CN108841985A - It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani - Google Patents
It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the nucleotide sequence group of Fusarium solani, including with downstream primer shown in upstream primer shown in SEQ ID No.2 and SEQ ID No.3, it and can also include the fluorescence probe as shown in SEQ ID No.4, the sequence being based on to primer is highly conserved, high degree of specificity, Fusarium solani is optionally expanded, so that it be come with the common other strain separatings of same Fusarium.Kit and PCR detection method based on this to primer, specificity, sensitivity and operation ease with higher.
Description
Technical field
The present invention relates to a kind of gene engineering technology fields more particularly to a kind of for detecting the nucleotide of Fusarium solani
Sequence group, kit and method.
Background technique
Fusarium solani (Fusarium solani) is the main pathogenic bacteria of plant root rot, mainly to Radix Astragali, Radix Salviae Miltiorrhizae,
The generations such as Radix Glycyrrhizae, Radix Isatidis harm.Root rot is a kind of fungus-caused phytopathy, which will cause butt rot, causes to plant
Object absorbs moisture and the function of nutrient gradually weakens, and last complete stool is dead, is mainly shown as whole strain yellow leaf, withers.Beancurd sheet
Sickle-like bacteria determines that it is macroconidium according to the presence of its shape, chlamydospore and long phial, however Morphological Identification cannot
Different biological species and the mitosis bacterial strain of gene diversity with common morphological characteristic are distinguished, such as according to traditional mirror
Determine method, is easy to appear deviation, and take a long time.
Although multiplex PCR detects Fusarium solani, specificity is good, accuracy is high, and PCR result needs to pass through agar
Sugared electrophoresis is analyzed, and influence factor is more.With the continuous development of Protocols in Molecular Biology, real-time fluorescence quantitative PCR is current
It is widely used to the detection of strain.It has time-consuming short, easy to operate, specific compared with conventional isolated culture method
Good feature, and result can be observed directly, and caused pollution in operating process can be effectively avoided.
Fusarium solani and other reaping hook category strains have higher homology, such as Fusarium oxysporum, fusarium tricinctum, standing grain
Paddy sickle-like bacteria needs to select highly conserved, special sequence as rake sequence for the high degree of specificity for realizing PCR reaction.
Summary of the invention
For overcome the deficiencies in the prior art, one of the objects of the present invention is to provide high specifics for detecting Beancurd sheet
The nucleotide sequence group of sickle-like bacteria.
The second object of the present invention is to provide a kind of for detecting the kit of Fusarium solani.
The third object of the present invention is to provide a kind of method for detecting Fusarium solani.
An object of the present invention adopts the following technical scheme that realization：
It is a kind of for detecting the nucleotide sequence group of Fusarium solani, including with upstream primer shown in SEQ ID No.2
With downstream primer shown in SEQ ID No.3.
It further, further include the probe as shown in sequence table SEQ ID No.4, the probe is fluorescence labeling probe.
Further, the fluorescent reporter group of 5 ' end labels of the probe is FAM, the fluorescent quenching group of 3 ' end labels
For BHQ.
The second object of the present invention adopts the following technical scheme that realization：
It is a kind of for detecting the kit of Fusarium solani, including upstream primer and SEQ as shown in SEQ ID No.2
Downstream primer shown in ID No.3 and the probe as shown in sequence table SEQ ID No.4.
Further, the fluorescent reporter group of 5 ' end labels of the probe is FAM, the fluorescent quenching group of 3 ' end labels
For BHQ.
It further, further include the standard items of positive plasmid, the standard items of the positive plasmid are with Fusarium solani
DNA is template, by downstream primer PCR amplification shown in the upstream primer as shown in SEQ ID No.2 and SEQ ID No.3 time
It receives, is obtained after being attached, transfect with pMD18-T plasmid.
The third object of the present invention is implemented with the following technical solutions：
A method of for detecting Fusarium solani, include the following steps：
1) standard items of positive plasmid are obtained：Using the DNA of Fusarium solani as template, by as shown in SEQ ID No.2
Upstream primer and SEQ ID No.3 shown in downstream primer PCR amplification recycling, after being attached, transfect with pMD18-T plasmid
Obtain the standard items of positive plasmid；
2) PCR is identified：Respectively using the DNA of sample to be tested, positive plasmid standard items as template, with ddH2O is negative right
According to, pass through downstream primer shown in the upstream primer as shown in SEQ ID No.2 and SEQ ID No.3 carry out PCR reaction examine.
