CN107142331A - A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae - Google Patents
A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae Download PDFInfo
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- CN107142331A CN107142331A CN201710572457.3A CN201710572457A CN107142331A CN 107142331 A CN107142331 A CN 107142331A CN 201710572457 A CN201710572457 A CN 201710572457A CN 107142331 A CN107142331 A CN 107142331A
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- 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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- 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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Abstract
The present invention provides a kind of primer and probe for being used to detect mycoplasma ovine pneumoniae, and the primer sequence is sense primer：5 ' CTTCGGGACTTATTGGAG 3 ', anti-sense primer：The probe sequences of 5 ' GATGCAAACTGATTTACTTG 3 ' are：5’‑ AAGACCGATTGTCAGGCCGA‑3’.The present invention designs specific primer and probe, first for mycoplasma ovine pneumoniae according to the GenBank KR270152.1 logged in p113 gene ordersp113Gene sets up real time fluorescence quantifying PCR method, and this method specificity is good, sensitivity is high, reproducible, the content available for mycoplasma ovine pneumoniae in detection clinical sample.
Description
Technical field
The present invention relates to a kind of primer and probe for being used to detect mycoplasma ovine pneumoniae, belong to biology field.
Background technology
Mycoplasma ovine pneumoniae (Mycoplasma ovipneumoniae, Mo) and it is to cause sheep and goat mycoplasma
The main pathogen of pneumonia (Mycoplasma pneumonia of goats and sheep, MPGS).MPGS infectiousness is strong, main
Will be by respiratory tract infection, also can vertical transmission, sick sheep and resistance to cross sheep be the sick major source of infection (He Ying etc., 2009;Ten thousand
Unitary etc., 2000).MPGS clinical symptoms show as hyperpyrexia, cough, breathe, it is gradual become thin, serosity occurs for lung and pleura
And fibrinous inflammation, case fatality rate is generally 30%~50%, up to 80% (Yang et al., 2014 having;
Rifatbegovic et al., 2011; Dassnayake et al., 2010; Besser et al., 2008)。
Current foreign countries Africa, Spain, Italy, the U.S., Jordan, domestic Sichuan, Guizhou, the Inner Mongol, Qinghai, Guangxi,
There is the pathogenetic report on the ground such as Fujian.The disease causes serious economic loss to sheep husbandry, therefore, needs foundation badly a kind of
Special, sensitive, the easy detection method of mycoplasma ovine pneumoniae.
TaqMan fluorescent PCRs are on the basis of regular-PCR, while adding a pair of specific primers in reaction system
A specific fluorescence probe is added, the technology of target nucleotide sequences is detected using fluorescent PCR detector, with special
Property strong, sensitivity it is high, it is quantitative accurate the advantages of.Found by consulting literatures, though the current country has for mycoplasma ovine pneumoniae
The real time fluorescence quantifying PCR method of detection, but primarily directed to the primer that 16S rRNA are designed, and only dye method fluorescence
Quantifying PCR method, there is not yet mycoplasma ovine pneumoniaeTaqMan fluorescent quantitative PCR detection methods.The foundation of the present invention can be with
Fill up the blank in the field.
The content of the invention
It is an object of the invention to provide a kind of primer and probe for being used to detect mycoplasma ovine pneumoniae.
To achieve the above object, the present invention uses following technical scheme：
Real-time fluorescence quantitative PCR primer for detecting mycoplasma ovine pneumoniae, the nucleotides sequence of primer is classified as：Sense primer：
5 '-CTTCGGGACTTATTGGAG -3 ', anti-sense primer：5’- GATGCAAACTGATTTACTTG -3’.
Probe for detecting mycoplasma ovine pneumoniae, sequence is： 5’- AAGACCGATTGTCAGGCCGA-3’.
Used probe is that mark fluorescent reporter group is distinguished at two ends（R）And fluorescent quenching group（Q）Oligonucleotides.
It can be used for detection mycoplasma ovine pneumoniae using the primer and probe of the present inventionp113Gene, be particularly suitable for use in base
In the detection of fluorescent quantitative PCR technique.By the optimization to reaction system and reaction condition, set up a kind of available for detection sheep
The quantitative fluorescent PCR method of mycoplasma pneumoniae.
Concrete operation method is as follows：
1st, design of primers：According to the mycoplasma ovine pneumoniae KR270152.1 genome sequences announced on GenBank, utilize
The softwares pair of Primer 6.0p113Gene order carries out primer and probe design, and probe synthesis carries out two ends fluorescence labeling simultaneously,
The fluorescent reporter group of the end of probe 5 ' mark is FAM, and the fluorescent quenching group of 3 ' end marks is Eclipse.
2nd, the optimization of reaction system：25 μ L optimal reaction systems of optimization are：Premix Ex Taq TM （Probe
qPCR）12.5 μ L, upstream and downstream primer（10 μmol/L）Each 0.5 μ L, probe（5 μmol/L）1 μ L, the μ L of template 2, the μ L of water 8.5.
Optimum reaction condition is：95 DEG C, 30s pre-degenerations；95 DEG C of 5s, 55 DEG C of 10s, 72 DEG C of 20s totally 45 circulations.
