CN112725488B - Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease - Google Patents
Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease Download PDFInfo
- Publication number
- CN112725488B CN112725488B CN202110153717.XA CN202110153717A CN112725488B CN 112725488 B CN112725488 B CN 112725488B CN 202110153717 A CN202110153717 A CN 202110153717A CN 112725488 B CN112725488 B CN 112725488B
- Authority
- CN
- China
- Prior art keywords
- quantitative pcr
- primer
- ehrlichiosis
- fluorescent quantitative
- lyme disease
- 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.)
- Active
Links
Images
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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer and a kit, wherein the ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer comprises a primer for detecting ehrlichiosis and a primer for detecting borrelia burgdorferi, and the kit contains the ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer. The method can be used for simultaneously detecting the Escherichia and the Borrelia burgdorferi, and has the advantages of high detection speed, high efficiency, accurate quantification, high sensitivity and strong specificity.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer and a kit.
Background
The multiplex fluorescence PCR method has high detection efficiency, namely, the multiplex fluorescence PCR can realize one tube for multiple detection, thereby simplifying the operation flow; the reagent cost and the personnel operation time are saved, and particularly, the detection cost and the operation time are obviously reduced when a large number of samples are detected. The SYBR Green I dye method has high cost performance of the fluorescent quantitative PCR, and can monitor the quantity of the double-stranded DNA existing in a PCR system by utilizing the linear relation between the fluorescent signal intensity of the SYBR Green I dye and the quantity of the double-stranded DNA and carry out differential diagnosis by utilizing a melting curve. The melting curve is a curve for reflecting the degradation degree of the double helix structure of DNA along with the temperature rise, the melting temperature when the double helix structure of the DNA is degraded by half is called as Tm value, the Tm value is related with the G-C value in the gene sequence, therefore, when the amplification product has corresponding different Tm peak values due to the difference of the G-C value, the specific product can be distinguished from other products through the characteristic peak, and the aim of differential diagnosis is achieved.
Ticks are ectoparasites that are obligate blood-sucking, are the second largest vectors to mosquitoes, can transmit bacteria, viruses, protozoa and the like, and cause serious harm to public health and animal husbandry development. A large part of emerging bacterial infectious diseases are transmitted by zoonotic vectors such as ticks and the like. The most prominent of these diseases are lyme disease, ehrlichiosis and bartonella disease.
Borrelia burgdorferi (b.g) is the causative agent of lyme disease. Lyme Disease (LD) is a zoonosis of multiple systems of organisms and multiple organs inflammatory reaction, is widely distributed in europe and asia, is the most common tick-borne disease in the united states, and has the characteristics of wide distribution, rapid transmission and high disability rate. Borrelia burgdorferi was first isolated in forest and sea counties of Heilongjiang in 1986, and existence of Borrelia burgdorferi in China was proved. At present, borrelia burgdorferi infection exists in more than 30 provinces (autonomous regions and direct prefectures) in China, and natural epidemic source areas exist in 20 provinces, cities and autonomous regions in China.
Members of the ehrlichia genus of the anaplasmataceae family cause ehrlichiosis in humans and animals. The ehrlichia species is an obligate intracellular parasitic bacterium, and besides wild animal hosts, livestock, companion animals and humans are also susceptible hosts. Since the discovery of the first strain of Escherichia in Algorilla in 1935, cases of Escherichia are continuously occurring in various places, and researchers detect Escherichia nucleic acid and antibody from dogs, mice, sheep, ticks and human bodies in China at present, which shows that natural epidemic sites of the disease may exist in China.