Further, in step 2), PCR reaction system is：10×PCR buffer 2.5μL,dNTP(2.5μM)0.5μ
L, upstream primer (1 μM) 0.5 μ L, downstream primer (1 μM) 0.5 μ L, 0.5 μ L of template, Taq enzyme (5U) 0.5 μ L and ddH2O supplies 25
μL；
PCR reaction condition is：94 DEG C of initial denaturation 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃
10min。
Further, in step 2), PCR amplification system includes the probe as shown in sequence table SEQ ID No.4.
Further, PCR reaction system：2 × premix, 10 μ L, upstream primer (10 μM) 0.2 μ L, downstream primer (10 μ
M) 0.2 μ L, probe (10 μM) 0.2 μ L, 0.3 μ L of template, ddH2O complements to 20 μ L.
PCR reaction condition：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 circulations.
Compared with prior art, the beneficial effects of the present invention are：
The present invention is provided to detect nucleotide sequence group, kit and the method for Fusarium solani, mainly to corruption
The Fs tef1 gene of the highly conserved specificity of skin sickle-like bacteria draws as rake sequence, design upstream as shown in SEQ ID No.2
Object and the downstream primer as shown in SEQ ID No.3, the amplification length of the sequence are 215bp, to Fusarium solani have compared with
Good specificity, can effectively distinguish Fusarium oxysporum, fusarium tricinctum, the Fusarium graminearum of itself and same Fusarium
Come；
The present invention is additionally provided simultaneously with the upstream primer as shown in SEQ ID No.2 and as shown in SEQ ID No.3
The corresponding probe of downstream primer, the probe can be applied to Fusarium solani in a step fluorescence quantitative analysis sample, have
Higher sensitivity and specificity；
Based on the kit and method of above-mentioned upstream and downstream primer and probe, there is relatively easily operability and higher spirit
Sensitivity, specificity.
Detailed description of the invention
Fig. 1 is the Blast result figure that embodiment 1 harrows sequence；
Fig. 2 is the Blast result figure of about 1 primer sequence of embodiment；
Fig. 3 is the canonical plotting of embodiment 5；
Fig. 4 is the sensitivity test curve of embodiment 7；
Wherein, a represents plasmid concentration as 1.00 × 107Copies/ μ L, b represent plasmid concentration as 1.00 × 106copies/
μ L, c represent plasmid concentration as 1.00 × 105Copies/ μ L, d represent plasmid concentration as 1.00 × 104Copies/ μ L, e represent matter
Grain concentration is 1.00 × 103Copies/ μ L, f represent plasmid concentration as 1.00 × 102Copies/ μ L, g represent plasmid concentration as
1.00×101Copies/ μ L and negative control.
Fig. 5 is the specific test curve of embodiment 7；
Wherein, a standard amplification curve is Fusarium solani genome, and it is respectively point that b representative, which does not occur standard amplification curve,
Fusarium oxysporum, fusarium tricinctum, Fusarium graminearum, negative control；
Fig. 6 is the specific test result figure of embodiment 8；
Wherein, it is right to be followed successively by Fusarium oxysporum, Fusarium solani, fusarium tricinctum, Fusarium graminearum, feminine gender by swimming lane 1-6
According to the Marker of, DL2000.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention, it should be noted that not
Under the premise of conflicting, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
The present invention designs PCR amplification primer and probe using highly conserved, special Fs tef1 gene as rake sequence, then
Fusarium solani Fs tef1 gene is expanded using round pcr, is connected to plasmid vector pMD18- using gene recombination technology
In T, recombinant plasmid pMD18-T-Fs tef1 is constructed, and carries out corresponding PCR identification and sequencing identification, is most quantitatively made afterwards
For the standard items to method for building up, lay the foundation for the method and assessment of next step.
The present invention provides a kind of for detecting the nucleotide sequence group of Fusarium solani, which includes with SEQ
ID No.1 is the primer pair for harrowing sequence design：
The upstream primer as shown in SEQ ID No.2；With
The downstream primer as shown in SEQ ID No.3.
Wherein, it is as follows to harrow sequence by SEQ ID No.1：
Wherein, the sequence of straight line underscore part is upstream primer sequence shown in SEQ ID No.2, and lower stroke of wave
The sequence of line part matches with downstream primer sequence shown in SEQ ID No.3.Upstream primer shown in SEQ ID No.2 and
The am-plified fragments size of downstream primer shown in SEQ ID No.3 is 215bp.
SEQ ID No.1 harrow sequence by ncbi database blast as the result is shown such as Fig. 1, with a variety of Beancurd sheet reaping hooks
The matching degree of the Fs tef1 gene order of bacterium isolated strains is high, similarity is up to 100%.
Upstream primer and downstream primer sequence are shown in Fig. 2 through Primer-Blast comparison result in NCBI.As shown in Figure 2, this
There is specificity to primer pair Fusarium solani.
The nucleotides sequence group includes the probe sequence as shown in SEQ ID No.4：Harrow gene point underscore part with it is described
Probe sequence is corresponding.
The fluorescent reporter group of probe sequence can preferably be designed as follows：
The fluorescent reporter group of 5 ' end labels is FAM, and the fluorescent quenching group of 3 ' end labels is BHQ.
The target sequence of the amplification to primer and probe is as shown in SEQ ID No.5.
Embodiment 1：The preparation of template DNA
It wants the present embodiment to extract Fusarium solani reference culture genomic DNA using bacterial genomes extracts kit, uses
Make the template of Fs tef1 gene PCR amplification.Its extracts kit used can be selected from the bacterium of hundred Imtech of Beijing production
Genome extraction kit, specific extracting method are as follows：
1) 1mL bacteria suspension is taken, 1.5mL centrifuge tube is added, 8000r/min is centrifuged 2 minutes, abandons supernatant；
2) 400 μ L Buffer Digestion suspension bacteria liquids precipitating is added, it is complete to cell that 65 DEG C of water-bath 1h are put into after mixing
It totally cleaves solution；
3) 200 μ L Buffer PB are added and mix -20 DEG C of standing 5min, centrifuging and taking supernatant；
4) 600 μ L isopropanols are added and mix -20 DEG C of standing 5min.Supernatant is abandoned in centrifugation；
5) -20 DEG C of standing 5min of 75% ethyl alcohol of 1mL are added, supernatant is abandoned in centrifugation；
6) ethanol washing with 75% is precipitated, it is dry, obtain template DNA；
7) it is dissolved in 50 μ L TE Buffer, -20 DEG C of preservations.
Embodiment 2：PCR amplification
The template DNA obtained with embodiment 1, with upstream primer sequence shown in SEQ ID No.2 and SEQ ID No.3 institute
The downstream primer sequence shown is expanded, and PCR reaction condition is：10×PCR buffer2.5μL,dNTP(2.5μM)0.5μL,
Upstream primer (1 μM) 0.5 μ L, downstream primer (1 μM) 0.5 μ L, 0.5 μ L of template DNA, Taq enzyme (5U) 0.5 μ L and ddH2O is supplied
25μL。
Wherein, Taq enzyme uses Beijing hundred Tyke Power Taq Plus DNAPolymerse, PCR amplification instrument Hang Zhoulang
The serial 96 grads PCR instrument of base scientific instrument Co., Ltd MG.
PCR amplification program is：
94 DEG C of initial denaturation 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃10min.5 μ L are taken to expand
Product carries out 2% agarose electrophoresis, detects PCR product size, then recycles examination using the DNA gel of Shanghai Sangon Biotech Company's production
The remaining pcr amplification product of agent box purification and recovery obtains recycling DNA.
Embodiment 3：Building, conversion and the acquisition of recombinant plasmid
A) plasmid connects
The recycling DNA and pMD18-T (Dalian treasured biotech firm) that embodiment 2 obtains is attached, using following connection
System is prepared：1 μ L of pMD18-T vector, recycling 4 μ L of DNA, Solution1 supply 10 μ L.It prepares and completes to be placed on
16 DEG C carry out staying overnight connection reaction, obtain connection product.
B) plasmid conversion identification
1) the DH5 α competent cell frozen is taken out from -70 DEG C of ultra low temperature freezer, being placed on ice chest solves it naturally
Freeze；
2) step A is taken) obtained 10 μ L of connection product is added in the DH5 α competent cell of 100 μ L；
3) heat shock 90s in 42 DEG C of water-baths sets cooled on ice 30min after heat shock immediately；
4) after LB liquid medium (without ampicillin) mixing of 800 μ L of pre-cooling is added into 1.5mL EP pipe,
37 DEG C of 140rpm jog culture 1h；
5) above-mentioned culture solution 8000rpm is centrifuged 1min, abandons supernatant, absorption residue is resuspended in cell and is coated on containing 0.1ng
It on the LB plate of Amp, faces up and places 30min, after bacterium solution is cultured base absorption completely, be inverted 37 DEG C of insulating boxs of culture dish
Overnight incubation；
6) next day is observed, and picks from the plate monoclonal colonies in 100 μ L LB liquid mediums (containing ampicillin)
In PCR pipe, 37 DEG C shaken cultivation 2-3 hours；2 μ L are drawn as template and carry out PCR identification, remaining bacterium solution is added to 20mL's
It carries out expanding in LB liquid medium and shake；
The identification reaction condition of PCR is as follows：10 × PCR buffer, 2.5 μ L, dNTP (2.5 μM) 0.5 μ L, upstream
Primer (10 μM) 0.5 μ L, downstream primer R (10 μM) 0.5 μ L, 0.5 μ L, 0.5 μ L of Taq enzyme (5U), ddH2O supplies 25 μ L；
The identification amplification program of PCR：94 DEG C of initial denaturation 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72
℃10min。
7) use the amplification of downstream primer shown in upstream primer sequence shown in SEQ ID No.2 and SEQ ID No.3 above-mentioned
Bacterium solution is diluted, PCR product uses 2% agarose gel electrophoresis, identifies positive transformant by detection PCR product size.
C) the extraction of positive recombinant plasmid：
Positive recombinant plasmid pMD18-T-Fstef1, measurement are extracted using the Plasmid Preparation kit of hundred Imtech production
Concentration and purity, while drawing a part of plasmid purification and supreme marine growth Engineering Co., Ltd is given to be sequenced, determine insertion piece
The gene order of section is consistent with aim sequence.
D the standard items of positive recombinant plasmid) are obtained：
1) take step C) the obtained 100 μ L transferred species of bacillus coli DH 5 alpha containing recombinant plasmid pMD18-T-Fs tef1 in
The LB liquid medium of 5mL, 37 DEG C of 200rpm shake training overnight；
2) take the bacterium solution transferred species of 1mL overnight incubation in 10mL LB liquid medium, 200rpm Zengjing Granule 2-3 hours, so
Plasmid is extracted using the Plasmid Preparation kit of hundred Imtech of Beijing production afterwards；
3) using hundred Tyke Biotechnology Co., Ltd ultramicron ultraviolet-uisible spectrophotometer (ND5000) of Beijing to mentioning
The plasmid taken is measured, and measures A260、A280, according to A260/A280Judge the purity of plasmid；
4) plasmid pMD18-T-Fs tef1 concentration (copy number) calculates
Molecular weight=2906bp × 660 (average molecular weight of each pair of base) of plasmid
Measuring plasmid concentration is 213.32ng/ μ L, plasmid purity A260/A280=1.91, because carrying out real time fluorescent quantitative
When PCR, need with " copy number " for unit, it is therefore desirable to by Conversion of measurement unit at copies/ μ L.
Plasmid copies/ μ L=Avgadro constant × plasmid molal quantity
Wherein Avgadro constant=6.02 × 1023copies/mol。
Therefore L=213.32 × 10 plasmid concentration copies/ μ extracted-9g/μL×6.02×1023copies/mol÷
(2906bp × 660g/bpmol)=6.69 × 1010copies/μL
It is 1.00 × 10 that 10 μ L plasmids, which add sterile water constant volume just to obtain concentration,10The plasmid of copies/ μ L, then by the plasmid into
A series of plasmid of concentration, i.e. the gradient standard items of positive plasmid just can be obtained in 10 multiple proportions gradient dilution of row, and saves in -20 DEG C
It is spare.
Embodiment 4：It is a kind of for detecting the kit of Fusarium solani
The kit includes that downstream shown in the upstream primer sequence as shown in SEQ ID No.2 and SEQ ID No.3 is drawn
Object, the probe as shown in SEQ ID No.4, wherein the fluorescent reporter group of 5 ' end labels of the probe is FAM, 3 ' end marks
The fluorescent quenching group of note is BHQ.
The kit includes the standard items for the positive plasmid that embodiment 3 obtains.