3rd, the preparation of positive criteria product：By PCR to FJ-CL01 plants of mycoplasma ovine pneumoniaep113Gene is expanded
Increase.20 μ L PCR amplification systems are：PremixTaq TMThe μ L of 2.0 plus dye of Version 10, upstream and downstream primer (10 μ
Mol/ μ L) each μ L of L, FJ-CL plants of DNA of 1 μ 2, sterilizing deionized water mend to 20 μ L.PCR programs：94℃ 2min；94℃ 30s、
55 DEG C of 15s, 72 DEG C of 15s, totally 30 circulations；72℃ 10min.With Gel Extraction kit PCR primer and by purpose
Fragment is connected on pMD19-T carriers, converts bacillus coli DH 5 alpha competent cell, and extracting plasmid does PCR and digestion identification, and
Deliver to precious bioengineering（Dalian）Co., Ltd is sequenced.To be used as standard items by the correct recombinant plasmid of sequencing identification.
4th, the foundation of standard curve：Performing PCR amplification is entered with the reaction system of optimization, using copy number as abscissa, with Ct values
Standard curve is set up for ordinate.
This method can be used for etiological diagnosis and the epidemiology survey of mycoplasma ovine pneumoniae, be that the early prevention of disease is established
Determine technical foundation.
Beneficial effect
The present invention is according to the GenBank KR270152.1's logged inp113Gene order, design specific primer and probe, it is first this
Set up for mycoplasma ovine pneumoniae p113 genesTaqMan real time fluorescence quantifying PCR methods, this method sensitivity is high, it is minimum can
10copies is detected, specificity is good, reproducible, the content available for mycoplasma ovine pneumoniae in detection clinical sample.
Brief description of the drawings
The amplification curve of Fig. 1 mycoplasma ovine pneumoniae real time fluorescence quantifying PCR methods.
Fig. 2 is the standard curve of the real-time fluorescence quantitative PCR of mycoplasma ovine pneumoniae.
Embodiment
The foundation of the fluorescence quantifying PCR method of the mycoplasma ovine pneumoniae of embodiment 1
First, material：
Mycoplasma capri goat pneumonia subspecies are presented by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences Chu Yue peaks, Escherichia coli,
Pasteurella, staphylococcus aureus etc. fly research by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute poultry diease laboratory Cheng Long
Member's present；FJ-CL01 plants of mycoplasma ovine pneumoniae, FJ-GT plants of Mycoplasma mycoides subsp.capri, Lai Shi acholeplasmas FJ-NP
Strain, FJ-HJ plants of Mycoplasma bovis, FJ-FZ plants of sheep of virus are the separation of this room, identification, preserved.
2nd, step
1st, instrument and reagent：PMD19-T carriers, SYBRPremix Ex TaqⅡ（2×）, DL2000 Marker be purchased from treasured
Bioengineering（Dalian）Co., Ltd；Quantitative fluorescent PCR pipe is purchased from Axygen（The U.S.）Company；Glue reclaim kit is purchased from healthy and free from worry
Life science（Wujiang）Co., Ltd；Small amount plasmid extraction agent box is purchased from Omega Bio-Tek（The U.S.）Company；
Mastercycler ep realplex quantitative fluorescent PCR instrument, eppendorf（Germany）Products.
2. the specificity of primer and probe
The specificity of primer and probe is the most important factor of this experiment institute method for building up.Logged according to GenBank
KR270152.1 p113 gene orders, are analyzed by comparing, and design primer and probe.
Primer sequence is：Sense primer：5 '-CTTCGGGACTTATTGGAG -3 ', anti-sense primer：5’-
GATGCAAACTGATTTACTTG -3’。
Probe sequence is： 5’- AAGACCGATTGTCAGGCCGA-3’.
3rd, the preparation of positive criteria product
By PCR to FJ-CL01 plants of mycoplasma ovine pneumoniaep113Gene is expanded.20 μ L PCR amplification systems are：
Premix Taq TMThe μ L of 2.0 plus dye of Version 10, upstream and downstream primer (10 μm of ol/ μ L) each L, FJ-CL plants of DNA2 μ of 1 μ
L, sterilizing deionized water are mended to 20 μ L.PCR programs：94℃ 2min；94 DEG C of 30s, 55 DEG C of 15s, 72 DEG C of 15s, totally 30 are followed
Ring；72℃ 10min.It is connected on pMD19-T carriers, converts with Gel Extraction kit PCR primer and by purpose fragment
Bacillus coli DH 5 alpha competent cell, extracting plasmid does PCR and digestion identification, and delivers to precious bioengineering（Dalian）Co., Ltd
Sequencing.To be used as standard items by the correct recombinant plasmid of sequencing identification.
4. the optimization of reaction condition
Using 25 conditioned response systemsPremix Ex Taq TM （Probe qPCR）12.5 μ L, upstream and downstream primer（10 μmol/
L）Each 0.5 μ L, probe（5 μmol/L）The μ L of positive criteria product 2 after 1 μ L, doubling dilution, moisturizing to the μ L of final volume 25, with
The Ct values for highest fluorescent value and minimum occur are index, respectively to annealing temperature（51~60 DEG C）, primer and probe concentration（5~
10 μmol/L）Optimize.
