CN114480663A - Primer pair, kit and detection method for detecting swine-origin differential ureaplasma - Google Patents
Primer pair, kit and detection method for detecting swine-origin differential ureaplasma Download PDFInfo
- Publication number
- CN114480663A CN114480663A CN202111346284.6A CN202111346284A CN114480663A CN 114480663 A CN114480663 A CN 114480663A CN 202111346284 A CN202111346284 A CN 202111346284A CN 114480663 A CN114480663 A CN 114480663A
- Authority
- CN
- China
- Prior art keywords
- ureaplasma
- swine
- primer pair
- differential
- primer
- 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
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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a fluorescent quantitative PCR primer pair for detecting swine-origin differential ureaplasma, which has the following nucleotide sequence: upstream primer P1: 5'-CGCGTAGCTGTACTGAGAGG-3', respectively; the downstream primer P2: 5'-CACGCGGCATTGCTTCATAA-3' are provided. The invention also provides a non-diagnostic purpose method for detecting the fluorescent quantitative PCR of the swine-origin differential ureaplasma based on the primer pair and application. The invention has the advantages of high detection accuracy of more than 85 percent, good specificity, low detection limit of 10 copies/mu L, capability of avoiding false negative and easy application and popularization. The method has the advantages of simple operation, high detection speed, no need of specific fluorescent labeled probes and low detection cost, and can be finished within 2 hours. The invention can effectively distinguish the swine-origin differential ureaplasma urealyticum from a plurality of common microorganisms, and has good application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of molecular biology detection, and specifically relates to a primer pair, a kit and a detection method for detecting swine-origin differential ureaplasma.
Background
Differential Ureaplasma (u.divesum) belongs to the genus Ureaplasma of the mycoplasma family, and was first found in the urogenital tract of the bovine population canada.
Early detection of U.Diversum depends on traditional bacteria isolation and culture technology, but because the bacterial colony formed by the bacteria is small (10-17 mu m) and invisible to naked eyes, the bacteria is dead due to severe culture conditions, high nutritional requirements and ammonia generated by self metabolism, the bacteria is difficult to subculture, and the separation rate and the detection accuracy are poor.
The development of molecular biology technology provides convenience for the detection of the pathogen, and improves the accuracy of clinical detection. Vasconce cells Cardoso M et al (Vet Microbiol. 2000; 72: 241-. Marques LM et al (Vet Microbiol.2013; 167: 670-. Even so, the positive detection rate of the bovine U.Diversum by people is still not ideal, and practical application is difficult.
Lobo, E et al (Rev. Salud anim. Vol.35No.1(2013):69), found porcine source U.Diversum for the first time in 2013. Later, the number of U.Diversium is very small, although it is reported in swine source. This leaves the understanding of swine source u.diversum still in the grope stage.
At present, people have not established an accurate and effective Real-time qPCR detection method for swine U.Diversium. Therefore, on the basis that a satisfactory positive detectable rate cannot be obtained by the Real-time qPCR detection method for bovine U.Diversum, it is particularly difficult to establish the Real-time qPCR detection method capable of efficiently detecting porcine U.Diversum.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fluorescent quantitative PCR primer pair for detecting the swine-origin differential ureaplasma, provide a method and a kit for detecting the non-diagnostic purpose of the fluorescent quantitative PCR of the swine-origin differential ureaplasma based on the primer pair, and provide the application of the primer pair for the non-diagnostic purpose in the aspect of detecting the swine-origin differential ureaplasma by using the fluorescent quantitative PCR method.
The invention aims to achieve the aim that the detection method based on the provided primer pair can enable the detection positive rate to reach more than 85 percent and has high specificity, and simultaneously can reduce the detection limit to 10 copies/. mu.L.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a fluorescent quantitative PCR primer pair for detecting swine-origin differential ureaplasma, wherein the nucleotide sequence of the primer pair is shown as follows:
upstream primer P1: 5'-CGCGTAGCTGTACTGAGAGG-3' (SEQ ID NO:1)
The downstream primer P2: 5'-CACGCGGCATTGCTTCATAA-3' (SEQ ID NO: 2).
