CN108642195A - A kind of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods - Google Patents
A kind of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods Download PDFInfo
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Abstract
The invention belongs to round pcr fields, and in particular to a kind of Aeromonas schubertii TaqMan MGB real-time fluorescence quantitative PCR detection methods.Designing specific primer and TaqMan-MGB probes, the standard curve of TaqMan-MGB probe for real-time fluorescence quantitative PCR detecting methods of foundation to the gene conserved regions rpoD of Aeromonas schubertii has preferable linear relationship；The TaqMan MGB real-time fluorescence quantitative PCR detection methods high sensitivity of foundation, high specificity, it can be preferably used for the detection of Aeromonas schubertii in cultivation water environment and Fish tissue, be of great significance to early diagnosis, epidemiological study and the Control Technology research etc. of Aeromonas schubertii disease.
Description
Technical field
The invention belongs to round pcr fields, and in particular to a kind of Aeromonas schubertii TaqMan-MGB real-time fluorescences are fixed
Measure PCR detection method.
Background technology
Aeromonas schubertii (Aeromonas schubertii) belongs to aeromonas section (Aeromonadaceae) gas list
Born of the same parents Pseudomonas (Aeromanas), is gram-Negative bacillus, and monopole flagellum moves extremely active.The bacterium be widely present in fresh water,
It can cause to infect in seawater, soil, fish and vertebrate enteron aisle, after human contact, be the important pathogenic bacteria of acute diarrhea.
Food, food poisoning sick sample and diarrhea patient in detected the bacterium, also have the report that the bacterium is separated on infected wound.
In aquaculture, Aeromonas schubertii, which can infect, causes murrel section fish (Channidae) to lead to its internal organ yeast-like symbiotes, the disease
It is common in the main breed variety such as snakehead, snakehead and hybridized snakehead fish, is to endanger one of murrel section fish culture most serious disease.Shu Bai
Special Aeromonas As Pathogen is frequently broken out in murrel section fish breeding process, due to the disease disease symptom early period unobvious, the course of disease of falling ill
It is short, the death rate is high, middle and later periods classical symptom be internal organ class tubercle, this and Nocard's bacillus, mycobacteria and rickettsia-like organism etc.
Caused fish tubercle disease symptoms are similar, easily cause mistaken diagnosis in the breeding process, and wrong medicine gives murrel section fish farming industry band
Carry out heavy losses.
Murrel section fish diseases caused by Aeromonas schubertii infection seriously constrain the sound development of aquaculture, at present still
The prevention that disease is carried out for the commercial vaccine of the bacterium is not developed.Epidemiological survey, early diagnosis and the environment of disease
The monitoring of middle pathogen has a very important role for the prevention of disease, and the foundation of the detection method of pathogen is above
The basis of research, conventional microbial physiology biochemical identification method operating process is cumbersome, time-consuming, and hardly results in accurate result,
Usually delay treatment diagnoses.
Therefore, the pathogen how is rapidly and accurately identified to efficiently control the state of an illness, is had in breeding production practice
It is significant, and the Quantitative Monitoring of pathogen is the basis of the disease early warning and Control Technology research.The diagnosis of conventional bacteria disease needs
Carry out pathogenicbacteria separation, culture, Physiology and biochemistry identification and regular-PCR identification etc., lack to pathogen carry out quickly, it is accurate,
The detection method for early warning of quantitative analysis.Fluorescence quantitative PCR detection technique has quick, sensitive, specific high, the detection range of linearity
It is wider, carry out accurate quantification analysis to amplified production, be less prone to the advantages that false positive, be just gradually applied to the micro- life of aquatic products cause of disease
In the detection and research of object.
Invention content
The object of the present invention is to provide a kind of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCRs detection sides
Method.Specific primer and TaqMan-MGB probes are designed to the gene conserved regions rpoD of Aeromonas schubertii, foundation
The standard curve of TaqMan-MGB probe for real-time fluorescence quantitative PCR detecting methods has preferable linear relationship；It is minimum to detect
To 36 rpoD gene copies；The coefficient of variation is 0.44% in 30 parallel sample repetitive test groups；To other 10 kinds of aquatic products
Common bacteria is cultivated without amplified reaction.The Aeromonas schubertii content in pond water and Fish tissue is examined using this method
Surveying result and count plate result has preferable consistency.Therefore, the TaqMan-MGB real-time fluorescence quantitative PCRs detection side of foundation
Method high sensitivity, high specificity can preferably be used for the detection of Aeromonas schubertii in cultivation water environment and Fish tissue, right
Early diagnosis, epidemiological study and Control Technology research of Aeromonas schubertii disease etc. are of great significance.