With the increasing economic level, the contact between human beings and the nature is gradually increased, and the diagnosis and the control of infectious diseases such as Lyme disease and Escherichia disease which are carried by tick worms attract attention. At present, the Escherichia disease is diagnosed mainly by carrying out Giemsa staining on blood cells and observing whether mulberries exist under a microscope, and the diagnosis of the Lyme disease has the current effective national standard, but the recommended pathogen separation and serological detection have the defects of high operation difficulty, long period and high requirements on operators, and a method and a standard for simultaneously diagnosing the two diseases are lacked at present. The Polymerase Chain Reaction (PCR) has good specificity and is simple to operate, so that the method becomes a widely applied laboratory test, and the establishment of a molecular biological diagnosis method for simultaneously diagnosing lyme disease and ehrlichiosis becomes a scientific problem to be solved urgently, and provides theoretical basis and technical support for the establishment of national standards.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer and a kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
a dual-fluorescence quantitative PCR primer for detecting ehrlichiosis and lyme disease comprises a primer for detecting ehrlichiosis and a primer for detecting borrelia burgdorferi;
the primer sequences for detecting the Escherichia are as follows:
groel-YG F：5’-AGCTGAATTGGGTACTGCTAAGAA-3’；
groel-YG R：5’-TACAGCAACACCACCAGAAAGT-3’；
the primer sequence for detecting the borrelia burgdorferi is as follows:
ospA-YG F：5’-GAGCAGACGGAACCAGAC-3’；
ospA-YG R：5’-CCTTCTTTAACCACCAAT-3’。
the invention also provides a double fluorescent quantitative PCR detection kit for simultaneously diagnosing the ehrlichiosis and the lyme disease, which contains the double fluorescent quantitative PCR primer for the ehrlichiosis and the lyme disease.
Further, in the double fluorescence quantitative PCR detection kit, a standard positive template and a negative control sample are also contained, wherein the standard positive template comprises an ehrlichia positive sample DNA template and a borrelia burgdorferi positive sample DNA template; negative control samples included DEPC H 2 O。
The invention has the beneficial effects that:
1. according to the invention, both Escherichia and Borrelia burgdorferi can be detected simultaneously.
2. The method is rapid and efficient in detection, conventional agarose gel electrophoresis detection is not needed, and result judgment can be performed through a self-contained program of a fluorescent quantitative PCR instrument after the reaction is finished.
3. By using the method, the sample to be detected can be accurately quantified by preparing the standard substance and drawing a standard curve according to the Ct values of the Escherichia and the Borrelia burgdorferi in the sample to be detected.
4. By using the invention, the minimum detection limit of both Escherichia and Borrelia burgdorferi is 1 × 10 1 copies/μL。
5. The invention has no response signal to the genome detection of Escherichia, borrelia burgdorferi, leptospira, rickettsia, escherichia coli and Pseudomonas aeruginosa, only generates fluorescence signal to the genome detection of Escherichia and Borrelia burgdorferi, and has difference of specific melting curve peak value (Tm value).
Drawings
FIG. 1 is a graph showing the real-time fluorescence quantitative PCR amplification curve of Escherichia coli obtained by optimizing the fluorescence quantitative PCR reaction conditions and system according to the embodiment of the present invention;
FIG. 2 is a standard curve of real-time fluorescence quantitative PCR of Escherichia coli in accordance with an embodiment of the present invention;
FIG. 3 is a melting curve of real-time fluorescence quantitative PCR of Escherichia coli obtained by optimizing the reaction conditions and system of fluorescence quantitative PCR according to the embodiment of the present invention;
FIG. 4 is a graph showing the PCR amplification curve of the real-time PCR for the borrelia burgdorferi obtained in the optimization of the PCR reaction conditions and system according to the embodiment of the present invention;
FIG. 5 is a standard curve of real-time fluorescent quantitative PCR of Borrelia burgdorferi in an embodiment of the invention;
FIG. 6 is a melting curve of real-time fluorescent quantitative PCR of Borrelia burgdorferi obtained in the optimization of the fluorescent quantitative PCR reaction conditions and system in the embodiment of the invention;
FIG. 7 is a melting curve of dual real-time fluorescent quantitative PCR in an embodiment of the present invention;
FIG. 8 shows the result of the dual fluorescent quantitative PCR specificity test in the example of the present invention;
FIG. 9 is a graph showing the amplification curve of the double fluorescent quantitative PCR obtained in the sensitivity detection in the example of the present invention;
FIG. 10 is a melting curve of the double real-time fluorescent quantitative PCR obtained in the sensitivity detection in the example of the present invention;
FIG. 11 shows the positive results of the detection of the clinical sample of the dual fluorescent quantitative PCR part in the clinical application of the embodiment of the present invention;
FIG. 12 shows the positive result of normal PCR detection of some clinical samples in clinical application according to the embodiment of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
1. Relevant test strains
The genomes of E.coli, B.burgdorferi, leptospira, rickettsia, E.coli and Pseudomonas aeruginosa for the experiments were identified and maintained in the infectious disease laboratory of the animal medical college of Jilin university.
