CN104195263A - Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR - Google Patents
Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR Download PDFInfo
- Publication number
- CN104195263A CN104195263A CN201410493854.8A CN201410493854A CN104195263A CN 104195263 A CN104195263 A CN 104195263A CN 201410493854 A CN201410493854 A CN 201410493854A CN 104195263 A CN104195263 A CN 104195263A
- Authority
- CN
- China
- Prior art keywords
- sybr green
- alpha fungus
- quantitative pcr
- primer
- curve
- 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
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR (polymerase chain reaction). The method comprises the following steps: (1) synthesizing a primer; (2) preparing a standard positive sample; (3) preparing samples to be detected; (4) performing SYBR Green I fluorogenic quantitative PCR; (5)drawing a standard curve and a solubility curve; (6) judging results. The method has the benefits that the accurate results can be obtained in three hours only in the whole process of detecting the rabbit trichophyton mentagrophyte by adopting the SYBR Green I fluorogenic quantitative PCR, and 96 samples can be detected in one time; compared with other methods, the method has the advantages of simplicity for operation, high detection speed, high sensitivity, high specificity, high flux and the like, and can provide guarantee for disease treatment and epidemic control.
Description
Technical field
The present invention relates to the detection method that a kind of rabbit alpha fungus infects, be specifically related to a kind of method of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, belong to biology field.
Background technology
Alpha fungus is that the most commonly encountered diseases of the many animals tineas such as rabbit is one of former, main infringement skin and appurtenant thereof, the symptoms such as performance scurf increases, forms a scab, loses hair or feathers, oozes out, folliculitis and gargalesthesia, cause rabbit malnutrition, growth retardation etc., bring tremendous economic loss to rabbit keeping.More seriously, this disease can also be transmitted to people, causes infectious diseases common to human beings and animals.So need to find one fast, detection method accurately, to treat in time and control epidemic situation.
At present, common detection method comprises: the direct Microscopic inspection of pathological material of disease, Isolation and culture of agent method.Although the direct Microscopic inspection of pathological material of disease is simple, quick, cannot differentiate the kind of fungi, and have the false negative rate of 5-15%; Although Isolation and culture of agent, by the form of bacterium colony, mycelia, spore, in conjunction with biochemical test, can be specified fungal species, length consuming time, is generally 3-4 week, and has 40% false negative rate.
Along with the development of technology, molecular biology method detects fungi infestation and has obtained certain application, comprises the content of chromosomal DNA G+C, total DNA homology, Mitochondrial DNA restriction fragment length Polymorphism Analysis, arbitrarily primed PCR, the analysis of random amplification DNA polymorphism, PCR fingerprint etc.Wherein, adopt rDNA district and the sequential analysis of rRNA small subunit, particularly ITS (analysis of internal transcribed spacer) district analyses and comparison coding rRNA small subunit (18SrRNA) gene more adapts to the detection to dermatophytes, at present, the ITS region sequence analysis in rDNA district is called to the gold standard of dermatophytosis qualification.This method is mainly to utilize the method for conventional PCR to increase to goal gene, then utilizes the methods such as electrophoresis observation, gene sequencing, sequential analysis, finally, by homology difference, dermatophytes cause of disease is made to diagnosis.But there is certain defect in this method, the sensitivity that is mainly manifested in conventional PCR be not high especially, need the qualification of PCR aftertreatment, electrophoresis need to use the aspects such as toxic EB dyestuff.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of detection time of short, visual result, susceptibility high, quantitatively accurately SYBR Green I fluorescence quantifying PCR method for the detection of rabbit alpha fungus.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
A method for SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, is characterized in that, comprises the following steps:
(1) primer is synthetic:
Upstream primer F:5'-GCAAAGAAGCCTGGAAGAAG-3',
Downstream primer R:5'-GGAGACCATCTGTGAGAGTTG-3;
(2) standard positive sample preparation: extract the plasmid DNA of standard positive recombinant bacterium, dilute for different copy number gradients;
(3) testing sample preparation: the step of pressing Solarbio test kit specification sheets is extracted testing sample DNA;
(4) SYBR Green I quantitative fluorescent PCR: be respectively template with standard positive sample and testing sample, F and R are that primer carries out the reaction of SYBR Green I quantitative fluorescent PCR;
(5) drawing standard curve and solubility curve: carry out SYBR Green I quantitative fluorescent PCR reaction result, drawing standard curve and solubility curve according to standard positive sample;
(6) result is judged: when testing sample SYBR Green I quantitative fluorescent PCR response curve is " S " type, and Ct value≤36.5, Tm value are between 86.48 DEG C～86.65 DEG C, in judgement testing sample, contain alpha fungus; If response curve is straight line, in judgement sample, do not contain alpha fungus.