Although some PCR detection methods are established for bovine-derived U.Diversium, as mentioned in the background art, the existing detection methods generally have the problem of poor accuracy and are difficult to be practically applied, wherein the key reason may be that an optimal primer pair is not designed yet. In the research process, a great deal of research is carried out on the primer pair of the swine U.Diversum. As shown in several comparative examples of the present invention, the primer pair designed in the investigation stage of the present invention generally has the problem of poor specificity, and the pig source u.diversum cannot be detected, not to mention that higher detection accuracy is obtained.
After repeated research, the inventors found that the primer pair of the present invention has high specificity to swine u.diversum. In addition, surprisingly, the accuracy of over 85 percent can be obtained by detecting the swine U.Diversum by using the primer pair disclosed by the invention, and the primer pair has a good application value; meanwhile, an excellent detection limit can be obtained, and false positive can be avoided to the greatest extent.
It is readily found that the present invention contributes to the prior art at least in that: compared with the prior art that only about 40-50% of detection accuracy can be obtained, the detection level of U.D. is remarkably improved; and a PCR detection method aiming at the swine U.Diversum and having high practical application value is obtained for the first time.
A method for detecting the non-diagnostic purpose of fluorescent quantitative PCR of swine-origin differential ureaplasma is carried out by applying the primer pair; the method comprises the following steps: extracting DNA of a sample to be detected; taking a sample DNA to be detected as a template, and carrying out PCR amplification by using the primer pair; recording the PCR amplification cycle number of each detection sample; and (4) judging the copy number of the swine source differential ureaplasma in the sample to be detected.
As an embodiment of the present invention, when PCR amplification is performed, the PCR reaction solution comprises a fluorescent quantitative PCR reagent, a DNA template, the primer pair and ddH2O。
As a preferred technical scheme of the invention, the fluorescent quantitative PCR reagent is 2 XTB Green Premix Ex Taq II.
In a preferred embodiment of the present invention, when performing PCR amplification, the reaction procedure is: denaturation at 95 ℃ for 30s, annealing at 95 ℃ for 5s, extension at 58 ℃ for 30s, and cyclic amplification for 40 times.
As one example of the present invention, in the case of PCR amplification, the PCR reaction solution is composed of 12.5. mu.L of 2 XTB Green Premix Ex Taq II, 2.0. mu.L of the upstream primer P11.0. mu.L of 100 nmol/. mu.L, 2.0. mu.L of the downstream primer P21.0. mu. L, DNA template of 100 nmol/. mu.L and the remaining volume of ddH per 25. mu.L of the PCR reaction solution2And (C) O.
And when the copy number of the swine-origin differential ureaplasma in the sample to be detected is judged, the judgment is carried out through the established standard curve.
Based on the primer pair, the invention also provides a fluorescent quantitative PCR detection kit for detecting the swine-origin differential ureaplasma, wherein the kit contains the primer pair as claimed in claim 1.
Based on the primer pair, the invention also provides the application of the primer pair in the aspect of detecting the swine-origin differential ureaplasma by using a fluorescent quantitative PCR method for non-diagnostic purposes.
For example, the use consists in: distinguishing swine-origin differential ureaplasma urealyticum from other pathogens and/or common pathogens by using the primers based on a fluorescent quantitative PCR method; the other pathogens are at least one of a blue-ear virus, a pseudorabies virus, a haemophilus parasuis, a streptococcus and a mycoplasma; the common pathogen is at least one of salmonella, escherichia coli and staphylococcus aureus.
The invention has the beneficial effects that:
1. the invention establishes a fluorescent quantitative PCR method for detecting the swine-origin differential ureaplasma, the detection accuracy of the method can reach more than 85 percent, the specificity is good, the detection limit is as low as 10 copies/mu L, the occurrence of false negative can be avoided, and the method is easy to apply and popularize.
2. The method has the advantages of simple operation, high detection speed, and low detection cost, and the whole operation process is within 2 hours, so that the detection time is greatly shortened, and meanwhile, a specific fluorescent labeled probe is not needed.