2. Primer design
And (3) selecting conserved sequences of an Escherichia groEL gene and an OSpA gene of the borrelia burgdorferi to design a double-fluorescent quantitative PCR primer for the ehrlichiosis and the lyme disease.
The primer sequences for detecting Ehrlichia are as follows:
groel-YG F:5 'AGCTGAATTGGGTACTGCATAAGAA-3'; as shown in SEQ ID NO. 1;
groel-YG R:5 'TACAGCAACACCAGAAAGT-3'; as shown in SEQ ID NO. 2;
the size of the target fragment is 196bp.
The primer sequences for detecting the borrelia burgdorferi are as follows:
ospA-YG F:5 'GAGCAGACGGAACCAGAC-3'; as shown in SEQ ID NO. 3;
ospA-YG R: 5-; as shown in SEQ ID NO. 4;
the size of the target fragment is 133bp.
The primers were synthesized by Biotechnology engineering (Shanghai) Co., ltd.
3. Extracting and purifying the genomic DNA of the sample to be detected
Reference to
The Genomic DNA Purification Kit instructions extract the genomes of Escherichia, borrelia burgdorferi, leptospira, rickettsia, escherichia coli and Pseudomonas aeruginosa, all of which are stored at-20 ℃ for later use.
4. Establishment of double real-time fluorescence quantitative PCR (polymerase chain reaction) diagnosis method for ehrlichiosis and lyme disease
4.1 construction of Positive Standard plasmid
The conserved region of heat shock protein groEL in E.coli genomic samples was amplified by the general PCR method (using Ehr-groEL-F5 ' -GTTGAAAAARACTGATGGTATGCA-3 ' (SEQ ID NO. 5) and Ehr-groEL-R5 ' -ACACGTTCTTAC-GYTCYTTAAC-3 ' (SEQ ID NO. 6) primers, reaction program: 94 ℃ 5min 94 ℃ 30s,53 ℃ 1min,72 ℃ 1.min,40 cycles; 72 ℃ 10 min) and the OSpA full length of the Boehringer's surface A protein (using OspAF5' -gacgagaaaacagcgtttcag-3 ' (SEQ ID NO. 7) and ospA R5 ' -ttttaagcgttttatttcata-3 ' (SEQ ID NO. 8) primers, reaction program: 94 ℃ 5min, 94 ℃ 30.55 ℃ 30 min; 30.30 ℃ 30 ℃ 5 ℃ 30 min; 5 ℃ cycle).
After the amplification product is identified as correct by 1% agarose gel electrophoresis, the gel is recovered and ligated
Easy vector is transformed into DH5 alpha competent cell, recombinant plasmid is extracted, T vector universal primer M13 primer is used for identification by PCR method, and successfully identified plasmid is sent to Shanghai bio-company for sequencing. After sequence alignment, the positive plasmids were named pT-groEL and pT-ospA, respectively.
The concentrations of positive standard pT-groEL and pT-ospA were measured by a microanalyzer, the copy numbers thereof were calculated and diluted to 1X 10 10 copies/. Mu.L, 10-fold serial dilutions were made, with plasmid contents of 1X 10 respectively 9 -1×10 1 The copies/mu L are subpackaged and stored at the temperature of 20 ℃ below zero for standby.
4.2 fluorescent quantitative PCR reaction conditions and system optimization
The reaction conditions were optimized by performing real-time fluorescent quantitative PCR reactions using positive standards (plasmids pT-groEL and pT-ospA) as templates and primers (groEL-YG F, groEL-YG R, ospA-YG F, ospA-YG R) at different annealing temperatures (46-62 ℃ C.) and concentrations of 0.2-1. Mu.M.
The optimal reaction system (20 mu L) is optimized to be PerfectStart TM Green qPCR SuperMix 10μL、ddH 2 The working concentration of O4. Mu.L, groEL and ospA upstream and downstream primers were all 1. Mu.M (total volume 4. Mu.L), and 1. Mu.L of each of the two positive standard plasmids. Negative controls were set with DEPC water as template.
The optimized optimal reaction conditions are pre-denaturation 30s at 94 ℃, denaturation 5s at 94 ℃, annealing 15s at 50 ℃, extension 30s at 72 ℃ and 40 cycles, and after the reaction is finished, a melting curve is collected.
And (4) judging a result: 2 specific peaks were generated on a smooth melting curve, corresponding to the Tm of E.coli and B.burgdorferi, respectively, which matched the Tm of the melting curve generated by single primer fluorescence quantification.
