CN106676195A - Fluorescent quantitative PCR detection primer for mycoplasma mycoides subsp. capri - Google Patents
Fluorescent quantitative PCR detection primer for mycoplasma mycoides subsp. capri Download PDFInfo
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- CN106676195A CN106676195A CN201710135665.7A CN201710135665A CN106676195A CN 106676195 A CN106676195 A CN 106676195A CN 201710135665 A CN201710135665 A CN 201710135665A CN 106676195 A CN106676195 A CN 106676195A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention provides a fluorescent quantitative PCR detection primer for mycoplasma mycoides subsp. capri. The primer has the following sequences: sense primer: 5'-TGTTGTTGGTGGGTCTGCTT-3', and anti-sense primer: 5'-ACTTTATCAGCCGCCATTTGA-3'. Based on the MMLC_1770 gene sequence of FQ377874.1 in GenBank entry, the invention designs the specific primer, and establishes the SYBR Green I real-time fluorescent quantitative PCR method for mycoplasma mycoides subsp. capri for the first time at home and abroad. The method has good specificity, high sensitivity and good repeatability, and can be used for detecting the content of mycoplasma mycoides subsp. capri in a clinical sample.
Description
Technical field
The present invention relates to a kind of real-time fluorescence quantitative PCRs of SYBR Green I for Mycoplasma mycoides subsp.capri are detected
Primer, belongs to biology field.
Background technology
Mycoplasma mycoides subsp.capri is one of member of thread mycoplasma cluster, be sheep mycoplasmal pneumonia cause of disease it
One.The incubation period average out to 18-20d of sheep mycoplasmal pneumonia, most fast 3-6d, up to 30-40d.The main table of its Clinical symptoms
Be now hyperpyrexia, gradual become thin and pulmonary parenchyma, leaflet interstitial and pleura occur serosity and fibrinous inflammation, lung of coughing
There is obvious liver to be changed into the breathing problem of principal character, can additionally cause mammitis, keratoconjunctivitis and septicaemia etc..Should
The incidence of disease of disease is 19%-90%, and the death rate is up to 40%.The ground such as current country Guizhou, Chongqing, the Inner Mongol, Qinghai, Guangxi, Fujian
There is the pathogenetic report.
At present the diagnostic method of sheep mycoplasmal pneumonia is less, and this belongs to silk mainly due to Mycoplasma mycoides subsp.capri
Shape mycoplasma cluster member, homology is higher between thread mycoplasma cluster member, there is cross reaction in serology.Currently-established
Diagnostic method mainly has Isolation and culture of agent identification, PCR etc..The former wastes time and energy, and the latter can not be quantitative.
The real-time fluorescence quantitative PCRs of SYBR Green I are on the basis of regular-PCR, fluorescent dye to be added in reaction system, using glimmering
Light PCR detectors detecting the technology of target nucleotide sequences, have the advantages that high specificity, it is quantitative accurately, sensitivity it is high.Pass through
Consulting literatures discovery, currently at home and abroad there is no the real-time fluorescences of SYBR Green I for Mycoplasma mycoides subsp.capri detection
Quantifying PCR method.The foundation of the present invention can fill up the blank in the field.
The content of the invention
It is an object of the invention to provide a kind of fluorescence quantitative PCR detection primer for Mycoplasma mycoides subsp.capri.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of real-time fluorescence quantitative PCR detection primers of SYBR Green I for Mycoplasma mycoides subsp.capri, the nucleosides of primer
Acid sequence is：
Upstream primer：5 '-TGTTGTTGGTGGGTCTGCTT-3 ',
Downstream primer：5 '-ACTTTATCAGCCGCCATTTGA-3 ',
Its purpose amplified fragments is 117 bp.
Can be used to detect the corresponding gene of Mycoplasma mycoides subsp.capri using the primer of the present invention, it is especially suitable
In the detection of the Real-Time Fluorescent Quantitative PCR Techniques of SYBR Green I.By the various optimizations to reaction system and reaction condition, build
A set of real time fluorescence quantifying PCR methods of SYBR Green I that can be used to detect Mycoplasma mycoides subsp.capri are found.Concrete operations
Method is as follows：
1st, design of primers：According to the Mycoplasma mycoides subsp.capri FQ377874.1 genome sequences announced on GenBank, utilize
The softwares of Primer 6.0 carry out design of primers to MLC_1770 gene orders, and using BLAST instruments line retrieval, preliminary identification are entered
Its specificity.