3. The invention can effectively distinguish the swine-origin differential ureaplasma urealyticum from microorganisms such as the blue-ear virus, the pseudorabies virus, the haemophilus parasuis, the streptococcus, the mycoplasma, the salmonella, the escherichia coli, the staphylococcus aureus and the like, and has good application value.
Drawings
FIG. 1 is a graph showing the results of a melting curve of a standard plasmid in example 1 of the present invention;
FIG. 2 is a graph showing the results of an amplification curve of a standard plasmid in example 1 of the present invention;
FIG. 3 is a graph showing the results of a calibration curve of a standard plasmid in example 1 of the present invention;
FIG. 4 is a graph showing the results of the specificity test in example 1 of the present invention;
FIG. 5 is a graph showing the results of the fluorescent quantitative PCR sensitivity test in example 1 of the present invention;
FIG. 6 is a graph showing the results of the fluorescent quantitative PCR assay of samples in example 1 of the present invention;
FIG. 7 is a verification diagram showing the specificity of the primers UD16S _124F/R and UD134_ 1F/R; wherein a1-a 10: amplifying a primer UD16S _ 124F/R; b1-b 10: amplifying a primer UD134_ 1F/R;
FIG. 8 is a verification chart of the specificity of the primer UD 16S-71F/R, UD 16S-87F/R; wherein, C1-C10: amplifying a primer UD 16S-87F/R; d1-d 10: amplifying a primer UD16S _ 71F/R; FIG. 9 is a diagram showing the verification of the specificity of the primer UD 16S-124F/R and the probe.
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
1. Establishment of swine-origin differential ureaplasma fluorescence quantitative PCR detection method
(1) Design of fluorescent quantitative PCR primer pair for detecting swine-origin differential ureaplasma
According to the gene sequence of the swine-origin differential ureaplasma genome 16S rRNA, a pair of primers is designed aiming at a conserved region, and the sequences are as follows:
upstream primer P1: 5'-CGCGTAGCTGTACTGAGAGG-3' (SEQ ID NO:1)
The downstream primer P2: 5'-CACGCGGCATTGCTTCATAA-3' (SEQ ID NO:2)
The expected amplified fragment size was 124 bp.
(2) Preparation of recombinant plasmid Standard
Extracting genome DNA of the swine-origin differential ureaplasma urealyticum according to the instruction of a DNA extraction kit, carrying out PCR amplification by using the upstream primer P1 and the downstream primer P2 designed in the step (1) by using the genome DNA as a template, cloning the PCR product in a pUC57 vector to construct a recombinant plasmid after recovering and purifying the PCR product, and displaying that the PCR product is 124bp through PCR identification and sequencing.
Taking the recombinant plasmid as a standard substance of fluorescence quantitative PCR, determining the concentration of the positive recombinant plasmid to be 100 ng/mu L, and according to a conversion formula of copy number of plasmid per microliter:
(3) Fluorescent quantitative PCR amplification and result analysis
A PCR reaction solution was prepared in the following composition with reference to the product manual of TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820A, Takara). Using a 25. mu.L PCR reaction system, 2 XTB Green Premix Ex Taq II 12.5. mu.L, upstream primer P1(100 nmol/. mu.L) and downstream primer P2(100 nmol/. mu.L) each 1.0. mu.L, nucleic acid template 2.0. mu.L, ddH, were added to 0.2mL eight-linked tubes2O8.5. mu.L. The PCR amplification reaction program is as follows: denaturation at 95 ℃ for 30 s; followed by annealing at 95 ℃ for 5s, extension at 58 ℃ for 30s (fluorescence was collected), and amplification was cycled 40 times.
Melting curve analysis, procedure is: 15s at 95 ℃; 15s at 60 ℃; dissolution curve analysis was performed at 60 ℃ to 95 ℃ at a melting rate of 0.02 ℃/s.
The results are shown in FIG. 1, and the peaked melting curve graph shows that a single peak Tm value range is formed, which is 86.17 + -0.22 ℃, and non-specific amplification is not caused.