Using the optimized reaction conditions and the dilution degree of 1 × 10 10 ～1×10 1 The amplification kinetics curves (see FIGS. 1 and 4) and the specific melting curves (see FIGS. 3 and 6) were obtained by using the two positive standard plasmids of copies/. Mu.L as templates. FIG. 1 is a graph showing the amplification curves of real-time fluorescence quantitative PCR of Escherichia, in which 1 to 10 are standard plasmid concentrations of 1X 10 10 - 1×10 1 An amplification curve of copies/mu L, 11 is an amplification curve of a negative control, and FIG. 3 is a melting curve of real-time fluorescence quantitative PCR of Escherichia coli; FIG. 4 is a graph showing the amplification curves of real-time fluorescent quantitative PCR of Borrelia burgdorferi, in which 1-10 are standard plasmid concentrations of 1X 10 10 - 1×10 1 The amplification curve of copies/. Mu.L, 11 is the amplification curve of negative control, and FIG. 6 is the melting curve of real-time fluorescent quantitative PCR of Borrelia burgdorferi.
4.3 creation of Standard Curve
Taking the common logarithm of the copy number (lgx) in each concentration standard template as an abscissa and the cycle number threshold (Ct value) as an ordinate, the linear equation of the obtained Ehrlichia real-time fluorescence quantitative PCR standard curve (shown in FIG. 2) is y = 3.1085x +37.99, R2= 0.9833, the linear equation of the Borrelia burgdorferi real-time fluorescence quantitative PCR standard curve (shown in FIG. 5) is y = y = 3.1142lx +35.367, and R2=0.9931, which indicates that the established standard curve of the real-time fluorescence quantitative PCR method has good linear relation.
4.4 melting Curve analysis
As described above, 2 specific peaks were generated on a smooth melting curve, which are Tm values of E.coli and B.burgdorferi, respectively, and matched with Tm values of melting curves generated by single primer fluorescence quantification, as shown in FIG. 7, in which 1 represents E.coli positive, 2 represents B.burgdorferi positive, 3 represents mixed E.coli and B.burgdorferi positive, and 4 represents a negative sample. And (4) judging a result: after the real-time fluorescent quantitative PCR reaction is finished, the analysis test result of the melting curve peak value (Tm value) is observed: if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (78.12 +/-0.40) ° C, the Escherichia is judged to be positive; if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (80.2 +/-0.14) DEG C, the detection sample is judged to be positive by the Borrelia burgdorferi; if both Tm = (76.61. + -. 0.34) ° C and Tm = (80.2. + -. 0.14) ° C, a peak appears, indicating a mixed infection of Escherichia and Borrelia burgdorferi.
4.5, specific detection
The double real-time fluorescent quantitative PCR diagnosis method is used for detecting the genomes of the Escherichia, the Borrelia burgdorferi, the Leptospira soma, the Rickettsia, the Escherichia coli and the Pseudomonas aeruginosa, only the experimental group has specific amplification, and other groups have no cross reaction, as shown in figure 8, wherein 1 is a positive detection result curve of the Escherichia and the Borrelia burgdorferi, and 2-4 represent detection result curves of the genomes of the Leptospira, the Rickettsia, the Escherichia coli and the Pseudomonas aeruginosa.
4.6 sensitivity detection
Dilution of gradient scale by double fluorescent quantitative PCR detectionThe standard plasmid is detected to obtain that the lower limit of detection of the Escherichia and the Borrelia burgdorferi are both 1 multiplied by 10 1 copies/. Mu.L, as shown in FIGS. 9-10. FIG. 9 shows the amplification curves of the dual fluorescent quantitative PCR, in which 1-10 are standard plasmid concentrations of 1X 10 10 -1×10 1 Amplification curve of copies/. Mu.L, 11 is the amplification curve of negative control. FIG. 10 shows melting curves of double real-time fluorescent quantitative PCR.
4.7 repeatability test
The melting curve has high contact ratio, stable corresponding Tm value, standard deviation smaller than 0.1 and variation coefficient smaller than 0.1%, which shows that the dual fluorescent quantitative PCR detection method using the dual fluorescent quantitative PCR primer has good amplification repeatability and good stability, and is shown in Table 1.
