CN114875163B - Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe - Google Patents
Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe Download PDFInfo
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- CN114875163B CN114875163B CN202210716891.5A CN202210716891A CN114875163B CN 114875163 B CN114875163 B CN 114875163B CN 202210716891 A CN202210716891 A CN 202210716891A CN 114875163 B CN114875163 B CN 114875163B
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- 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
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- 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 a specific primer and a probe for detecting mouse rhino-rosella and application thereof, wherein the nucleotide sequences of an upstream primer and a downstream primer are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The specific probe and the primer are applied to the preparation of a kit for detecting the mouse rhinobacter bacteria, so that the specific detection of the mouse rhinobacter bacteria is realized. The invention provides a specific primer and a probe for detecting the mouse rhinorrhoea bacteria for the first time, which are used for preparing a kit and detecting the mouse rhinorrhoea bacteria, and the detection is 10 2 copise/. Mu.L to 10 7 Within the interval of copise/mu L, the linear relation is good, the detection method is simple, and the specificity is high.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a specific primer and a probe for detecting mouse rhinobacter and application thereof.
Background
Rochelia (Rothia) is a gram-positive bacterium, facultative anaerobic, non-sporulating, non-motile bacterium. Currently, the genus comprises six species, rothia aeria, rothia amarae, rothia dentocariosa, rothia mucoginosa, rothia nasimurium and Rothia terrae, respectively. Of these, norovirus (r. Nasimurium) was first reported in 2000 and isolated from healthy nasal cavities.
In recent years, with the increasingly serious abuse phenomenon of antibiotics, the problem of bacterial drug resistance becomes a hot point of scientific research, and the increasing of multiple drug-resistant bacteria, particularly the appearance of 'super bacteria', increases the treatment difficulty and the medical cost and further leads to the embarrassing situation that people face no drug and can be saved. The murine rhinococcus has been considered as a symbiotic flora existing in the upper respiratory tract and the intestinal tract of human, poultry and mice, has serious pan-drug resistance and is often infected with staphylococcus aureus in a synergistic manner. The multiple drug resistance and the synergistic infectivity of the murine rhinococcus indicate that the bacterium has the transmission risk, affects the public health and needs to be paid attention. However, the existing method for detecting the norathyriella rhinis is relatively complex, lacks corresponding primers, probes and kits, and needs to be further solved.
Disclosure of Invention
Aiming at the problems that the detection method of the mouse rhinobacter is complex and a corresponding primer, a probe and a kit are lacked in the prior art, the invention provides a specific primer, a probe and application thereof for detecting the mouse rhinobacter and application thereof.
The invention is realized by the following technical scheme:
a specific primer and a probe for detecting the mouse rhinoceros are disclosed, wherein the nucleotide sequences of an upstream primer and a downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
Further, the 5 'end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.
Further, the fluorescent group is FAM, and the quenching group is BHQ1.
In the invention, the specific primer and the probe are applied to the preparation of the kit for detecting the mouse rhinobacter nasi.
In the invention, the specific primer and the probe are applied to the detection of the roseburia murinus.
Further, the PCR amplification reaction system for detecting the mouse rhinotracheitis is 20 mu L, and the PCR amplification reaction system comprises Premix Ex Taq TM 10 μ L, upstream primers 0.2 μ M, 1 μ L, downstream primers 0.2 μ M, 1 μ L, probes 0.1 μ M, 0.5 μ L, template 5 μ L, ddH 2 And O is complemented.
Further, the air conditioner is provided with a fan, the PCR amplification reaction program in the detection of the murine rhinoceros bacteria is 95 ℃ 600s,95 ℃ 15s,60 ℃ 60s and 40 cycles.
Advantageous effects
The invention provides specific primers and probes for detecting the mouse rhinoceros for the first time, can be used for preparing a kit and detecting the mouse rhinoceros, and is 10 DEG 2 copise/. Mu.L to 10 7 Within the interval of copise/mu L, the linear relation is good, the detection method is simple, and the specificity is high.
Drawings
FIG. 1 is a fluorescence quantitative PCR amplification curve of Racing species bacterium murinus;
FIG. 2 shows the standard curve of fluorescence quantitative PCR of Ralstonia murinus.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
The invention provides a specific primer and a probe for detecting mouse rhinorrhea bacteria, wherein the nucleotide sequence is shown in the following table 1, the nucleotide sequences of an upstream primer and a downstream primer are respectively shown in SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the probe is shown in SEQ ID No. 3;
TABLE 1 nucleotide sequence table of specific primers and probes for detecting Ralstonia murinus
The 5 'end of the probe is marked with FAM fluorescent group, and the 3' end is marked with BHQ1 quenching group.