(4) Establishment of a Standard Curve
8 dilution gradients (5.35X 10) of recombinant plasmids were selected8Copy/. mu.L-5.35X 101Copy/. mu.L), each dilution is repeated three times, plasmid DNA of each concentration gradient is used as a template, and the established fluorescent quantitative PCR is adopted for amplification to obtain a kinetic amplification curve (figure 2), so that a standard curve is generated.
The results are shown in FIG. 3, where the standard curve equation is y-3.607x +35.11, correlation coefficient R20.999; the results showed a good linear relationship.
(5) Specificity test
The specificity of the method is verified by carrying out fluorescent quantitative PCR reaction by using the DNA of the infected material for diagnosing the swine source differential urea protozoon, the wild strain of the mycoplasma hyopneumoniae (owned by the inventor's laboratory), the vaccine strain of the mycoplasma hyopneumoniae (168 strain), the haemophilus parasuis (serotype 4), the streptococcus (serotype 2), the pseudorabies virus (Bartha-K61 strain), the salmonella (ATCC13076), the escherichia coli (ATCC25922) and the staphylococcus aureus (ATCC6538) and the cDNA of the blue ear virus (JXA1-R strain) as templates.
As shown in FIG. 4, only the swine-origin differential ureaplasma infection pathogens showed amplification curves, and none of the other pathogenic nucleic acids were amplified. Indicating that the established method has good specificity.
(6) Sensitivity test
Recombinant plasmid was selected for 7 dilution gradients (10)7Copy/. mu.L-101Copy/. mu.L), using plasmid DNA of each concentration gradient as a template, and performing amplification by using established fluorescent quantitative PCR to determine the lowest detection limit, and determining a standard to form a kinetic amplification curve.
As shown in FIG. 5, the fluorescent quantitative PCR amplification showed a clear amplification curve at template concentrations as low as 10 copies/. mu.L.
2. Sample testing experiment
ddH using pig-derived heteroureaplasma as positive control2And O is negative control, and 21 samples of lung tissues and lung lavage fluid are detected by adopting the fluorescent quantitative PCR method for quantitatively detecting the swine-origin differential ureaplasma established in the research.
The specific detection method comprises the following steps:
(1) extracting DNA of a sample to be detected: according to a DNA extraction kit;
(2) taking a sample DNA to be detected as a nucleic acid template, carrying out fluorescent PCR amplification by using an upstream primer P1 and a downstream primer P2 designed in the step (1) of the experiment part 1, wherein the fluorescent quantitative PCR amplification refers to a product instruction of TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820A, Takara) and the PCR is prepared according to the following componentsReaction solution: using a 25. mu.L PCR reaction system, 2 XTB Green Premix Ex Taq II 12.5. mu.L, 1.0. mu.L each of the upstream primer P1(100 nmol/. mu.L) and the downstream primer P2(100 nmol/. mu.L), 2.0. mu.L of the nucleic acid template, and ddH were added to 0.2mL eight tubes2O8.5. mu.L. The PCR amplification reaction program is as follows: 30s at 95 ℃; followed by denaturation at 95 ℃ for 30 s; followed by annealing at 95 ℃ for 5s, extension at 58 ℃ for 30s (fluorescence was collected), and amplification was cycled 40 times.
(3) And (3) judging standard: the positive sample is judged as the sample with obvious amplification curve and Ct value less than 35. And calculating the copy number of the sample to be detected according to the Ct value which is the established standard curve.
The sample test results are shown in fig. 6, and the fluorescence quantitative PCR detection positive rate of 21 lung tissue and lung lavage fluid samples is 85.71%, which indicates that the detection method has high sensitivity and can obviously reduce the false negative of clinical detection.
Comparative example 1
Sequencing aiming at microbial diversity to obtain partial fragments of the porcine differential ureaplasma hyoscyami 16S rRNA gene, and designing 4 pairs of specific primers by using primer 5.0 (Table 1).