TABLE 1 different concentration reproducibility analysis of Dual fluorescent quantitative PCR
4.8 determination of results of double fluorescent quantitative PCR detection method
After the real-time fluorescent quantitative PCR reaction is finished, observing the analysis test result of the peak value (Tm value) of the dissolution curve: if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (83.09 +/-0.10) ° C, the Escherichia is judged to be positive; if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (86.95 +/-0.08) ° C, the detection sample is judged to be positive by the Borrelia burgdorferi; if both Tm = (83.09. + -. 0.10) ° C and Tm = (86.95. + -. 0.08) ° C, a peak appears, indicating mixed infection of Escherichia and Borrelia burgdorferi.
5. Clinical application
The detection results of 25 Duochidion rhynchophyllus genomic DNA samples are shown in Table 2, wherein the Ct value of the positive sample is 11.08-20.49, the detection rate of Escherichia by using the double fluorescence quantitative PCR detection method is 28%, the detection rate of Borrelia burgdorferi is 60% (shown in figure 11), and the detection rate of the sample is 12% higher than that of the sample by using the common PCR method (shown in figure 12). In FIG. 12, A is the result of dsb gene identification in an Escherichia positive sample, and B is the result of 5s-23s spacer sequence identification in B. In A, M represents Trans 2000DNA marker,1 represents a negative control, and 2-6 represent Escherichia coli positive samples. In B, M represents a Trans 2000DNA marker,1-10 represents a part of a Borrelia burgdorferi positive sample, and 11 represents a negative control.
TABLE 2 clinical sample test results
Various changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.
Sequence listing
<110> university of Jilin
<120> double fluorescent quantitative PCR primer for ehrlichiosis and lyme disease and kit
<130> 123
<141> 2021-02-04
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 1
agctgaattg ggtactgcta agaa 24
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 2
tacagcaaca ccaccagaaa gt 22
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence ()
<400> 3
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence ()
<400> 4
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 5
gttgaaaara ctgatggtat gca 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 6
acacgrtctt tacgytcytt aac 23
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 7
gacgagaaaa acagcgtttc ag 22
<210> 8
<211> 29
<212> DNA
<213> Artificial sequence ()
<400> 8
ttattttaaa gcgtttttaa tttcatcaa 29
Claims (3)
1. A dual-fluorescence quantitative PCR primer for detecting ehrlichiosis and lyme disease is characterized by comprising a primer for detecting ehrlichiosis and a primer for detecting borrelia burgdorferi;
the primer sequences for detecting the Escherichia are as follows:
groel-YG F：5’-AGCTGAATTGGGTACTGCTAAGAA-3’；
groel-YG R：5’-TACAGCAACACCACCAGAAAGT-3’；
the primer sequence for detecting the borrelia burgdorferi is as follows:
ospA-YG F：5’-GAGCAGACGGAACCAGAC-3’；
ospA-YG R：5’-CCTTCTTTAACCACCAAT-3’。
2. a dual fluorescent quantitative PCR detection kit for simultaneous diagnosis of ehrlichiosis and lyme disease, comprising the ehrlichiosis and lyme disease dual fluorescent quantitative PCR primer of claim 1.