Example 1
Detection of murine rhinobyon (reaction condition optimization):
to make a mouse Bordetella nasalis: (A), (B)Rothia nasimurium) The recombinant plasmid is taken as a template, and fluorescence quantitative PCR detection is carried out;
20 mu L of fluorescent quantitative PCR amplification reaction system, premix Ex Taq is adopted TM 10 μ L, 1 μ L of forward primer, 1 μ L of backward primer, 0.5 μ L of probe, and template: (Rothia nasimuriumRecombinant plasmid of (3) 5. Mu.L, supplemented ddH 2 O to 20 mu L, and respectively optimizing the annealing temperature (56-60 ℃), the cycle number (30-40), the primer concentration (0.2-1.0 mu M) and the probe concentration (0.1-0.5 mu M) by taking the highest fluorescence value and the smallest Ct value as indexes
The optimized 20 μ L optimal fluorescent quantitative PCR amplification reaction system is shown in the following table 2, and the fluorescent quantitative PCR amplification reaction conditions are shown in the following table 3: premix Ex Taq TM 10. Mu.L, 1. Mu.L of forward primer (0.2. Mu.M), 1 μ L of downstream primer (0.2 μ M), 0.5 μ L of probe (0.1 μ M), template: (Rothia nasimuriumRecombinant plasmid of (4) 5. Mu.L of ddH 2 O2.5. Mu.L, annealing temperature 60 ℃, cycle number 40, optimal PCR amplification reaction system and specific amplification curve of PCR method under optimal amplification reaction conditions are shown in FIG. 1.
TABLE 2 fluorescent quantitative PCR amplification reaction System
TABLE 3 fluorescent quantitative PCR amplification reaction conditions
The optimized conditions are adopted to respectively detect nucleic acid samples of the mouse rhinorrhea bacteria, the escherichia coli, the pasteurella, the staphylococcus aureus, the pseudomonas aeruginosa, the klebsiella pneumoniae and the candida albicans, the specificity is evaluated, and the result shows that the detection results of other samples are negative except for the amplified signal of the mouse rhinorrhea bacteria.
Example 2
Establishing a standard curve
Plasmid standards of Rothia nasimurium were diluted 10-fold in a gradient of 1X 10 0 、1×10 1 、1×10 2 、1×10 3 、1×10 4 、1×10 5 、1×10 6 、1×10 7 copies/. Mu.L. The PCR amplification was performed using the optimized system (Table 2) and conditions (Table 3) with dilutions of different concentrations as templates. Finally, taking the logarithm value of the concentration of the plasmid standard substance as an X axis and the Ct value as a Y axis, drawing a standard curve, as shown in figure 2, and displaying the result: ct and copy number of the fluorescent quantitative PCR method to the mouse rhino-rosella is 10 2 copise/. Mu.L to 10 7 Within the interval of copise/muL, the linear relation of the standard curve of Rothia nathimurium is good, and the linear equation of the obtained copy number (X) and the Ct value (Y) is Y = -2.5055X +35.01.
Sequence listing
<110> Shandong province animal epidemic disease prevention and control center (Shandong province zoonosis and zoonosis adjustment monitoring center), qingdao animal protection national engineering research center, inc
<120> specific primer and probe for detecting mouse rhinoroteus bacteria and application thereof
<130> 20220623
<160> 7
<170> PatentIn version 3.5
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<212> DNA
<213> Artificial Sequence
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<223> R.n GroEL1 Forward
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gcaggcaggc gtygata 17
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<212> DNA
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<223> R.n GroEL1 Probe
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agatacaccg tcgttggtaa tg
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<212> DNA
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<223> R.n GroEL1 Reverse
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agcttgctaa cgctgtcaag gtga 24
Claims (7)
1. A specific primer and a probe for detecting Ralstonia murinus are characterized in that the nucleotide sequences of an upstream primer and a downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. The specific primer and probe for detecting the norrhabdus rhinis as claimed in claim 1, wherein the 5 'end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group.
3. The specific primer and probe for detecting norovirus bacterium murinus according to claim 2, wherein the fluorescent group is FAM and the quenching group is BHQ1.
4. An application of the specific primer and probe of 1~3 in preparing the kit for detecting mouse rhinoceros bacteria.
5. Use of specific primers and probes according to any of claims 1~3 for the detection of roseburia murinus for non-disease diagnosis purposes.
6. The use of claim 5, wherein the PCR amplification reaction system for detecting mouse rhinotracheitis is 20 μ L, and comprises Premix Ex Taq TM 10 μ L, upstream primers 0.2 μ M, 1 μ L, downstream primers 0.2 μ M, 1 μ L, probes 0.1 μ M, 0.5 μ L, template 5 μ L, ddH 2 And (4) complementing O.
7. The use according to claim 5, wherein the PCR amplification reaction program for detecting the mouse rhinobacter is 95 ℃ 600s,95 ℃ 15s,60 ℃ 60s and 40 cycles.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210716891.5A CN114875163B (en) | 2022-06-23 | 2022-06-23 | Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe |
| NL2034931A NL2034931A (en) | 2022-06-23 | 2023-05-26 | Specific primer and probe for detecting Rothia nasimurium and application thereof |
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| CN202210716891.5A CN114875163B (en) | 2022-06-23 | 2022-06-23 | Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe |
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| CN114875163A CN114875163A (en) | 2022-08-09 |
| CN114875163B true CN114875163B (en) | 2023-03-24 |
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| WO2012095714A1 (en) * | 2011-01-13 | 2012-07-19 | Bigtec Private Limited | Probes and primers for detecting typhoid |
| CN102286617B (en) * | 2011-07-19 | 2013-06-05 | 中国疾病预防控制中心传染病预防控制所 | Real-time fluorescence quantitative polymerase chain reaction (PCR) kit for incorporeity and rickettsia |
| CN108374051A (en) * | 2018-03-02 | 2018-08-07 | 蔡先全 | Detect the primer of C.perfringens and groEL genes, kit and method in food |
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