The technical scheme is that：
A kind of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods, include the following steps：
(1) special using Primer5 Software for Design according to the Rpod gene orders of Aeromonas schubertii in GenBank
Property primer and probe, primer and probe sequence are：
Rpod-1F:5'ATGGAGCAAACCCCGCAGTC 3'
Rpod-1R:5'CGAGAACTTGTAGCCACGACGG 3'
Rpod-2F：5'CGGCTCCGAACTGGGTG 3'
Rpod-2R：5'CGAGCCACTTCCGGATCC 3'
Rpod-probe:5'CGTCACCGTCGTCGTCGTTAC 3'
(2) prepared by standard items template：DNA with Aeromonas schubertii separation strains WL-2 is template, Rpod-1F, Rpod-
1R is that primer carries out PCR amplification, and amplified production is connect after glue recovery purifying with pMD-18T carriers, is transformed into bacillus coli DH 5
In α, PCR detect positive colony and be sequenced it is correct after, extraction recombinant plasmid through spectrophotometric determination recombinant plasmid concentration, according to
Formula：Molecule copy number (copies μ L-1)=6.02 × 1023(copies·mol-1) × plasmid concentration (g μ L-1)/plasmid
Molecular weight (gmol-1) calculate recombinant plasmid DNA copy number, 10 times of multiple proportions are carried out to recombinant plasmid with EASY Dilution
Dilution, makes its concentration be followed successively by 3.6 × 108~3.6 × 101copies·μL-18 groups of standard items；
(3) standard curve is established：Respectively 3.6 × 10 are followed successively by with concentration in step (2)8~3.6 × 101copies·μL-18 groups of standard items be template, using Rpod-2F, Rpod-2R as primer, be added probe Rpod-probe, Premix (Takara)
Ex TaqTMTaqMan-MGB real-time fluorescence quantitative PCR reactions are carried out with ROXReference Dye II (50 ×), obtain standard items
Standard curve between copy number and Ct values；
(4) sample detection：Bacterium or tissue gene group DNA in sample to be tested are extracted, and using this DNA as template, with Rpod-
2F, Rpod-2R are primer, and probe Rpod-probe, Premix (Takara) Ex Taq are addedTMWith ROX Reference Dye
II (50 ×) carry out TaqMan-MGB real-time fluorescence quantitative PCR reactions, according to detected sample to be tested Ct values, according to standard
Curve calculates DNA of bacteria molecule in sample to be tested and copies.
Preferably, PCR amplification condition is in step (2)：94 DEG C of pre-degenerations 3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s,
Totally 30 cycles, last 72 DEG C of extensions 8min.
Preferably, the recombinant plasmid extracted in step (2) absorbance at 260nm and 280nm through spectrophotometric determination
Ratio is converted to recombinant plasmid concentration.
Preferably, TaqMan-MGB real-time fluorescence quantitative PCR reaction systems are in step (3) and step (4)：Premix
Ex TaqTM10 μ L, ROX Reference Dye II (50 ×), 0.4 μ L, PCR Forward Primer and PCR Reverse
(final concentration is 0.20 μm of olL to each 0.4 μ L of Primer-1), 0.6 μ L of probe Rpod-probe (final concentration of 0.30 μm of ol
L-1), DNA profiling 2 μ L, ddH26.6 μ L of O, total system are 20 μ L.
Preferably, TaqMan-MGB real-time fluorescence quantitative PCR reaction conditions are in step (3) and step (4)：The first step,
95℃30s；Second step, 40 cycles, 95 DEG C of 5s, 60 DEG C of 34s (collecting FAM fluorescence signals).
Preferably, sample to be tested is cultivating pool water in step (4), the extraction sides bacterial genomes DNA in the sample to be tested
Method is：It takes pond water sample 10000r/min to centrifuge 5min, discards supernatant, extracted with bacterial genomes DNA extraction kit
Genomic DNA in precipitation.
Preferably, sample to be tested is Fish tissue in step (4), tissue gene group DNA extraction method in the sample to be tested
For：Acquisition fish body liver, spleen or nephridial tissue are ground, and genomic DNA is extracted with tissue gene group DNA extraction kit.
The Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods are in detection pond water and fish body
The application of Aeromonas schubertii content.
Beneficial effects of the present invention are as follows：
RpoD gene conserved regions design specific primer and TaqMan-MGB of the present invention for Aeromonas schubertii
Probe, TaqMan-MGB probe for real-time fluorescence quantifying PCR methods of foundation avoid Standard PCR detection, and there are false positives and PCR
The drawbacks such as pollution.TaqMan-MGB probes are improved on the basis of TaqMan probe, are increased at the ends 3' of probe
The quenching group of MGB (mi-nor groove binder) molecule, probe uses non-fluorescence quenching group, to substantially reduce
Real-time fluorescence PCR reacts background signal.TaqMan-MGB fluorescence quantifying PCR methods that this research is established are minimum to can detect 36
A copy, 2 orders of magnitude sensitiveer than regular-PCR method are easy to, in the pathogenetic early detection pathogenic bacterial infection of disease, make in time
Early warning and alert gets the time for the prevention and control of the disease.The annealing temperature of probe is improved 10 DEG C or so by the modification of MGB simultaneously, is improved
Specificity in PCR reaction process.Since difference is smaller between each bacterium of Aeromonas, common 16S Rna gene sequencing
Technology is difficult to identify kind, and rpoD genes are the genes of 70 factors of coding RNA polymerase σ, the conservative in bacterium kind compared with
Height is widely used in the molecular biology classification of pathogen, in identification research, and the present invention is according to the rpoD of Aeromonas schubertii
The fluorescent quantitative PCR detection method that gene order is established, all to Aeromonas schubertii type strain ATCC43700 and WL-2 bacterial strain
With typical amplification curve；With belong to Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria and other common aquatic products
It is yellow to cultivate pathogen Streptococcusagalactiae, Nocard's bacillus, Pseudomonas fluorescens, Edwardsiella tarda, Channel-catfish tardas, column
The equal no cross reaction of bacillus, Plesiomonas shigelloides, it is consistent with negative control result, it is stronger to show that established method has
Specificity.The generation of bacterial disease is mostly due to infection host when pathogenic microorganism is in environment acute variation or poor environment
It is caused.Therefore the dynamic change for studying pathogen in breeding environment and host can provide theoretical direction for fish culture, together
When for disease provide early warning.Aeromonas schubertii TaqMan-MGB fluorescence quantifying PCR methods that this research and utilization is established
In the analog culture pond water sample and Fish tissue sample that detect Aeromonas schubertii content with count plate result
Consistency is higher, shows that the fluorescence quantifying PCR method established can preferably be applied in cultivation water environment and Fish tissue easypro primary
In the monitoring of special Aeromonas.
The TaqMan-MGB fluorescent quantitative PCR detection methods that this research is established have high sensitivity, repeatability strong, special
Property good, the characteristics of being widely used, can be quickly and accurately quantitative to cause of disease, for murrel section fish Aeromonas schubertii disease
Early diagnosis and epidemiological study and Control Technology research etc. are of great significance.
Description of the drawings
Attached drawing 1 is that TaqMan-MGB real time fluorescent quantitatives specific primer Rpod-2F, Rpod-2R PCR expand in embodiment 1
Increase result；
Attached drawing 2 is Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR examination criteria curves in embodiment 1；
Attached drawing 3 is Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR sensitivity technique results in embodiment 1
Amplification curve diagram, wherein during the value of Δ Rn indicates PCR, the amount of probe degradation；
Attached drawing 4 is 30 repetition experiment knots of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCRs in embodiment 1
The amplification curve diagram of fruit, wherein during the value of Δ Rn indicates PCR, the amount of probe degradation；
Attached drawing 5 is Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR specific detection results in embodiment 1
Amplification curve diagram, wherein during the value of Δ Rn indicates PCR, the amount of probe degradation.
Specific implementation mode
With reference to embodiment, technical scheme of the present invention is described in further detail, but do not constituted pair
Any restrictions of the present invention.