3. The dual fluorescent quantitative PCR detection kit of claim 2, further comprising a standard positive template and a negative control sample, wherein the standard positive template comprises an Ehrlichia positive sample DNA template and a Borrelia burgdorferi positive sample DNA template; negative control samples included DEPC H 2 O。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110153717.XA CN112725488B (en) | 2021-02-04 | 2021-02-04 | Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110153717.XA CN112725488B (en) | 2021-02-04 | 2021-02-04 | Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112725488A CN112725488A (en) | 2021-04-30 |
| CN112725488B true CN112725488B (en) | 2023-03-24 |
Family
ID=75595746
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110153717.XA Active CN112725488B (en) | 2021-02-04 | 2021-02-04 | Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725488B (en) |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7390626B2 (en) * | 2005-02-25 | 2008-06-24 | Immunosciences Lab., Inc. | Methods and kit for diagnosing tick borne illnesses |
| US8685406B2 (en) * | 2009-11-13 | 2014-04-01 | The Board Of Regents Of The University Of Texas System | Compositions of and method of using heat shock protein peptides |
| WO2013113326A1 (en) * | 2012-01-31 | 2013-08-08 | Curevac Gmbh | Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen |
| CN102703587B (en) * | 2012-05-18 | 2013-11-27 | 中国疾病预防控制中心传染病预防控制所 | Loop-mediated isothermal amplification method for detecting lyme disease spirochete |
| EP2889382A1 (en) * | 2013-12-30 | 2015-07-01 | Centrum Badan DNA Sp. z o.o. | Oligonucleotides, use thereof, methods and a kit for detection of DNA Borrelia burgdorferi, Bartonella henselae, Babesia divergens, Anaplasma phagocytophilum |
| US11326213B2 (en) * | 2015-01-21 | 2022-05-10 | T2 Biosystems, Inc. | NMR methods and systems for the rapid detection of tick-borne pathogens |
| NL2014614B1 (en) * | 2015-04-10 | 2017-01-25 | Pld Biochemistry B V | Method and means for increasing antibodies against Borrelia burgdorferi and increasing antigen of Borrelia burgdorferi. |
| CN105624300A (en) * | 2016-02-03 | 2016-06-01 | 吉林大学 | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting pathogenic leptospira canicola |
| US20190093149A1 (en) * | 2017-09-27 | 2019-03-28 | The United States Of America, As Represented By The Secretary Of Agriculture | Assays for detecting multiple tick-borne pathogens |
-
2021
- 2021-02-04 CN CN202110153717.XA patent/CN112725488B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112725488A (en) | 2021-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111074000B (en) | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain from double gene deletion strain | |
| CN111876502B (en) | Method for identifying Brucella S2 vaccine strain by dual real-time fluorescent quantitative PCR and kit used by same | |
| CN110408727B (en) | CPA primer group for detecting J subgroup avian leukosis virus, CPA nucleic acid test strip kit and application thereof | |
| CN110734994A (en) | Specific primer pair, probe and detection kit for detecting aeromonas hydrophila | |
| CN103484571A (en) | LAMP (loop-mediated isothermal amplification) detection primer group, detection kit and detection method for infectious hypodermal and hematopoietic necrosis virus | |
| CN107312875B (en) | Primer group of loop-mediated isothermal amplification method for detecting porcine circovirus type 3 | |
| CN108048600B (en) | Fluorescent quantitative PCR detection method for infectious bovine rhinotracheitis virus | |
| CN111154903A (en) | Primer group for Brucella RPA amplification, visual detection kit and application | |
| CN103509880A (en) | LAMP detection kit of highly-pathogenic porcine reproductive and respiratory syndrome viruses | |
| CN112725488B (en) | Double fluorescent quantitative PCR (polymerase chain reaction) primer and kit for ehrlichiosis and lyme disease | |
| CN113136459A (en) | LAMP primer for detecting IBRV virus and method thereof | |
| CN110607398B (en) | RT-LAMP kit for fluorescent visual rapid detection of porcine epidemic diarrhea virus | |
| CN110656189B (en) | Fluorescent PCR (polymerase chain reaction) kit for single-tube multiplex rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum | |
| CN109762910B (en) | Primer and kit for simultaneously detecting two types of echinococcosis | |
| CN116656845A (en) | Triple fluorescent quantitative PCR detection kit for diagnosing brucella vaccine immunity and natural infection and detection method thereof | |
| CN115181803A (en) | Taqman probe qPCR detection primer group for detecting chaulmoogra and application | |
| CN107653345B (en) | Real-time fluorescence quantitative PCR method for simian B virus, universal primer, MGB probe and kit thereof | |
| CN107699639B (en) | Primer and method for identifying bovine rotavirus and enterotoxigenic escherichia coli | |
| RU2700449C1 (en) | Method for detecting dna of rhinotracheitis virus (bovine herpes virus 1, bohv-1) in cattle | |
| RU2700477C1 (en) | Test system for detecting the genome of the causative agent of bordetella bronchiseptica dna in farm livestock | |
| CN110373486B (en) | PCR primer, PCR method and kit for detecting staphylococcus cohnii | |
| CN109897907B (en) | RAA fluorescent primer, probe and detection method for detecting borrelia burgdorferi | |
| CN111321246A (en) | Canine distemper virus nano PCR detection kit and application thereof | |
| RU2703405C1 (en) | Method for detection of dna of the bordetlelosis (bordetella bronchiseptica) agent genome in farm animals | |
| CN112725533B (en) | Primer group for real-time fluorescent quantitative PCR identification of duck type 1 adenovirus and duck type 4 adenovirus |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |