pMD19-simple vector, wherein the reaction system is as follows:
After formulation was complete, ligation was performed overnight at 16 ℃.
(3) transformation and PCR identification of pMD19-simple ligation products
(a) DH 5. alpha. competent cells were removed from a super-cold freezer at-80 ℃ and thawed on ice.
(b) mu.L of the ligation product was added to 100. mu.L of DH 5. alpha. competent cells and ice-cooled for 30 min.
(c) heat shock at 42 deg.C for 90s, and placing on ice for 2 min.
(d) Adding LB liquid medium without antibiotic 1ml, rotating at 37 deg.C 150, and culturing for 90 min.
(e) 100 μ L of the suspension was spread on ampicillin-resistant LB plates and cultured overnight at 37 ℃ in an inverted state.
(f) Single colonies were picked from the plates and cultured in 500. mu.L of ampicillin-resistant LB liquid medium in 1.5ml EP tube at 37 ℃ for 5-6 hours with shaking at 220 rpm.
(g) Taking 1 mu L as a template to carry out PCR identification on the bacterial liquid. The positive bacteria solution was added to 5ml of LB liquid medium and the mixture was subjected to amplification.
(4) The plasmid was extracted from the clone identified as positive, and the concentration of the plasmid was measured using a NanoDrop 2000/2000C (Thermo Scientific, America) ultramicro spectrophotometer and converted to copy/. mu.L, which was used as a plasmid standard.the constructed plasmid standard was serially diluted to a final concentration of 1.0X 10 7 -1.0X 10 1 copies/. mu.L for susceptibility testing.
3. Sensitivity testing
And (3) performing a minimal detection experiment by using the diluted standard plasmid as a template and the established TaqMan fluorescent quantitative PCR reaction condition.
Reaction system:
The reaction tube is placed in a fluorescent quantitative PCR instrument, and the reaction procedure is as follows: activating and pre-denaturing Taq enzyme at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, denaturation at 55 ℃ for 30s, and 40 cycles; fluorescence signals were collected at 55 ℃.
3. Determination of detection result
The lowest plasmid concentration of which the Ct value is less than 35 is taken as the lower detection limit of the kit.
The results are shown in FIG. 3, and the results show that the detection sensitivity of the amplification primers and the detection probes used in the invention is 10 copies per reaction, i.e., the kit can detect samples with concentration of more than 10 copies per microliter.
example 4: application evaluation of TaqMan real-time fluorescence quantitative PCR detection kit
(1) Clinical samples
200 anticoagulated blood were collected from tick-bitten patients hospitalized at the second hospital of Lanzhou university, and the collection of samples was informed by consent of the patient or family and approved by the ethical Committee of the second hospital of Lanzhou university. The extraction of blood DNA was carried out using the QIAamp DNA Mini Kit (Qiagen, Germany), and the detailed procedures were carried out according to the instructions.
(2) Comparative detection of samples
200 parts of genome DNA samples are firstly detected by combining nested PCR with DNA sequencing technology, and the result shows that 200 parts of samples are negative to the Babesia dunken. Then, the Duncan Babesia dunking TaqMan fluorescent quantitative PCR kit is used for detection, and positive and negative controls are set simultaneously, and the result shows that (figure 4) the fluorescent quantitative PCR detection result is completely consistent with the nested PCR combined gene sequencing result.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> Duncan Babesia dunnii identification and detection kit and detection method
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (upstream primer BWA-F)
<400> 1
gcagttaaaa agctcgta 18
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (downstream primer BWA-R)
<400> 2
aaacactcta attttctc 18
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence (Probe BWA-P)
<400> 3
ctctggcggt ggttctcc 18
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (upstream primer PIRO-F140)
<400> 4
ccatggataa ccgtgctaat tg 22
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (downstream primer R1380)
<400> 5
catctaaggg catcacagac c 21
Claims (4)
1. A Duncan Babesia dunnii detection kit is characterized in that the detection kit comprises two amplification primers with sequences of SEQ ID No.1 and SEQ ID No.2, and a probe primer with a sequence of SEQ ID No.3 and with a 5 '-end mark and a 3' -end mark HBQ 1.
2. The Duncan Babesia dunnii detection kit according to claim 1, wherein the detection kit further comprises a detection reagent MasterMix for identification.
3. The Duncan Babesia dunnii test kit of claim 2, wherein the test kit further comprises a standard positive plasmid for fluorescent quantitative detection, a standard negative control and conventional reagents required.