In following embodiment：Artificial challenge is healthy hybridized snakehead fish (80-120g) with experiment fish, is purchased from Guangzhou Jing Yang fries
；Genome DNA extracting reagent kit is purchased from Beijing TIANGEN companies.
Embodiment 1
A kind of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods, include the following steps：
1, the foundation of PCR amplification method
1.1 design of primers
According to the Rpod gene orders (CP013067.1) of Aeromonas schubertii in GenBank, application
Primer5 Software for Design specific primer and probe, pair of primers (Rpod-1F, Rpod-1R) expand Rpod Gene Partial sequences
Row, amplified production length are 1302bp, are used for construction recombination plasmid, and template is provided to make standard curve；Second pair of primer
(Rpod-2F, Rpod-2R) and MGB probes (Rpod-probe) are used for fluorescent quantitative PCR, and amplified production length is
129bp.Primer and probe is synthesized by Guangzhou Ai Ji Bioisystech Co., Ltd.Primer and probe sequence is：
Rpod-1F:5'ATGGAGCAAACCCCGCAGTC 3'
Rpod-1R:5'CGAGAACTTGTAGCCACGACGG 3'
Rpod-2F：5'CGGCTCCGAACTGGGTG 3'
Rpod-2R：5'CGAGCCACTTCCGGATCC 3'
Rpod-probe:5'CGTCACCGTCGTCGTCGTTAC 3'
It is prepared by 1.2 standard items templates
It is template with the DNA of WL-2, Rpod-1F, Rpod-1R are that primer carries out PCR amplification.Response parameter is 94 DEG C of pre- changes
Property 3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, totally 30 cycle, it is last 72 DEG C extension 8min.Amplified production recycles pure through glue
It connect, is transformed into bacillus coli DH 5 alpha with pMD-18T carriers (TaKaRa) after change, Ai Jisheng is sent after PCR detection positive colonies
Object Technology Co., Ltd. is sequenced.Correct recombinant plasmid is sequenced after measured after the absorbance at 260 and 280nm, according to following
Method calculates the DNA copy number of recombinant plasmid.Molecule copy number (copies μ L-1)=6.02 × 1023(copies·mol-1)
× plasmid concentration (g μ L-1)/plasmid molecule amount (gmol-1).It is right with EASY Dilution (for Real Time PCR)
Standard items carry out 10 times of doubling dilutions, for building standard curve.
The foundation of 2, TaqMan-MGB quantitative fluorescent PCRs and condition optimizing
Using TaqMan-MGB probe techniques, with reference to Premix Ex TaqTM(Probe qPCR) specification, by upstream and downstream
Primer Rpod-2F and Rpod-2R, probe Rpod-probe, Premix (Takara) Ex TaqTMWith ROX Reference Dye
II (50 ×) are added in reaction system, with QuantStudioTM 6Flex types fluorescence quantitative PCR instrument (American AB I companies) into
Row PCR amplification.Each component concentration and response procedures are optimized, primer and probe concentration is screened, it is minimum to obtain
Ct values and highest fluorescence intensity value added standard.Select 0.2,0.4,0.6,0.8 μm of olL-1Primer concentration and 0.2,
0.4、0.6、0.8μmol·L-1Concentration and probe concentration, screen the optium concentration of primer and probe.Respectively choose 56 DEG C, 58 DEG C, 60
DEG C, 62 DEG C be annealing temperature, screen optimum annealing temperature.
3, standard curve is established
Recombinant plasmid is subjected to 10 times of gradient dilutions, its concentration is made to be followed successively by 3.6 × 108~3.6 × 101copies·μL-1As standard items template, each dilution set 3 it is parallel.The reaction system of optimization and under the conditions of carry out real time fluorescent quantitative
PCR amplification draws standard curve after reaction.
Sensibility, repeatability and the specific test of 4, TaqMan-MGB fluorescent quantitative PCR detection methods
The sensitivity tests of 4.1 TaqMan-MGB fluorescent quantitative PCR detection methods
Recombinant plasmid is subjected to 10 times of gradient dilutions, its concentration is made to be followed successively by 3.6 × 106~3.6 × 100copies μ L-1As standard items template, with ddH2O replaces template as negative control, and it is fixed to carry out fluorescence with the reaction system of optimization and condition
Measure PCR reaction, according between samples copy number and Ct values linear relationship and related coefficient determine minimum dfetectable quantity.Simultaneously with drawing
Object Rpod-2F, Rpod-2R carry out common PCR reaction, calculate the minimum template concentrations that 2 kinds of methods can be detected out, compare two
The sensibility of kind method.