Specifically, which includes following component：
(2 × premix is the production of hundred Imtech of Beijing to 2 × premix, and full name is 2 × real-time PCR
Premixture (probe)), it is final concentration of 10 μM of upstream primer, final concentration of 10 μM of downstream primer, 10 μM final concentration of
Probe, the obtained positive plasmid of 3 step 4) of embodiment gradient standard items (series of concentrations is：1.00×107copies/μL、
1.00×106copies/μL、1.00×105copies/μL、1.00×104copies/μL、1.00×103copies/μL、
1.00×102copies/μL、1.00×101Copies/ μ L), positive control (concentration be 6.07 × 1010The reality of copies/ μ L
Apply the positive recombinant plasmid of 3 step 3) of example preparation) and ddH2O, ddH2O is used as reagent and negative control.
Embodiment 5：The drafting of standard curve
The gradient standard items of the positive plasmid obtained using 3 step 4) of embodiment is templates, using as shown in SEQ ID No.2
Upstream primer sequence and SEQ ID No.3 shown in downstream primer, the probe as shown in SEQ ID No.4, carry out PCR expansion
Increase, with ddH2O is as negative control.
PCR reaction system is as follows：2 × premix, 10 μ L, upstream primer (10 μM) 0.2 μ L, downstream primer (10 μM) 0.2 μ
L, probe (10 μM) 0.2 μ L, 0.3 μ L of template, ddH2O complements to 20 μ L.
PCR reaction condition is as follows：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 are followed
Ring.
The preparation method of standard curve：Using the logarithm of the plasmid concentration of standard items as abscissa, obtained using ct value as ordinate
To standard curve, Fig. 3 is as a result referred to.Standard curve original equation is y=ax+b, and the equation of this standard curve is Y=-
3.089x+40.83。
Fig. 3 is the standard curve of standard items of the present invention.From the figure 3, it may be seen that standard items standard curve is smooth, related coefficient is high,
Specially R2=0.997, meet the requirement of real-time fluorescence quantitative PCR detection.
Embodiment 6：A method of for detecting Fusarium solani
Gradient standard items (the series of concentrations of the positive plasmid obtained respectively with sample to be tested DNA and 3 step 4) of embodiment
For：1.00×107copies/μL、1.00×106copies/μL、1.00×105copies/μL、1.00×104copies/μ
L、1.00×103copies/μL、1.00×102copies/μL、1.00×101Copies/ μ L) it is template, use embodiment 5
Obtained kit carries out fluorescent quantitative PCR.
PCR reaction system is as follows：2 × premix, 10 μ L, upstream primer (10 μM) 0.2 μ L, downstream primer (10 μM) 0.2 μ
L, probe (10 μM) 0.2 μ L, 0.3 μ L of template, ddH2O complements to 20 μ L.
Reaction condition：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 circulations.It draws
Standard curve carries out rapid quantitative detection by the Ct value of standard curve and sample to be tested.
Embodiment 7：Method validation
The kit for detecting Fusarium solani that is obtained using embodiment 5, to embodiment 6 obtain for detecting corruption
The method of skin sickle-like bacteria carries out sensitivity, specific test
A) sensitivity test
(series of concentrations is the gradient standard items of the positive plasmid obtained respectively with 3 step 4) of embodiment：1.00×
107copies/μL、1.00×106copies/μL、1.00×105copies/μL、1.00×104copies/μL、1.00×
103copies/μL、1.00×102copies/μL、1.00×101Copies/ μ L) it is template, with ddH2O is negative control, into
Row PCR reaction.
PCR reaction condition：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 circulations.
The fluorescence signal according to detected by instrument handles through software and obtains fluorescence curve, observes the signal of fluorescence curve, as a result reference
Fig. 4, data are shown when plasmid concentration reaches 1.00 × 102Still there is fluorescence signal when copies/ μ L, when plasmid concentration reaches
1.00×101There is no fluorescence signal when copies/ μ L.Therefore the sensitivity of the method for the present invention is 1.00 × 102copies/μL。
B) specific test
Reaping hook bacteria microorganism Fusarium oxysporum, fusarium tricinctum, the Fusarium graminearum infected using common clinical carries out special
Specific assay.
Positive recombinant plasmid with the preparation of 3 step 3) of embodiment is positive template, with Fusarium oxysporum, fusarium tricinctum,
Fusarium graminearum is test template, with ddH2O is negative control, carries out PCR reaction, and wherein PCR reaction system is：2×
10 μ L of premix, upstream primer (10 μM) 0.2 μ L, downstream primer (10 μM) 0.2 μ L, probe (10 μM) 0.2 μ L, 0.3 μ of template
L、ddH2O complements to 20 μ L.