TABLE 1 porcine differential Urea primers sequence information Table
And performing primer specificity verification by taking the DNAs of the swine-origin differential ureaplasma hyopneumoniae, the wild strain of the mycoplasma hyopneumoniae, the vaccine strain of the mycoplasma hyopneumoniae, the haemophilus parasuis, the streptococcus, the pseudorabies virus, the salmonella, the escherichia coli and the staphylococcus aureus and the cDNA of the porcine reproductive and respiratory syndrome virus as templates. PCR reaction solutions were prepared according to the following composition and experiments were carried out on 4 sets of primer pairs (UD 16S-71F/R, UD 16S-124F/R, UD 16S-87F/R and UD 134-1F/R) with reference to the product manual of "Primulaceae gold Mix (green)". Using a 20. mu.L PCR reaction system, 10. mu.L of 2 Xgold Mix (green), 1.0. mu.L each of the upstream primer (100 nmol/. mu.L) and the downstream primer, 2.0. mu.L of the nucleic acid template, and ddH were added to a 0.2mL EP tube2O6. mu.L. The PCR amplification reaction program is as follows: 2min at 98 ℃; performing cyclic amplification for 30 times at 98 ℃ for 10s, Tm for 10s and 72 ℃ for 10 s; 72 ℃ for 2 min.
As shown in FIGS. 7 and 8, the UD16S _71F/R, UD16S _87F/R and UD134_1F/R primers have poor specificity, and are not suitable for identifying swine-origin differential ureaplasma.
Comparative example 2
A partial fragment of the porcine-derived differential ureaplasma hyorhinis 16S rRNA gene was obtained by sequencing for microbial diversity, and primers and probes were designed using Primer Express 3.0 (Table 2).
TABLE 2 pig origin differential ureaplasma urealyticum primer and Probe sequence information Table
And performing primer and probe specificity verification by taking the DNAs of the swine-origin differential ureaplasma hyopneumoniae, the wild strain of the mycoplasma hyopneumoniae, the vaccine strain of the mycoplasma hyopneumoniae, the haemophilus parasuis, the streptococcus, the pseudorabies virus, the salmonella, the escherichia coli and the staphylococcus aureus and the cDNA of the porcine reproductive and respiratory syndrome virus as templates. A PCR reaction solution was prepared in the following composition with reference to Premix Ex TaqTM (Probe qPCR), product manual of Bulk (RR390L, Takara). Using a 20. mu.L PCR reaction system, 2 XPrimx Ex TaqTM 10. mu.L, 0.4. mu.L each of the forward primer (100 nmol/. mu.L) and the reverse primer (100 nmol/. mu.L), 0.8. mu.L of the probe (100 nmol/. mu.L), 2.0. mu.L of the nucleic acid template, and ddH2O 6.4.4. mu.L were placed in 0.2mL eight tubes. The PCR amplification reaction program is as follows: 30s at 95 ℃; subsequently, amplification was performed 45 times in cycles at 95 ℃ for 5 seconds and at 58 ℃ for 30 seconds (fluorescence was collected).
The results are shown in FIG. 9, which shows that the probe has poor specificity and is not suitable for identifying porcine-derived differential ureaplasma.
Sequence listing
<110> animal husbandry veterinary institute of Guizhou province
<120> primer pair, kit and detection method for detecting swine-origin differential ureaplasma urealyticum
<141> 2021-11-15
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (10)
1. A fluorescent quantitative PCR primer pair for detecting swine-origin differential ureaplasma is characterized in that the nucleotide sequence of the primer pair is as follows:
upstream primer P1: 5'-CGCGTAGCTGTACTGAGAGG-3'
The downstream primer P2: 5'-CACGCGGCATTGCTTCATAA-3' are provided.
2. A method for the non-diagnostic purpose of fluorogenic quantitative PCR for the detection of porcine-derived differential ureaplasma, characterized in that it is carried out using the primer pair according to claim 1; the method comprises the following steps: extracting DNA of a sample to be detected; taking a sample DNA to be detected as a template, and carrying out PCR amplification by using the primer pair; recording the PCR amplification cycle number of each detection sample; and judging the copy number of the swine-origin differential ureaplasma in the sample to be detected.