The reperformance test of 4.2 TaqMan-MGB fluorescent quantitative PCR detection methods
It repeats 30 progress real-time fluorescence quantitative PCR detections in one experiment to same sample, passes through the Ct values in group
The coefficient of variation carrys out the repeatability of entry evaluation this method.
The specific test of 4.3 TaqMan-MGB fluorescent quantitative PCR detection methods
Extraction bacterial strain Aeromonas schubertii type strain ATCC43700, Aeromonas veronii (A.veronii) respectively
JZ09, Aeromonas sobria (A.sobria) SJ-1, Streptococcusagalactiae (Streptococcus agalactiae) 1535, promise card
Salmonella (Nocardia seriolae) WL-24, Pseudomonas fluorescens (Pseudomonas fluorescens) BD-1, slow love
Moral Fahrenheit bacterium (Edwardsiella tarda) HG-2, Channel-catfish tarda (Edwardsiella ictaluri) zbl141, column
Shape Flavobacterium (Flavobacterium columnare) CY-1, Plesiomonas shigelloides (Plesiomonas
Shigelloides) J-1 plants of HP-1, Aeromonas hydrophila bacterium solution genomic DNAs, with the real time fluorescence quantifying PCR method of foundation
Specific amplification is carried out, while setting ddH2O is the negative control of masterplate and positive control that WL-2 is template to evaluate this method
Specificity.
The application of 5, TaqMan-MGB fluorescent quantitative PCR detection methods
The validity for evaluating Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR detection methods, is utilized respectively
This method detects the Aeromonas schubertii content in analog culture pond water and Fish tissue sample, verifies the accurate of this method
Rate.
The detection of Aeromonas schubertii content in 5.1 pond waters
With the WL-2 bacterium solutions of BHI medium cultures 12h, bacterial concentration is first diluted to 1 × 10 with sterile water9CFU·mL-1
(Maxwell turbidimetry for Determination is carried out at the same time count plate confirmation) afterwards, then be added in pond water with after 10 times of gradient dilutions, keep its dense
Degree is followed successively by 1 × 108~1 × 103CFU·mL-1As analog culture pond water sample, it is not added with the pond water and ddH of WL-2 bacterium2O
Respectively as negative and blank control, all samples extract genomic DNA as masterplate, are examined with fluorescence quantifying PCR method
It surveys, according to detected sample Ct values, DNA of bacteria molecule in sample is calculated according to standard curve and is copied, and be actually added into
Amount of bacteria compare, verify the accuracy of fluorescence quantifying PCR method.
The detection of Aeromonas schubertii content in 5.2 Fish tissues
Utilize 1.5 × 106CFU·mL-1WL-2 bacterium solutions, the healthy hybridized snakehead fish (200 μ L/ tails) of intraperitoneal injection infection, in attacking
2d acquires liver, spleen and nephridial tissue and is ground after poison, extracts genomic DNA, and it is real-time to carry out TaqMan-MGB by the method for optimization
Fluorescence quantitative PCR detection calculates DNA of bacteria molecule in sample and copies.Meanwhile sick sample tissue being carried out to conventional bacterium physics and chemistry
CHARACTERISTICS IDENTIFICATION, 16S rRNA gene orders compare and count plate, relatively determine the accuracy of this method.
6 results and analysis
The PCR amplification of 6.1 RpoD genes and clone
Aeromonas schubertii DNA carries out PCR amplification using primer Rpod-1F, Rpod-1R, obtains 1302bp's or so
Segment after the segment is cloned into carrier pMD18-T, confirms identical as expected Insert Fragment through sequencing.It is passed through after recombinant plasmid extraction
A concentration of 799 μ gmL of spectrophotometric determination-1, A260/A280 values are 1.95.
