CN108070665A - One kind is used to detect Mannheimia haemolytica real-time fluorescence PCR primer - Google Patents
One kind is used to detect Mannheimia haemolytica real-time fluorescence PCR primer Download PDFInfo
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- CN108070665A CN108070665A CN201810149979.7A CN201810149979A CN108070665A CN 108070665 A CN108070665 A CN 108070665A CN 201810149979 A CN201810149979 A CN 201810149979A CN 108070665 A CN108070665 A CN 108070665A
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- mannheimia haemolytica
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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
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- 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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- 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
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention provides a kind of primer for I real-time fluorescence quantitative PCRs of SYBR Green detection Mannheimia haemolytica, and the primer sequence is sense primer：5 ' AAGCCGTGGTTGATACTATT 3 ', anti-sense primer：5’‑CCGCCTGCCATTACTAAG‑3’.The present invention designs specific primer, establishes I real time fluorescence quantifying PCR methods of SYBR Green of Mannheimia haemolytica gcp genes for the first time according to the gcp gene orders of the GenBank AY839679 logged in, and this method specificity is good, high sensitivity, reproducible.
Description
Technical field
The present invention relates to one kind to be used for I real-time fluorescence quantitative PCR detection primers of Mannheimia haemolytica SYBR Green,
Belong to biology field.
Background technology
Mannheimia haemolytica is a kind of conditioned pathogen for infecting a variety of domestic animals and wild animal, main infection ox,
The ruminants such as sheep.The bacterium is the main pathogen of ox, sheep pneumonia and newborn lamb septicemia, has seriously endangered the strong of sheep husbandry
Kang Fazhan, the global ox that dies of illness every year there are about 30% to have direct or indirect relation, only economic loss caused by north america with the bacterium
More than 1,000,000,000 dollars.
At present to the etiological diagnosis technical aspect of Mannheimia haemolytica, existing bacteria distribution identification method and general both at home and abroad
The report of logical PCR method though being related to the fluorescence quantifying PCR method that Mannheimia belongs to specificity, so there are no hemolytic Mans
The report of I real time fluorescence quantifying PCR methods of bacillus gcp gene SYBR Green.Bacteria distribution identification method is longer in the presence of taking,
And infection early stage separates more difficult, and various bacteria such as pasteurella multocida, sea in animal body after antibiosis extract for treating
Algae Pasteurella etc. can inhibit the growth of the bacterium, make it that interest rate be divided to be far below actual infection rate.Regular-PCR there are sensibility and
The shortcomings of specificity is relatively low, makes it be subject to certain restrictions in practical applications.I real-time fluorescence quantitative PCRs of SYBR Green are
On the basis of regular-PCR, fluorescent dye is added in the reaction system, and target nucleotide sequence is detected using fluorescent PCR detector
The technology of row has many advantages, such as high specificity, quantitative accurate, high sensitivity.It is found by consulting literatures, currently both at home and abroad not yet
There are I real time fluorescence quantifying PCR methods of SYBR Green for Mannheimia haemolytica gcp genes.The foundation of the present invention can be with
Fill up the blank in the field.
The content of the invention
The object of the present invention is to provide a kind of for detecting the real-time fluorescence PCR primer of Mannheimia haemolytica.
In order to achieve the above objectives, the present invention uses following technical scheme：
A kind of I real-time fluorescence quantitative PCR detection primers of SYBR Green for Mannheimia haemolytica gcp genes, primer
Nucleotides sequence is classified as：
Sense primer：5 '-AAGCCGTGGTTGATACTATT -3 ',
Anti-sense primer：5’- CCGCCTGCCATTACTAAG -3’.
It can be used for being detected the gcp genes of Mannheimia haemolytica using the primer of the present invention, be particularly suitable for
The detection of I Real-Time Fluorescent Quantitative PCR Techniques of SYBR Green.By the various optimizations to reaction system and reaction condition, establish
A set of SYBR Green I real time fluorescence quantifying PCR methods that can be used for detection Mannheimia haemolytica gcp genes.
Concrete operation method is as follows：
1st, design of primers：According to the Mannheimia haemolytica AY839679 genome sequences announced on GenBank, Beacon is utilized
7.9 softwares of Designer carry out design of primers to gcp gene orders, are retrieved using BLAST instruments, its spy of preliminary identification
The opposite sex.