PCR reaction condition：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 circulations.
The fluorescence signal according to detected by instrument handles through software and obtains fluorescence curve, observes the signal of fluorescence curve, and analysis is special
Property.Testing result is as shown in table 1,
1 specific test result table of table
As a result Fig. 5 is referred to：Using genomic DNA as template, only Fusarium solani test positive, remaining microorganism are yin
Property, show that the present invention has specificity well.
Embodiment 8：Primer specificity test
Positive recombinant plasmid with the preparation of 3 step 3) of embodiment is positive template, with Fusarium oxysporum, fusarium tricinctum,
Fusarium graminearum is test template, is drawn with downstream shown in upstream primer sequence shown in SEQ ID No.2 and SEQ ID No.3
Object detects Fusarium solani specific primer probe fluorescent quantitation as a result, with ddH by 2% agarose gel2O is negative right
According to progress PCR reaction.
Pcr amplification reaction condition is：10 × PCR buffer, 2.5 μ L, dNTP (2.5 μM) 0.5 μ L, upstream primer (1 μM)
0.5 μ L, downstream primer (1 μM) 0.5 μ L, 0.5 μ L of template, Taq enzyme (5U) 0.5 μ L and ddH2O supplies 25 μ L.
PCR amplification condition is：94 DEG C of initial denaturation 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃
10min。
Fig. 6 is specificity experiments gel imaging proof diagram of the present invention.Wherein, swimming lane 1-6 is followed successively by Fusarium oxysporum, Beancurd sheet
Sickle-like bacteria, fusarium tricinctum, Fusarium graminearum, negative control, DL2000 Marker.
As the result is shown：Only Fusarium solani detection amplified band is the correct purpose band of size, remaining microorganism is not
Purpose band is amplified, shows that the present invention has specificity well.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Sequence table
<110>Suzhou Bai Yuan gene technology Co., Ltd
<120>It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani
<130> 0000
<141> 2018-06-29
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 714
<212> DNA
<213>It harrows sequence ()
<400> 1
gaggacaaga ctcacctcaa cgtcgtcgtc atcggccacg tcgactctgg caagtcgacc 60
accgtaagtc aagcccccat cgcgatctgc ttatctcggg tcgtggaacc ccgcctggta 120
tctcgggcgg ggtactcatc agtcacttca tgctgacaat catctacaga ccggtcactt 180
gatctaccag tgcggtggta tcgacaagcg aaccatcgag aagttcgaga aggttggtga 240
catctccccc gatcgcgcct tgctattcca catcgaattc cctcttctgc gacacgctct 300
gcgcccgctt ctcccgagtc ccaaaaattt tgcggttcga ccgtaatttt tttttggtgg 360
ggcatttacc ccgccactcg agcgacgttg gacgagccct gaaccctgca cacaaaaaac 420
accaaaccct cttggcgcgc atcacgtggt tcacaacaga cactaacagg ttcaacaata 480
ggaagccgct gagctcggta agggttcctt caagtacgcc tgggtccttg acaagctcaa 540
ggccgagcgt gagcgtggta tcaccatcga tattgctctc tggaagttcg agactccccg 600
ctactatgtc accgtcattg gtatgtcgcc ctcatctctc tcaatcacgt ctcatcacta 660
acaatcaaca gacgcccccg gccaccgtga tttcatcaag aacatgatca ctgg 714
<210> 2
<211> 17
<212> DNA
<213>Upstream primer ()
<400> 2
ccctcttctg cgacacg 17
<210> 3
<211> 18
<212> DNA
<213>Downstream primer ()
<400> 3
gctcagcggc ttcctatt 18
<210> 4
<211> 26
<212> DNA
<213>Probe ()
<400> 4
ctgcgcccgc ttctcccgag tcccaa 26
<210> 5
<211> 215
<212> DNA
<213>Target sequence ()
<400> 5
ccctcttctg cgacacgctc tgcgcccgct tctcccgagt cccaaaaatt ttgcggttcg 60
accgtaattt ttttttggtg gggcatttac cccgccactc gagcgacgtt ggacgagccc 120
tgaaccctgc acacaaaaaa caccaaaccc tcttggcgcg catcacgtgg ttcacaacag 180
acactaacag gttcaacaat aggaagccgc tgagc 215
Claims (10)
1. a kind of for detecting the nucleotide sequence group of Fusarium solani, which is characterized in that including shown in SEQ ID No.2
Downstream primer shown in upstream primer and SEQ ID No.3.