The foundation of 6.2 TaqMan-MGB real-time fluorescence quantitative PCRs and condition optimizing
After recombinant plasmid carries out regular-PCR amplification using fluorescence quantification PCR primer Rpod-2F, Rpod-2R, product is through fine jade
Sepharose electrophoretic analysis, amplification are as shown in Fig. 1, wherein M. standard DNAs DL2000；1-6. plasmid concentrations are distinguished
It is 3.6 × 106~3.6 × 101copies·μL-1.There is a specific band in 150bp or so, with expected fragment length
(129bp) is almost the same, shows that the primer can be used for quantitative fluorescent PCR reaction.By the excellent of quantitative fluorescent PCR reaction condition
Change, it is final to determine that reaction system is：Premix Ex TaqTM10 μ L, ROX Reference Dye II (50 ×) 0.4 μ L, PCR
(final concentration is 0.20 μm of olL to each 0.4 μ L of Forward Primer and PCR Reverse Primer-1), probe Rpod-
0.6 μ L of probe (final concentration of 0.30 μm of olL-1), DNA profiling 2 μ L, ddH26.6 μ L of O, total system are 20 μ L.PCR amplification
Program：The first step, 95 DEG C of 30s；Second step, 40 cycles, 95 DEG C of 5s, 60 DEG C of 34s (collecting FAM fluorescence signals).
The foundation of 6.3 standard curves
To choose 3.6 × 10 after 10 times of gradient dilution recombinant plasmids8~3.6 × 101copies·μL-18 dilutions
Fluorescent quantitative PCR is carried out, amplification curve is obtained, is Y-axis mapping by X-axis, Ct values of recombinant plasmid copy number, what is obtained relaxes
Bert Aeromonas TaqMan-MGB real-time fluorescence quantitative PCR examination criteria curves are as shown in Fig. 2, which is
Y=-3.25x+35.85, coefficient R2=0.9998, amplification efficiency 103% illustrates real-time fluorescence quantitative PCR 108
~101copies·μL-1There is preferable linear relationship in range.
Sensitivity, repeatability and the specific test result of 6.4 TaqMan-MGB quantitative fluorescent PCRs
6.4.1 to recombination 10 times of gradient dilutions of plasmid standard after, obtain concentration and be followed successively by 3.6 × 106~3.6 ×
100copies·μL-1Standard items sample carries out regular-PCR and TaqMan-MGB fluorescent quantitative PCRs respectively to sample.
The amplification curve diagram such as attached drawing 3 of the Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCR sensitivity technique results arrived
It is shown, the results show that quantitative fluorescent PCR expands recombinant plasmid, it is minimum to can detect 36 DNA of bacteria molecule copy numbers；
And as can be seen that regular-PCR method is minimum 3.6 × 10 in attached drawing 23copies·μL-1Place can detect band；Therefore, it relaxes
100 times sensitiveer than regular-PCR method of Bert Aeromonas TaqMan-MGB real-time fluorescence quantitative PCRs.
6.4.2 for statistical analysis to 30 detected Ct values of same positive repetition, obtained Shu Baite gas
The amplification curve diagram of monad TaqMan-MGB 30 repetition test results of real-time fluorescence quantitative PCR is as shown in Fig. 4, as a result table
Bright, the amplification curve of 30 Duplicate Samples is substantially coincident near threshold line, and Ct value range of readings is 17.77~17.07, standard
Deviation is 0.085, the coefficient of variation 0.44%.
6.4.3 with strains A TCC43700, WL-2, JZ09, SJ-1, J-1,1535, WL-24, BD-1, HG-2, zbl141,
CY-1, HP-1 bacterium solution genomic DNA are template, carry out real-time fluorescence quantitative PCR specific detection, obtained Shu Baite gas unit cells
The amplification curve diagram of bacterium TaqMan-MGB real-time fluorescence quantitative PCR specific detection results is as shown in Fig. 5, the results showed that,
ATCC43700 and WL-2 has typical amplification curve, and fluorescence signal value is high, and testing result is the positive, and remaining bacterium and feminine gender are right
According to no amplification curve, amplified reaction does not occur.
6.5 the application of TaqMan-MGB fluorescent quantitative PCR detection methods
6.5.1 in the water of analog culture pond Aeromonas schubertii content detection
Maxwell turbidimetry for Determination a concentration of 1 × 109CFU·mL-1WL-2 bacterium solutions, reality is measured by count plate after dilution
Border a concentration of 0.96 × 109CFU·mL-1.Using the fluorescence quantifying PCR method detection analog culture pond water sample result of foundation
1 is see the table below, the results show that passing through Shu Baite gas in the calculated 5 pond water samples of Ct values after fluorescence quantifying PCR method detection
The content of monad and the amount of being actually added into are almost the same, and it is minimum can detect 13 bacteriums in pond water, WL- is not added
The pond water and dd H of 2 bacterium2Aeromonas schubertii is not detected in O.