2nd, the optimization of reaction system：25 μ L optimal reaction systems of optimization are：SYBR® Premix Ex Taq TM
(TIiRNaseH Plus) 12.5 μ L, upstream and downstream primer（10 μmol/L）Each 0.5 μ L, the μ L of template 2, water complements to 25 μ L.Most
Good reaction condition is：95 DEG C, 30s denaturations；95 DEG C, 5s, 60 DEG C, 30s, totally 40 circulations.
3rd, the preparation of positive criteria product：Take 1 mL Mycoplasma mycoides subsp.capris nutrient solution to add in 1.5 mL centrifuge tubes,
10000r/min is centrifuged 3min, abandons supernatant collects thalline, DNA is extracted using raw work tissue DNA extracts kit, using protein
Nucleic acid instrument determines its concentration, is converted into copy number, standby as positive criteria product.
4th, the foundation of calibration curve：Performing PCR amplification is entered with the reaction system for optimizing, with copy number as abscissa, with Ct values
Calibration curve is set up for ordinate, positive standard is judged.
The method can be used for the etiological diagnosis of Mycoplasma mycoides subsp.capri and epidemiology survey, be that the early stage of disease prevents
Offer detection method is provided.
Beneficial effect：
The MLC_1770 gene orders of the FQ377874.1 that the present invention is logged according to GenBank, design specific primer, in the country
The real time fluorescence quantifying PCR method of Mycoplasma mycoides subsp.capri is initially set up outward, and the method specificity is good, sensitivity is high, weight
Renaturation is good, can be used to detect the content of Mycoplasma mycoides subsp.capri in clinical sample.
Description of the drawings
The amplification curve of the real time fluorescence quantifying PCR methods of Fig. 1 Mycoplasma mycoides subsp.capri SYBR Green I.
Fig. 2 is the calibration curve of the real-time fluorescence quantitative PCRs of SYBR Green I of Mycoplasma mycoides subsp.capri.
Specific embodiment
The foundation of the quantitative fluorescent PCR methods of SYBR Green I of the Mycoplasma mycoides subsp.capri of embodiment 1
First, material：
Mycoplasma mycoides subsp.capri（GenBank accession number：KU870648）
Mycoplasma capri goat pneumonia subspecies are presented by Lanzhou research institute of the Chinese Academy of Sciences, Escherichia coli, Pasteurella, golden yellow
Staphylococcus, haemophilus parasuis etc. are by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute poultry diease laboratory and swine disease laboratory
Present；Continuous mycoplasma pneumoniae, sheep of virus are that this section office separates, preserves.
2nd, step
1st, instrument and reagent：Mastercyclereprealplex quantitative fluorescent PCRs instrument is purchased from eppendorf companies；SYBR® Premix Ex Taq TM (TIi RNaseH Plus), DL2000 Marker are purchased from precious bioengineering（Dalian）Limited public affairs
Department；Quantitative fluorescent PCR pipe is purchased from Axygen.
2. the specificity of primer
The specificity of primer is the most important factor of this experiment institute method for building up.The FQ377874.1's logged according to GenBank
MLC_1770 gene orders, by comparing analysis, design primer.
3rd, the preparation of positive criteria product
DNA with extraction takes 1 mL Mycoplasma mycoides subsp.capris nutrient solution and adds in 1.5 mL centrifuge tubes as template,
10000r/min is centrifuged 3min, abandons supernatant collects thalline, DNA is extracted using raw work tissue DNA extracts kit, using protein
Nucleic acid instrument determines its concentration, is converted into copy number, standby as positive criteria product.
4th, the optimization of reaction condition
Using 25 μ L reaction systems SYBR® Premix Ex Taq TM (TIiRNaseH Plus) 12.5 μ L, PCR upstream primers
（10μmol/L）0.5 μ L, PCR downstream primers（10μmol/L）0.5 μ L, the μ L of positive criteria product 2 after doubling dilution, moisturizing is extremely
The μ L of final volume 25, non-specific peak is occurred without as index with the Ct values for minimum occur and in melting curve analysis, right respectively
Annealing temperature（55~68 DEG C）And primer concentration（0.2~1.0 μm of ol/L）It is optimized.
Positive criteria product is carried out into continuous 10 times to be serially diluted（10-1~10-8）, expanded with the condition for optimizing, to copy
Shellfish number is abscissa, and calibration curve is set up as ordinate with Ct values.
The amplification curve and calibration curve of positive criteria product are shown, the quantitative fluorescent PCR methods of SYBR Green I are to silk
The CT of shape mycoplasma goat subspecies is with copy number 1.12 × 102~1.12 × 109Have linear well in copy/reaction range
Relation, coefficient correlation is 0.996, and amplification efficiency is 162%.Amplification curve is shown in Fig. 1, and calibration curve is shown in Fig. 2.