The method of aforesaid a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, in step (2), the OD260/OD280 of the plasmid DNA of standard positive recombinant bacterium is between 1.8～2.0.
The method of aforesaid a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, in step (2), copy number gradient is 1.0 × 10
99 gradients of～1.0 × 10 copy/μ L.
The method of aforesaid a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, in step (4), SYBR Green I quantitative fluorescent PCR reaction system is: the SYBR Premix Ex Taq12.5 μ L of 2X, the each 1.0 μ L of upstream primer F, downstream primer R of 10pmol/L, DNA profiling 2.0 μ L, complement to 25.0 μ L with sterilizing ultrapure water.
The method of aforesaid a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, in step (4), SYBR Green I quantitative fluorescent PCR response procedures is: 94 DEG C of denaturation 60s; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 40 circulations.
Usefulness of the present invention is: adopt the whole process of method of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus only to need just can draw for 3 hours accurate result, and once can carry out the detection of 96 samples, compared with additive method have simple to operate, detection speed is fast, highly sensitive, high specificity, flux advantages of higher, can provide safeguard for the treatment of disease and the control of epidemic situation.
Brief description of the drawings
Fig. 1 is the SYBR Green I quantitative fluorescent PCR typical curve obtaining in a specific embodiment of the present invention;
Fig. 2 is the SYBR Green I fluorescent quantitative PCR solubility curve obtaining in a specific embodiment of the present invention;
Fig. 3 is the one group of SYBR Green I fluorescence quantitative PCR detection result obtaining in a specific embodiment of the present invention.
Embodiment
1. primer is synthetic
The alpha fungus gene order of having delivered with reference to GenBank, designs a pair of Auele Specific Primer:
Upstream primer F:5'-GCAAAGAAGCCTGGAAGAAG-3',
Downstream primer R:5'-GGAGACCATCTGTGAGAGTTG-3.
Expection amplified fragments size 288bp.
2. standard positive sample preparation:
(1) accurate bacterial strain DNA extraction
Alpha fungus type strain is seeded in to potato liquid of glucose substratum, and 28 DEG C of shaking tables are cultivated 72h, centrifugal collection mycelia, and refrigerator freezing saves backup.Mycelia is thawed, by liquid nitrogen grinding, then press the step of Solarbio test kit specification sheets and extract DNA.
(2) structure of standard positive recombinant bacterium
Taking extract reference culture DNA as template, F and R are that primer carries out pcr amplification.PCR reaction system is: Mix 25 μ L, and the each 1.0 μ L of primer, template DNA 4.0 μ L, add ddH
2o supplies 25 μ L.Response procedures is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C are extended 50s, totally 35 circulations; 72 DEG C are extended 10min again, in 4 DEG C of termination reactions, save backup.
Get 5 μ L PCR products electrophoresis on 1% sepharose, go out to observe band at 288bp, then correct by Shanghai Sheng Gong biotechnology service company sequencing.
PCR product, after sepharose reclaims purifying, is connected with pMD18-T carrier, transforms intestinal bacteria, obtain recombinant bacterium.
Recombinant bacterium is accredited as to the positive through double digestion and PCR, obtains standard positive recombinant bacterium.
(3) extract the plasmid DNA that standard positive is recombinated, dilute for different copy number gradients
The step of pressing Solarbio test kit specification sheets is extracted the DNA of standard positive recombinant bacterium, measures OD260 value and OD280 value, and the plasmid DNA that OD260/OD280 value is recombinated between 1.8～2.0 Zhunyang property can be used for subsequent experimental.
Calculate plasmid concentration according to OD260 value, and be converted into copy number, then become 1.0 × 10 with 10 times of gradient dilutions
99 gradients of～1.0 × 10 copy/μ L.Copy number=(concentration × avogadros constant)/(molecular-weight average × total length of a base pair).Standard positive sample taking the different copy numbers of dilution carries out SYBR Green I quantitative fluorescent PCR as template.