Aeromonas schubertii content results in the fluorescence quantitative PCR detection pond water of table 1
The detection of Aeromonas schubertii content in 6.52 Fish tissues
The fluorescence quantifying PCR method established with this experiment, to hybridized snakehead fish liver, spleen and the nephridial tissue of artificial challenge 2d respectively into
Row detection see the table below 2, Conventional bacteria separation identification by the copy number and count plate result of the calculated corresponding gene of Ct values
The results show that separated bacterium is Aeromonas schubertii.The gene for three tissues that fluorescence quantifying PCR method is measured is copied
Shellfish number is consistent with count plate result.
Table 2 infects Aeromonas schubertii content results in Fish tissue with fluorescence quantitative PCR detection
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (9)
1. a kind of primer and probe of Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCRs detection, which is characterized in that
Its sequence is：
Rpod-1F:5'ATGGAGCAAACCCCGCAGTC 3'
Rpod-1R:5'CGAGAACTTGTAGCCACGACGG 3'
Rpod-2F：5'CGGCTCCGAACTGGGTG 3'
Rpod-2R：5'CGAGCCACTTCCGGATCC 3'
Rpod-probe:5'CGTCACCGTCGTCGTCGTTAC 3' 。
2. primer and probe described in claim 1 is detected in Aeromonas schubertii TaqMan-MGB real-time fluorescence quantitative PCRs
Application in method.
3. application according to claim 2, which is characterized in that Aeromonas schubertii TaqMan-MGB real time fluorescent quantitatives
PCR detection method includes the following steps：
(1) prepared by standard items template：DNA with Aeromonas schubertii separation strains WL-2 is template, and Rpod-1F, Rpod-1R are
Primer carries out PCR amplification, and amplified production connect with pMD-18T carriers after glue recovery purifying, is transformed into bacillus coli DH 5 alpha,
After PCR detects positive colony and is sequenced correctly, extraction recombinant plasmid is through spectrophotometric determination recombinant plasmid concentration, according to public affairs
Formula：Molecule copy number=6.02 × 1023× plasmid concentration/plasmid molecule amount calculates the DNA copy number of recombinant plasmid, uses EASY
Dilution carries out 10 times of doubling dilutions to recombinant plasmid, its concentration is made to be followed successively by 3.6 × 108~3.6 × 101copies·μL-18 groups of standard items；
(2) standard curve is established：Respectively 3.6 × 10 are followed successively by with concentration in step (2)8~3.6 × 101copies·μL-18
Group standard items are template, and using Rpod-2F, Rpod-2R as primer, probe Rpod-probe, Premix Ex Taq is addedTMAnd ROX
Reference Dye II carry out TaqMan-MGB real-time fluorescence quantitative PCR reactions, obtain between standard items copy number and Ct values
Standard curve；
(3) sample detection：Extract bacterium or tissue gene group DNA in sample to be tested, and using this DNA as template, with Rpod-2F,
Rpod-2R is primer, and probe Rpod-probe, Premix Ex Taq is addedTMIt is carried out with ROX Reference Dye II
TaqMan-MGB real-time fluorescence quantitative PCRs react, and according to detected sample to be tested Ct values, are calculated and are waited for according to standard curve
DNA of bacteria molecule copies in sample.
4. application according to claim 3, which is characterized in that PCR amplification condition is in step (1)：94 DEG C of pre-degenerations
3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, totally 30 recycle, last 72 DEG C of extensions 8min.
5. application according to claim 3, which is characterized in that the recombinant plasmid extracted in step (1) is through spectrophotometer
It measures the absorbance ratio at 260nm and 280nm and is converted to recombinant plasmid concentration.
6. application according to claim 3, which is characterized in that TaqMan-MGB real-time fluorescences in step (2) and step (3)
Quantitative PCR reaction system is：Premix ExTaqTM10 μ L, ROX Reference Dye, II 0.4 μ L, PCR Forward
Primer and PCR Reverse Primer 0.6 μ L of each 0.4 μ L, probe Rpod-probe, DNA profiling 2 μ L, ddH2O 6.6μ
L, total system are 20 μ L.