5th, standard curve is set up
With EASY Dilution by the standard items gradient dilution (10 of structure9、108、107、106、105、104、103、102、
101Copies/ μ L) as template, expanded, using copy number as abscissa, built using Ct values as ordinate with the condition of optimization
Day-mark directrix curve.
Fluorescence quantifying PCR method is to mycoplasma ovine pneumoniae to be shown to the amplification curve and standard curve of positive criteria product
Lowest detection be limited to 10copies, and Ct with copy number 1 × 101~1 × 109Have linear well in the range of copy/μ L
Relation, coefficient R2For 0.999, amplification efficiency is 102%.Amplification curve is shown in Fig. 1, and standard curve is shown in Fig. 2.
6 specific detections
6.1 specific detection
With the condition of optimization respectively to mycoplasma ovine pneumoniae, mycoplasma capri goat pneumonia subspecies, Escherichia coli, Pasteur's bar
Bacterium, staphylococcus aureus, haemophilus parasuis, Lai Shi acholeplasmas, Mycoplasma bovis, Mycoplasma mycoides subsp.capri and sheep
Blue tongue virus nucleic acid samples are detected that the specificity to this method is evaluated.Testing result shows, only pneumonia of sheep branch
Pathogen nucleic acid sample is the positive, and remaining nucleic acid samples testing result is feminine gender.
6.2 repeatabilities and repeatability are assessed
3 repeating pipes are set to same positive criteria product, are detected with the real-time fluorescence quantitative PCR of foundation, its repeatability is evaluated,
The coefficient of variation in calculating group；Positive criteria product is placed in -20 DEG C of preservations, examined again respectively at the 7th, 14,21d, its reproduction is evaluated
Property, calculate its between-group variation coefficient.Calculate group in a coefficient of variation be 0.58%~1.29%, between-group variation coefficient be 0.19%~
1.22%, favorable repeatability.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Fujian Academy animal and veterinary research institute
<120>A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
cttcgggact tattggag 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gatgcaaact gatttacttg 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
aagaccgatt gtcaggccga 20
Claims (3)
1. a kind of primer for being used to detect mycoplasma ovine pneumoniae, it is characterised in that：The primer sequence is：
Sense primer：5 '-CTTCGGGACTTATTGGAG -3 ',
Anti-sense primer：5’- GATGCAAACTGATTTACTTG -3’.
2. a kind of be used to detect the probe of mycoplasma ovine pneumoniae, it is characterised in that the probe sequence is：
5’- AAGACCGATTGTCAGGCCGA-3’。
3. a kind of kit for being used to detect mycoplasma ovine pneumoniae, it is characterised in that：The kit comprising claim 1 and
Primer and probe described in 2.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710572457.3A CN107142331A (en) | 2017-07-14 | 2017-07-14 | A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae |
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| CN201710572457.3A CN107142331A (en) | 2017-07-14 | 2017-07-14 | A kind of primer and probe for being used to detect mycoplasma ovine pneumoniae |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502676A (en) * | 2017-10-20 | 2017-12-22 | 福建省农业科学院畜牧兽医研究所 | A kind of primer for being used to detect mycoplasma ovine pneumoniae |
| CN107988340A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of PCR amplification primer of quick detection mycoplasma ovine pneumoniae and its application |
| CN108823324A (en) * | 2018-06-12 | 2018-11-16 | 福建省农业科学院畜牧兽医研究所 | Mycoplasma ovine pneumoniae and Mycoplasma mycoides subsp.capri double fluorescent quantitative method |
| CN112760392A (en) * | 2020-11-17 | 2021-05-07 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
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| US7889334B2 (en) * | 2005-03-15 | 2011-02-15 | University Of Georgia Research Foundation, Inc. | Surface enhanced Raman spectroscopy (SERS) systems for the detection of bacteria and methods of use thereof |
| CN106434994A (en) * | 2016-11-30 | 2017-02-22 | 石河子大学 | Rapid detecting mycoplasma ovipneumoniae method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502676A (en) * | 2017-10-20 | 2017-12-22 | 福建省农业科学院畜牧兽医研究所 | A kind of primer for being used to detect mycoplasma ovine pneumoniae |
| CN107988340A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of PCR amplification primer of quick detection mycoplasma ovine pneumoniae and its application |
| CN107988340B (en) * | 2017-12-13 | 2021-05-14 | 广西壮族自治区兽医研究所 | PCR amplification primer for rapidly detecting mycoplasma ovipneumoniae and application thereof |
| CN108823324A (en) * | 2018-06-12 | 2018-11-16 | 福建省农业科学院畜牧兽医研究所 | Mycoplasma ovine pneumoniae and Mycoplasma mycoides subsp.capri double fluorescent quantitative method |
| CN112760392A (en) * | 2020-11-17 | 2021-05-07 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
| CN112760392B (en) * | 2020-11-17 | 2021-08-27 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma ovipneumoniae and application |
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