3. testing sample preparation
By the inoculation to be measured of clinical separation, at potato liquid of glucose substratum, 28 DEG C of shaking tables are cultivated 72h, centrifugal collection mycelia, and refrigerator freezing saves backup.Mycelia is thawed, by liquid nitrogen grinding, then press the step of Solarbio test kit specification sheets and extract DNA, carry out SYBR Green I quantitative fluorescent PCR as testing sample group template.
4.SYBR Green I quantitative fluorescent PCR
Be respectively template with standard positive sample testing sample, F and R are primer, carry out the reaction of SYBR Green I quantitative fluorescent PCR.SYBR Green I quantitative fluorescent PCR reaction system is: the SYBR Premix Ex Taq12.5 μ L of 2X, and the each 1.0 μ L of upstream primer F, downstream primer R of 10pmol/L, DNA profiling 2.0 μ L, complement to 25.0 μ L with sterilizing ultrapure water; Response procedures is: 94 DEG C of denaturation 60s; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 40 circulations.
5. drawing standard curve and solubility curve:
Carry out SYBR Green I quantitative fluorescent PCR reaction result according to standard positive sample, adopt software automatic analysis drawing standard curve (seeing Fig. 1) and solubility curve (seeing Fig. 2).
6. result is judged:
Testing sample SYBR Green I quantitative fluorescent PCR response curve is " S " type (seeing Fig. 3), and combined standard curve and solubility curve draw: Ct value≤36.5, and Tm value is between being between 86.48 DEG C～86.65 DEG C, so contain alpha fungus in judgement testing sample.
Susceptibility and the specificity confirmatory experiment of 7.SYBR Green I quantitative fluorescent PCR
(1) sensitivity test
Get respectively 1.0 × 10 of dilution
99 gradient standard positive sample 1 μ L of～1.0 × 10 copy/μ L are that template is carried out SYBR Green I quantitative fluorescent PCR and regular-PCR, measure the susceptibility of SYBR Green I quantitative fluorescent PCR, and contrast with regular-PCR.Found that the method can detect the standard positive sample of 10 copies/μ L, and conventional PCR can only detect 10
3the standard positive sample of copy/μ L, the susceptibility of SYBR Green I quantitative fluorescent PCR is 100 times of conventional PCR.
(2) specificity experiment
Carry out SYBR Green I quantitative fluorescent PCR as template with the DNA of the clinical sample of rabbit alpha fungus, Sabouraudites lanosus, acrothesium floccosum, hungmao mentagrophyte, Candida albicans isolated strains and rabbit alpha fungus.Result shows: the response curve of the clinical sample of the strain isolated of rabbit alpha fungus and rabbit alpha fungus is " S " type, and CT value is between 18～25; And the response curve of the isolated strains of Sabouraudites lanosus, acrothesium floccosum, hungmao mentagrophyte, Candida albicans is straight line, negative result (seeing Fig. 3).Repeat to test the Ct value variation coefficient and be less than 5%, show the high specificity of SYBR Green I quantitative fluorescent PCR.
Claims (5)
1. a method for SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus, is characterized in that, comprises the following steps:
(1) primer is synthetic:
Upstream primer F:5'-GCAAAGAAGCCTGGAAGAAG-3',
Downstream primer R:5'-GGAGACCATCTGTGAGAGTTG-3;
(2) standard positive sample preparation: extract the plasmid DNA of standard positive recombinant bacterium, dilute for different copy number gradients;
(3) testing sample preparation: the step of pressing Solarbio test kit specification sheets is extracted testing sample DNA;
(4) SYBR Green I quantitative fluorescent PCR: be respectively template with standard positive sample and testing sample, F and R are that primer carries out the reaction of SYBR Green I quantitative fluorescent PCR;
(5) drawing standard curve and solubility curve: carry out SYBR Green I quantitative fluorescent PCR reaction result, drawing standard curve and solubility curve according to standard positive sample;
(6) result is judged: when testing sample SYBR Green I quantitative fluorescent PCR response curve is " S " type, and Ct value≤36.5, Tm value are between 86.48 DEG C～86.65 DEG C, in judgement testing sample, contain alpha fungus; If response curve is straight line, in judgement sample, do not contain alpha fungus.
2. the method for a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus according to claim 1, is characterized in that, in step (2), the OD260/OD280 of the plasmid DNA of standard positive recombinant bacterium is between 1.8～2.0.
3. the method for a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus according to claim 1 and 2, is characterized in that, in step (2), copy number gradient is 1.0 × 10
99 gradients of～1.0 × 10 copy/μ L.