7. application according to claim 3, which is characterized in that TaqMan-MGB real-time fluorescences in step (2) and step (3)
Quantitative PCR reaction condition is：The first step, 95 DEG C of 30s；Second step, 40 cycles, 95 DEG C of 5s, 60 DEG C of 34s；Collect FAM fluorescence letter
Number.
8. application according to claim 3, which is characterized in that sample to be tested is cultivating pool water in step (3), this is to be measured
Bacterial genomes DNA extraction method is in sample：It takes pond water sample 10000r/min to centrifuge 5min, discards supernatant, with thin
Genomic DNA in bacterium genome DNA extracting reagent kit extraction precipitation.
9. application according to claim 3, which is characterized in that sample to be tested is Fish tissue in step (3), this waits for test sample
Tissue gene group DNA extraction method is in product：Acquisition fish body liver, spleen or nephridial tissue are ground, and are extracted with tissue gene group DNA
Kit extracts genomic DNA.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129461A (en) * | 2019-04-19 | 2019-08-16 | 南开大学 | To the gene liquid chip and detection method of three kinds of serotype O antigens genotypings of Plesiomonas shigelloides |
| CN110878365A (en) * | 2019-11-22 | 2020-03-13 | 广东海洋大学深圳研究院 | Method for detecting pathogenic bacteria of fish sarcoidosis by multiple PCR |
| CN112239777A (en) * | 2019-07-16 | 2021-01-19 | 汕头大学 | Qualitative and quantitative detection method for carotenoid producing bacteria |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965438A (en) * | 2012-11-16 | 2013-03-13 | 中国水产科学研究院珠江水产研究所 | Duplex polymerase chain reaction (PCR) detection primer group, kit and method for pathogenic channa source aeromonas schubertii |
| CN105420373A (en) * | 2015-12-22 | 2016-03-23 | 于辉 | Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas |
| CN105734165A (en) * | 2016-05-11 | 2016-07-06 | 辽宁大学 | Aeromonas schubertii specific primer and application thereof in turbot farming process |
| CN108179203A (en) * | 2017-12-20 | 2018-06-19 | 中国水产科学研究院珠江水产研究所 | The primer and probe and kit and detection method of a kind of murrel Aeromonas schubertii fluorescence quantitative PCR detection |
-
2018
- 2018-06-26 CN CN201810669723.9A patent/CN108642195A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965438A (en) * | 2012-11-16 | 2013-03-13 | 中国水产科学研究院珠江水产研究所 | Duplex polymerase chain reaction (PCR) detection primer group, kit and method for pathogenic channa source aeromonas schubertii |
| CN105420373A (en) * | 2015-12-22 | 2016-03-23 | 于辉 | Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas |
| CN105734165A (en) * | 2016-05-11 | 2016-07-06 | 辽宁大学 | Aeromonas schubertii specific primer and application thereof in turbot farming process |
| CN108179203A (en) * | 2017-12-20 | 2018-06-19 | 中国水产科学研究院珠江水产研究所 | The primer and probe and kit and detection method of a kind of murrel Aeromonas schubertii fluorescence quantitative PCR detection |
Non-Patent Citations (2)
| Title |
|---|
| J Y LIU等: "First case of Aeromonas schubertii infection in the freshwater cultured snakehead fish, Ophiocephalus argus (Cantor), in China", 《JOURNAL OF FISH DISEASES》 * |
| 王清印: "《从产量到质量 海水养殖业发展的必然趋势》", 31 October 2009, 海洋出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129461A (en) * | 2019-04-19 | 2019-08-16 | 南开大学 | To the gene liquid chip and detection method of three kinds of serotype O antigens genotypings of Plesiomonas shigelloides |
| CN112239777A (en) * | 2019-07-16 | 2021-01-19 | 汕头大学 | Qualitative and quantitative detection method for carotenoid producing bacteria |
| CN110878365A (en) * | 2019-11-22 | 2020-03-13 | 广东海洋大学深圳研究院 | Method for detecting pathogenic bacteria of fish sarcoidosis by multiple PCR |
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