2nd, the optimization of reaction system：25 μ L optimal reaction systems of optimization are：SYBR Premix Ex Taq 2×
12.5 μ L, upstream and downstream primer（20 μmol/L）Each 1.0 μ L, 2 μ L of template, water complement to 25 μ L.Optimum reaction condition is：95
DEG C, 30s pre-degenerations；95 DEG C, 5s, 58 DEG C, 15s, 72 DEG C, 20s, totally 40 cycle.
3rd, the preparation of positive criteria product：1 mL Mannheimia haemolyticas culture solution is taken to add in 1.5 mL centrifuge tubes,
10000r/min centrifuges 3min, abandons supernatant and collects thalline, extracts DNA using raw work tissue DNA extracts kit, utilizes protein
Nucleic acid instrument measures its concentration, is converted into copy number, spare as positive criteria product.
4th, the foundation of standard curve：PCR amplification is carried out with the reaction system of optimization, using copy number as abscissa, with Ct values
Standard curve is established for ordinate, judges positive standard.
This method can be used for etiological diagnosis and the epidemiology survey of Mannheimia haemolytica, be established for the early prevention of disease
Determine technical foundation.
The advantage of the invention is that：
The present invention designs specific primer according to the gcp gene orders of the GenBank AY839679 logged in, in both at home and abroad first
I real time fluorescence quantifying PCR methods of SYBR Green of Mannheimia haemolytica gcp genes are established, this method specificity is good, clever
It is sensitivity high (minimum detectable 6.2 copies/ μ L), reproducible.
Description of the drawings
The amplification curve of I real time fluorescence quantifying PCR methods of Fig. 1 Mannheimia haemolytica gcp gene SYBR Green.
Fig. 2 is the standard curve of I real-time fluorescence quantitative PCRs of Mannheimia haemolytica gcp gene SYBR Green.
Specific embodiment
The foundation of 1 Mannheimia haemolytica SYBR Green of embodiment, I quantitative fluorescent PCR methods
First, material：
Mannheimia haemolytica, Escherichia coli, pasteurella multocida, staphylococcus aureus, salmonella, streptococcus, Wei
Family name clostridium is separated by laboratory and identified.
2nd, step
1st, instrument and reagent：Mastercycler ep realplex quantitative fluorescent PCRs instrument is purchased from eppendorf companies；
SYBR Premix Ex Taq 2 ×, DL2000 Marker be purchased from precious bioengineering（Dalian）Co., Ltd；Fluorescent quantitation
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.According to the gcp of the GenBank AY839679 logged in
Gene order is analyzed by comparing, and designs primer.
The nucleotides sequence of primer is classified as：
Sense primer：5 '-AAGCCGTGGTTGATACTATT -3 ',
Anti-sense primer：5’- CCGCCTGCCATTACTAAG -3’.
3rd, the preparation of positive criteria product
Using the DNA of extraction as template, 1 mL Mannheimia haemolyticas culture solution is taken to add in 1.5 mL centrifuge tubes, 10000r/min
3min is centrifuged, supernatant is abandoned and collects thalline, DNA is extracted using raw work tissue DNA extracts kit, is measured using protein nucleic acid instrument
Its concentration, is converted into copy number, spare as positive criteria product.
4. the optimization of reaction condition
Using 25 μ L reaction systems (SYBR Premix Ex Taq 2 ×), 12.5 μ L, PCR sense primers（20μmol/L）1.0μ
L, PCR anti-sense primer（20μmol/L）1.0 μ L, the 2 μ L of positive criteria product after doubling dilution, moisturizing is to 25 μ L of final volume, to go out
Show minimum Ct values and do not occur non-specific peak in melting curve analysis for index, respectively to annealing temperature（50~65
℃）And primer concentration（0.2~1.0 μm of ol/L）It optimizes.
Positive criteria product is carried out continuous 10 times to be serially diluted（10-1~10-8）, expanded with the condition of optimization, to copy
Shellfish number is abscissa, and standard curve is established using Ct values as ordinate.
The amplification curve and standard curve of positive criteria product are shown, I quantitative fluorescent PCR methods of SYBR Green are to molten
The Ct of courageous and upright Mannheimia is with copy number 6.2 × 101~6.2 × 108There is good linear relationship in copy/reaction range,
Related coefficient is 0.999, amplification efficiency 126%.Amplification curve is shown in Fig. 1, and standard curve is shown in Fig. 2.