5th, specific detection
5.1 specific detection
Mycoplasma capri goat pneumonia subspecies, Escherichia coli, Pasteurella, Staphylococcus aureus are detected respectively with the condition of optimization
Bacterium, haemophilus parasuis, continuous mycoplasma pneumoniae, sheep of virus nucleic acid samples are detected, its specificity are evaluated.Inspection
Survey result and be feminine gender.
5.2 repeatable and repeatability assessments
3 repeating pipes are set to same positive criteria product, is detected with the real-time fluorescence quantitative PCRs of SYBR Green I, evaluate it
Repeatability,
The coefficient of variation in calculating group；Positive criteria product is placed in into -20 DEG C of Refrigerator stores, is examined again respectively at the 7th, 14,21d, evaluated
Its repeatability, calculates its between-group variation coefficient.It is 0.60%~2.46% to calculate the coefficient of variation in group, and between-group variation coefficient is
1.07%~2.67%, favorable repeatability.
The foregoing is only presently preferred embodiments of the present invention, all impartial 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 Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>A kind of fluorescence quantitative PCR detection primer for Mycoplasma mycoides subsp.capri
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
tgttgttggt gggtctgctt 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
actttatcag ccgccatttg a 21
Claims (2)
1. a kind of fluorescence quantitative PCR detection primer for Mycoplasma mycoides subsp.capri, it is characterised in that：The nucleotides of primer
Sequence is：
Upstream primer：5 '-TGTTGTTGGTGGGTCTGCTT-3 ',
Downstream primer：5’- ACTTTATCAGCCGCCATTTGA-3’.
2. application of the detection primer as claimed in claim 1 in detection Mycoplasma mycoides subsp.capri reagent is prepared.
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| CN201710135665.7A CN106676195B (en) | 2017-03-08 | 2017-03-08 | Fluorescent quantitative PCR detection primer for mycoplasma filiformis goat subspecies |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967840A (en) * | 2017-05-31 | 2017-07-21 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescence quantitative PCR detection primer for mycoplasma capri goat pneumonia subspecies |
| 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 |
| CN108977507A (en) * | 2018-08-16 | 2018-12-11 | 福建省农业科学院畜牧兽医研究所 | For detecting the RPA primer of Mycoplasma mycoides subsp.capri |
| CN109666753A (en) * | 2019-03-01 | 2019-04-23 | 福建省农业科学院畜牧兽医研究所 | A kind of triple fluorescent quantitative PCR primer and probe detecting 3 kinds of sheep pathogenic mycoplasmas |
| CN111235289A (en) * | 2020-03-15 | 2020-06-05 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma capricolum goat pneumonia subspecies |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434919A (en) * | 2016-09-28 | 2017-02-22 | 福建省农业科学院畜牧兽医研究所 | Primers for triple PCR of three types of sheep pathogenic mycoplasmas and detection method |
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2017
- 2017-03-08 CN CN201710135665.7A patent/CN106676195B/en active Active
Patent Citations (1)
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| CN106434919A (en) * | 2016-09-28 | 2017-02-22 | 福建省农业科学院畜牧兽医研究所 | Primers for triple PCR of three types of sheep pathogenic mycoplasmas and detection method |
Non-Patent Citations (5)
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| JIANG,J.X等: "GenBank: KU870648.1", 《NCBI-GENBANK》 * |
| THIAUCOURT,F等: "GenBank: FQ377874.1", 《NCBI-GENEBANK》 * |
| 王绍伟等: "绵羊和山羊嗜血支原体感染的检测及分子鉴定", 《中国兽医科学》 * |
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| 黄海碧: "绵羊肺炎支原体检测方法的建立及黏附蛋白的研究", 《中国博士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967840A (en) * | 2017-05-31 | 2017-07-21 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescence quantitative PCR detection primer for mycoplasma capri goat pneumonia subspecies |
| 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 |
| CN108977507A (en) * | 2018-08-16 | 2018-12-11 | 福建省农业科学院畜牧兽医研究所 | For detecting the RPA primer of Mycoplasma mycoides subsp.capri |
| CN109666753A (en) * | 2019-03-01 | 2019-04-23 | 福建省农业科学院畜牧兽医研究所 | A kind of triple fluorescent quantitative PCR primer and probe detecting 3 kinds of sheep pathogenic mycoplasmas |
| CN111235289A (en) * | 2020-03-15 | 2020-06-05 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma capricolum goat pneumonia subspecies |
| CN111235289B (en) * | 2020-03-15 | 2022-04-22 | 中国农业科学院兰州兽医研究所 | Specific PCR primer for detecting mycoplasma capricolum goat pneumonia subspecies |
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