4. The method for using the Duncan Babesia dunnii test kit as claimed in claim 1, 2 or 3, wherein the method comprises the following steps:
(1) extracting the genomic DNA of a sample to be detected; (2) and (2) taking the extracted genome DNA as a template, and amplifying by using an amplification reaction system and amplification conditions established by the primer and the probe, wherein the amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min for one cycle; 5s at 95 ℃, 30s at 55 ℃ and 40 cycles; (3) and (3) forming a Ct curve by using the amplification product, and judging whether the sample is positive to the Duncan Babesia dunnii according to the Ct value of the sample to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910775862.4A CN110551839A (en) | 2019-08-21 | 2019-08-21 | Duncan babesia dongchensis identification and detection kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910775862.4A CN110551839A (en) | 2019-08-21 | 2019-08-21 | Duncan babesia dongchensis identification and detection kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110551839A true CN110551839A (en) | 2019-12-10 |
Family
ID=68737819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910775862.4A Pending CN110551839A (en) | 2019-08-21 | 2019-08-21 | Duncan babesia dongchensis identification and detection kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110551839A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111893201A (en) * | 2020-07-29 | 2020-11-06 | 深圳市疾病预防控制中心 | Reagent and kit for detecting 4 babesia by isothermal amplification and application of reagent and kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120015360A1 (en) * | 2009-12-07 | 2012-01-19 | Ibis Biosciences, Inc. | Compositions for use in identification of babesia bioagents |
| WO2014197607A1 (en) * | 2013-06-05 | 2014-12-11 | The Regents Of The University Of California | Detection of tick-borne diseases |
| CN105861651A (en) * | 2016-04-01 | 2016-08-17 | 南京农业大学 | Babesia canis PCR detection kit containing internal amplification control and use method of Babesia canis PCR detection kit |
| CN106636347A (en) * | 2015-01-24 | 2017-05-10 | 中国农业科学院兰州兽医研究所 | Primer pairs and kit capable of detecting and distinguishing indeterminate Sinkiang strain of ovine babesia |
| CN107148565A (en) * | 2014-10-24 | 2017-09-08 | 莫纳什大学 | Method and system for detecting the pathogen in blood |
-
2019
- 2019-08-21 CN CN201910775862.4A patent/CN110551839A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120015360A1 (en) * | 2009-12-07 | 2012-01-19 | Ibis Biosciences, Inc. | Compositions for use in identification of babesia bioagents |
| WO2014197607A1 (en) * | 2013-06-05 | 2014-12-11 | The Regents Of The University Of California | Detection of tick-borne diseases |
| CN107148565A (en) * | 2014-10-24 | 2017-09-08 | 莫纳什大学 | Method and system for detecting the pathogen in blood |
| CN106636347A (en) * | 2015-01-24 | 2017-05-10 | 中国农业科学院兰州兽医研究所 | Primer pairs and kit capable of detecting and distinguishing indeterminate Sinkiang strain of ovine babesia |
| CN105861651A (en) * | 2016-04-01 | 2016-08-17 | 南京农业大学 | Babesia canis PCR detection kit containing internal amplification control and use method of Babesia canis PCR detection kit |
Non-Patent Citations (2)
| Title |
|---|
| MELISA WILSON 等: "Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani", 《EXP PARASITOL》 * |
| 魏金龙 等: "巴贝斯虫感染对宿主红细胞及免疫系统的影响", 《中国动物传染病学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111893201A (en) * | 2020-07-29 | 2020-11-06 | 深圳市疾病预防控制中心 | Reagent and kit for detecting 4 babesia by isothermal amplification and application of reagent and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Garrido et al. | Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis | |
| CN106399590B (en) | Universal nucleic acid isothermal detection reagent for respiratory tract infection related adenovirus | |
| CN108384899B (en) | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof | |
| Rahumatullah et al. | Triplex PCR using new primers for the detection of Toxoplasma gondii | |
| CN110004240B (en) | Real-time fluorescence detection kit and test strip detection kit for mycoplasma gallisepticum based on RPA and application of kit and test strip detection kit | |
| Nunan et al. | Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei | |
| CN110628956A (en) | Dual-fluorescence PCR primer and kit for identifying ASFV strain and CD2v gene deletion strain | |
| CN113151522A (en) | LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof | |
| CN114540526B (en) | Primers, probes and method for typing detection of five input plasmodium | |
| CN107365843B (en) | LAMP primer combination for detecting two main parasites causing calf diarrhea and application thereof | |
| CN104561344A (en) | Primer pairs and kit capable of detecting and distinguishing different breeding sheep babesia | |
| CN114790490A (en) | Molecular marker capable of distinguishing Brucella melitensis and detection method | |
| CN107974515B (en) | Constant-temperature rapid detection kit for tilapia lake Luo virus | |
| CN110551839A (en) | Duncan babesia dongchensis identification and detection kit and detection method | |
| CN110607398B (en) | RT-LAMP kit for fluorescent visual rapid detection of porcine epidemic diarrhea virus | |
| CN114574628B (en) | Detection kit and application thereof | |
| CN108410991B (en) | Detection method for distinguishing tick interspecific and intraspecies specificity | |
| CN108103152B (en) | Rapid detection method for Listonella anguillarum | |
| CN107604101B (en) | Novel pigeon adenovirus real-time fluorescent quantitative PCR detection kit | |
| RU2511440C2 (en) | Method of quantitative determination of fixed rabies virus "moskva 3253" | |
| CN112029910B (en) | Nucleic acid detection method for SARS-CoV-2 virus | |
| CN104593493B (en) | A kind of real-time fluorescence quantitative PCR kit for detecting sheep Babesia | |
| KR101395938B1 (en) | Pcr diagnosis using specific primer for bacteria that cause diseases of allomyrina dichotoma | |
| CN112708685A (en) | Eva Green fluorescent quantitative PCR kit for detecting ehrlichia and detection method | |
| CN109439782B (en) | Kit for identifying and detecting babesia bovis and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20191210 |