3. The method of claim 2, wherein the PCR reaction solution comprises a fluorescent quantitative PCR reagent, a DNA template, the primer pair and ddH when PCR amplification is performed2O。
4. The method of claim 3, wherein the fluorescent quantitative PCR reagent is 2 XTB Green Premix Ex Taq II.
5. The method of claim 4, wherein the reaction sequence is: denaturation at 95 ℃ for 30s, annealing at 95 ℃ for 5s, extension at 58 ℃ for 30s, and cyclic amplification for 40 times.
6. The method according to claim 5, wherein the PCR reaction solution comprises 2 XTB GreenPremix Ex Taq II 12.5. mu.L, upstream primer P11.0. mu.L at a concentration of 100 nmol/. mu.L, downstream primer P21.0. mu. L, DNA template 2.0. mu.L at a concentration of 100 nmol/. mu.L and ddH in a residual volume per 25. mu.L2And (C) O.
7. The method according to any one of claims 2 to 6, wherein the determination of the copy number of the swine-origin differential ureaplasma in the test sample is performed by using a standard curve established.
8. A fluorescent quantitative PCR detection kit for detecting swine-origin differential ureaplasma, characterized by comprising the primer pair according to claim 1.
9. Use of a primer pair according to claim 1 for non-diagnostic purposes in the detection of swine-origin differential ureaplasma using a fluorescent quantitative PCR method.
10. Use according to claim 9, characterized in that it consists in distinguishing between differential ureaplasma hyorhinis and other and/or common pathogens on the basis of a fluorescent quantitative PCR method using said primers; the other pathogen is at least one of a blue ear virus, a pseudorabies virus, haemophilus parasuis, streptococcus and mycoplasma; the common pathogen is at least one of salmonella, escherichia coli and staphylococcus aureus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111346284.6A CN114480663A (en) | 2021-11-15 | 2021-11-15 | Primer pair, kit and detection method for detecting swine-origin differential ureaplasma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111346284.6A CN114480663A (en) | 2021-11-15 | 2021-11-15 | Primer pair, kit and detection method for detecting swine-origin differential ureaplasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114480663A true CN114480663A (en) | 2022-05-13 |
Family
ID=81492107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111346284.6A Pending CN114480663A (en) | 2021-11-15 | 2021-11-15 | Primer pair, kit and detection method for detecting swine-origin differential ureaplasma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480663A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014058876A1 (en) * | 2012-10-08 | 2014-04-17 | Cornell University | Compositions and methods for diagnosis and prophylaxis of mastitis in ruminants |
| WO2014088088A1 (en) * | 2012-12-07 | 2014-06-12 | 三菱化学メディエンス株式会社 | Method for detecting and identifying bacterium belonging to genus mycoplasma or ureaplasma |
| CN106399559A (en) * | 2016-11-16 | 2017-02-15 | 广东省农业科学院动物卫生研究所 | Primer pair and kit for detecting mycoplasma hyopneumoniae and Syto9 fluorescent quantitative PCR-HRM detecting method |
| CN108379576A (en) * | 2010-12-22 | 2018-08-10 | 拜耳知识产权有限责任公司 | The immune response enhanced in ox species |
| JP2018126089A (en) * | 2017-02-08 | 2018-08-16 | 学校法人日本大学 | Primer sets for detecting ureaplasma bacteria and uses thereof |
| RU2683029C1 (en) * | 2018-06-01 | 2019-03-26 | Федеральное бюджетное учреждение науки "Санкт-Петербургский научно-исследовательский институт эпидемиологии и микробиологии им. Пастера Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека" (ФБУН НИИ эпидемиологии и микробиологии имени Пастера) | Method for detecting and quantifying dna content of ureaplasma diversum by pcr method in real time in adult cattle material |
-
2021
- 2021-11-15 CN CN202111346284.6A patent/CN114480663A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379576A (en) * | 2010-12-22 | 2018-08-10 | 拜耳知识产权有限责任公司 | The immune response enhanced in ox species |
| WO2014058876A1 (en) * | 2012-10-08 | 2014-04-17 | Cornell University | Compositions and methods for diagnosis and prophylaxis of mastitis in ruminants |