4. the method for a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus according to claim 3, it is characterized in that, in step (4), SYBR Green I quantitative fluorescent PCR reaction system is: the SYBR Premix Ex Taq12.5 μ L of 2X, the each 1.0 μ L of upstream primer F, downstream primer R of 10pmol/L, DNA profiling 2.0 μ L, complement to 25.0 μ L with sterilizing ultrapure water.
5. the method for a kind of SYBR Green I fluorescence quantitative PCR detection rabbit alpha fungus according to claim 4, is characterized in that, in step (4), SYBR Green I quantitative fluorescent PCR response procedures is: 94 DEG C of denaturation 60s; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410493854.8A CN104195263A (en) | 2014-09-24 | 2014-09-24 | Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410493854.8A CN104195263A (en) | 2014-09-24 | 2014-09-24 | Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104195263A true CN104195263A (en) | 2014-12-10 |
Family
ID=52080625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410493854.8A Pending CN104195263A (en) | 2014-09-24 | 2014-09-24 | Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104195263A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878118A (en) * | 2015-06-25 | 2015-09-02 | 河北工程大学 | Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection |
| CN111206114A (en) * | 2020-03-03 | 2020-05-29 | 杭州缔蓝生物技术有限公司 | Primer and kit for fluorescence PCR (polymerase chain reaction) detection of nine dermatophytes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004835A (en) * | 2014-05-15 | 2014-08-27 | 河北工程大学 | Multiplex PCR detection method of rabbit skin fungi |
-
2014
- 2014-09-24 CN CN201410493854.8A patent/CN104195263A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004835A (en) * | 2014-05-15 | 2014-08-27 | 河北工程大学 | Multiplex PCR detection method of rabbit skin fungi |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878118A (en) * | 2015-06-25 | 2015-09-02 | 河北工程大学 | Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection |
| CN111206114A (en) * | 2020-03-03 | 2020-05-29 | 杭州缔蓝生物技术有限公司 | Primer and kit for fluorescence PCR (polymerase chain reaction) detection of nine dermatophytes |
| CN111206114B (en) * | 2020-03-03 | 2023-10-03 | 杭州缔蓝生物技术有限公司 | Nine primers and kit for fluorescence PCR detection of dermatophytes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103866034A (en) | Multiple real-time fluorescence quantification PCR (polymerase chain reaction) detection kit and detection method for helicobacter pylori in gastric juice | |
| CN101712987B (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus plantarum in probiotic dairy products | |
| CN106987626B (en) | Primer and probe for rapidly detecting various fungi and identifying strains and application thereof | |
| CN104059977A (en) | Salmonella serotype identification method and kit thereof | |
| CN103468811A (en) | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit | |
| CN111690760A (en) | Method for accurately detecting cryptococcus neoformans | |
| CN101712989B (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus casei in probiotic dairy products | |
| CN109762940A (en) | For detecting the primer sets and kit of infectious spleen and kidney necrosis virus Yu mandarin fish rhabdovirus | |
| CN101712986B (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus acidophilus in probiotic dairy products | |
| CN103333903A (en) | Target sequence, primer and probe for detecting helicobacter pylori and kit thereof | |
| CN102312005A (en) | Multiplex PCR primer for synchronously detecting vibrio parahaemolyticus and vibrio alginolyticus, and design method thereof | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| CN104004835B (en) | A kind of rabbit dermatophyte composite PCR detecting method | |
| CN104195263A (en) | Method for detecting rabbit trichophyton mentagrophyte by SYBR Green I fluorogenic quantitative PCR | |
| CN112359125A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN102154487A (en) | Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis | |
| CN103014164B (en) | Duplex fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for shellfish Bonamia and Perkinsus | |
| CN101712988B (en) | Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products | |
| CN105200122A (en) | Quantitative detection kit for wheat stripe rust and application thereof | |
| CN104946638A (en) | Multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof | |
| CN105331677A (en) | Primer group used for multiple PCR detection by aiming at salmonella and Shigella in food, kit and detection method thereof | |
| CN114317808A (en) | Quantitative detection kit, detection method and application of thermophilic fungi | |
| CN101649352A (en) | Quick qualitative and quantitative measuring method of fermented lactobacillus in probiotic milk products | |
| CN111500751A (en) | Detection method and kit for rapidly detecting high-toxicity Klebsiella pneumoniae | |
| CN104894232A (en) | Citrobacter freundii fluorescent quantitative PCR diagnostic reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141210 |