6 specific detections
6.1 specific detection
Mannheimia haemolytica, Escherichia coli, pasteurella multocida, Staphylococcus aureus are detected respectively with the condition of optimization
Bacterium, salmonella, streptococcus, clostridieum welchii nucleic acid samples are detected, its specificity is evaluated.Testing result shows,
In addition to Mannheimia haemolytica has amplified signal, remaining is without amplified signal.
6.2 repeatabilities and reproducibility assessment
3 repeating pipes are set to same positive criteria product, is detected with I real-time fluorescence quantitative PCRs of SYBR Green, evaluates it
Repeatability.
The coefficient of variation in calculating group；Positive criteria product is placed in -20 DEG C of refrigerators to preserve, examines, comments again respectively at the 3rd, 6,9d
Its reproducibility of valency calculates its between-group variation coefficient.It is 0.78%~1.23% that the interior coefficient of variation must be organized by, which calculating, and between-group variation coefficient is
0.72%~1.03 %, favorable repeatability.
The foregoing is merely 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>Lin Yusheng
<120>One kind is used to detect Mannheimia haemolytica real-time fluorescence PCR primer
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
aagccgtggt tgatactatt 20
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
ccgcctgcca ttactaag 18
Claims (2)
1. one kind is used for I real-time fluorescence quantitative PCR detection primers of Mannheimia haemolytica SYBR Green, it is characterised in that：Draw
The nucleotides sequence of object is classified as：
Sense primer：5 '-AAGCCGTGGTTGATACTATT -3 ',
Anti-sense primer：5’- CCGCCTGCCATTACTAAG -3’.
2. a kind of detection kit for including primer described in claim 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559784A (en) * | 2018-07-06 | 2018-09-21 | 陕西省动物研究所 | A kind of while three kinds of pathogens of detection triple PCR detection primer, probe, kit and detection methods |
| CN113249503A (en) * | 2021-05-21 | 2021-08-13 | 华中农业大学 | LAMP primer group and method for detecting mannheimia haemolytica |
| CN115044686A (en) * | 2022-05-11 | 2022-09-13 | 华中农业大学 | Real-time fluorescent quantitative PCR primer pair and probe combination for simultaneously detecting seven BRDC pathogens |
| CN115896313A (en) * | 2022-07-08 | 2023-04-04 | 云南省畜牧兽医科学院 | Primer and probe for mannheimia bacteria specificity TaqMan real-time fluorescence quantitative PCR, detection method and application |
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| CN101638687A (en) * | 2009-08-26 | 2010-02-03 | 北京市农林科学院 | Gene chip, kit and method for detecting common pathogenic bacteria of piglets |
| CN106399555A (en) * | 2016-11-10 | 2017-02-15 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection |
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| CN101638687A (en) * | 2009-08-26 | 2010-02-03 | 北京市农林科学院 | Gene chip, kit and method for detecting common pathogenic bacteria of piglets |
| CN106399555A (en) * | 2016-11-10 | 2017-02-15 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method as well as standard substance and detection kit for real-time fluorescent quantitative PCR detection |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559784A (en) * | 2018-07-06 | 2018-09-21 | 陕西省动物研究所 | A kind of while three kinds of pathogens of detection triple PCR detection primer, probe, kit and detection methods |
| CN113249503A (en) * | 2021-05-21 | 2021-08-13 | 华中农业大学 | LAMP primer group and method for detecting mannheimia haemolytica |
| CN115044686A (en) * | 2022-05-11 | 2022-09-13 | 华中农业大学 | Real-time fluorescent quantitative PCR primer pair and probe combination for simultaneously detecting seven BRDC pathogens |
| CN115044686B (en) * | 2022-05-11 | 2023-11-10 | 华中农业大学 | Real-time fluorescent quantitative PCR primer pair and probe combination for simultaneously detecting seven BRDC pathogens |
| CN115896313A (en) * | 2022-07-08 | 2023-04-04 | 云南省畜牧兽医科学院 | Primer and probe for mannheimia bacteria specificity TaqMan real-time fluorescence quantitative PCR, detection method and application |
| CN115896313B (en) * | 2022-07-08 | 2023-09-12 | 云南省畜牧兽医科学院 | Primers and probes for real-time fluorescent quantitative PCR (polymerase chain reaction) of specific TaqMan of bacteria of genus Mannheimia, detection method and application |
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Application publication date: 20180525 |