| WO2014088088A1 (en) * | 2012-12-07 | 2014-06-12 | 三菱化学メディエンス株式会社 | Method for detecting and identifying bacterium belonging to genus mycoplasma or ureaplasma |
| CN106399559A (en) * | 2016-11-16 | 2017-02-15 | 广东省农业科学院动物卫生研究所 | Primer pair and kit for detecting mycoplasma hyopneumoniae and Syto9 fluorescent quantitative PCR-HRM detecting method |
| JP2018126089A (en) * | 2017-02-08 | 2018-08-16 | 学校法人日本大学 | Primer sets for detecting ureaplasma bacteria and uses thereof |
| RU2683029C1 (en) * | 2018-06-01 | 2019-03-26 | Федеральное бюджетное учреждение науки "Санкт-Петербургский научно-исследовательский институт эпидемиологии и микробиологии им. Пастера Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека" (ФБУН НИИ эпидемиологии и микробиологии имени Пастера) | Method for detecting and quantifying dna content of ureaplasma diversum by pcr method in real time in adult cattle material |
Non-Patent Citations (2)
| Title |
|---|
| LUCAS M. MARQUES等: "A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum", VETERINARY MICROBIOLOGY, vol. 167, pages 670 - 674 * |
| 张永强等: "牛支原体肺炎流行RT-PCR 方法的初步诊断", 中国动物检疫, vol. 28, pages 48 - 55 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| 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 | |
| CN111705163A (en) | Novel coronavirus rapid detection kit based on thermal convection PCR | |
| CN110735006A (en) | African swine fever TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and kit | |
| CN110305975B (en) | RPA kit for rapidly detecting mycoplasma synoviae and application thereof | |
| CN109371148B (en) | Fluorescent PCR kit for identifying three porcine respiratory bacteria and quantitative detection method | |
| CN112941211A (en) | Multiplex fluorescence quantitative PCR detection kit for streptococcus suis type 2 virulence genes and detection method thereof | |
| CN112592992A (en) | Primer probe set and kit for combined detection of mycoplasma pneumoniae and chlamydia pneumoniae based on fluorescent RMA method | |
| CN113862393A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN110699470A (en) | Dual PCR primer, kit, application and method for detecting salmonella and identifying pullorum disease/gallinarum serotype | |
| CN114480663A (en) | Primer pair, kit and detection method for detecting swine-origin differential ureaplasma | |
| CN113186359B (en) | Multiplex PCR rapid diagnosis kit for detecting and parting pig astrovirus | |
| JP3525259B2 (en) | Detection of Pectinatus spp. | |
| CN112195258B (en) | Multiplex PCR detection kit for multiple pathogenic bacteria of waterfowl and application thereof | |
| CN113430288A (en) | Composite PCR typing kit for distinguishing three streptococcus suis serotypes and application thereof | |
| CN109628621B (en) | Real-time quantitative LAMP primer group and kit for detecting Klebsiella pneumoniae | |
| CN109666750B (en) | Multiplex real-time fluorescence PCR detection primer probe set and detection method for identifying streptococcus suis and pasteurella multocida | |
| CN107604102B (en) | Double Real time PCR detection kit for pigeon TTV and novel pigeon adenovirus | |
| K Ibraheim et al. | Conventional and molecular detection of Pasteurella multocida in outbreak of respiratory tract infection of sheep and goats in Basrah Province | |
| CN113718044A (en) | Primer, probe and kit for fluorescent quantitative detection of swine mycoplasma, mycoplasma microformis and new mycoplasma haemophilus | |
| KR20090122554A (en) | Primer set and probe for detecting salmonella typhimurium | |
| CN111020040A (en) | Multiple fluorescence quantitative PCR detection primer group and kit for pathogenic bacteria in dairy products and application of primer group and kit | |
| CN110499374B (en) | Triple PCR primer group for detecting Klebsiella pneumoniae, kit and application | |
| CN108753999B (en) | Nucleic acid, kit and detection method for detecting salmonella paratyphi of pigeon | |
| CN110512017B (en) | Mouse corynebacterium fluorescent quantitative PCR detection kit and primer |
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 |