2. as described in claim 1 for detecting the nucleotide sequence group of Fusarium solani, which is characterized in that further include such as sequence
Probe shown in list SEQ ID No.4, the probe are fluorescence labeling probe.
3. as claimed in claim 2 for detecting the nucleotide sequence group of Fusarium solani, which is characterized in that the probe
The fluorescent reporter group of 5 ' end labels is FAM, and the fluorescent quenching group of 3 ' end labels is BHQ.
4. a kind of for detecting the kit of Fusarium solani, which is characterized in that draw including the upstream as shown in SEQ ID No.2
Downstream primer and the probe as shown in sequence table SEQ ID No.4 shown in object and SEQ ID No.3.
5. the kit of detection Fusarium solani as claimed in claim 4, which is characterized in that 5 ' end labels of the probe
Fluorescent reporter group is FAM, and the fluorescent quenching group of 3 ' end labels is BHQ.
6. the kit of detection Fusarium solani as claimed in claim 4, which is characterized in that further include the standard of positive plasmid
Product, the standard items of the positive plasmid are using the DNA of Fusarium solani as template, by the upstream as shown in SEQ ID No.2
The recycling of downstream primer PCR amplification shown in primer and SEQ ID No.3, obtains after being attached, transfect with pMD18-T plasmid
's.
7. a kind of method for detecting Fusarium solani, which is characterized in that include the following steps：
1) standard items of positive plasmid are obtained：Using the DNA of Fusarium solani as template, by as shown in SEQ ID No.2
The recycling of downstream primer PCR amplification shown in primer and SEQ ID No.3 is swum, is obtained after being attached, transfect with pMD18-T plasmid
The standard items of positive plasmid；
2) PCR is identified：Respectively using the DNA of sample to be tested, positive plasmid standard items as template, with ddH2O is negative control, is led to
It crosses downstream primer shown in the upstream primer as shown in SEQ ID No.2 and SEQ ID No.3 and carries out PCR reaction inspection.
8. the method as claimed in claim 7 for detecting Fusarium solani, which is characterized in that in step 2), PCR reactant
System is：10 × PCR buffer, 2.5 μ L, dNTP (2.5 μM) 0.5 μ L, upstream primer (1 μM) 0.5 μ L, downstream primer (1 μM)
0.5 μ L, 0.5 μ L of template, Taq enzyme (5U) 0.5 μ L and ddH2O supplies 25 μ L；
PCR reaction condition is：94 DEG C of initial denaturation 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃10min.
9. the method as claimed in claim 7 for detecting Fusarium solani, which is characterized in that in step 2), PCR amplification body
System includes the probe as shown in sequence table SEQ ID No.4.
10. the method as claimed in claim 7 for detecting Fusarium solani, which is characterized in that PCR reaction system：2×
10 μ L of premix, upstream primer (10 μM) 0.2 μ L, downstream primer (10 μM) 0.2 μ L, probe (10 μM) 0.2 μ L, 0.3 μ of template
L、ddH2O complements to 20 μ L；
PCR reaction condition：94 DEG C initial denaturation 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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|---|---|---|---|
| CN201810701043.0A CN108841985A (en) | 2018-06-29 | 2018-06-29 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani |
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| CN108841984A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium oxysporum |
| CN109355424A (en) * | 2018-12-07 | 2019-02-19 | 江苏省农业科学院 | A kind of detection method and its application of the lotus rhizome rot disease pathogen based on Lamp technology |
| CN109825628A (en) * | 2019-03-28 | 2019-05-31 | 南京林业大学 | A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection Fusarium solani |
| CN111549165A (en) * | 2020-05-06 | 2020-08-18 | 兰州百源基因技术有限公司 | Primer, probe, kit and method for RT-QPCR (reverse transcription-quantitative polymerase chain reaction) detection of fusarium solani |
| CN113005218A (en) * | 2021-04-02 | 2021-06-22 | 山西农业大学 | LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani |
| CN113999926A (en) * | 2021-02-24 | 2022-02-01 | 四川省农业科学院植物保护研究所 | Primer group, kit and application of kit in rapid identification of xanthoxylum |
| CN116042900A (en) * | 2022-12-29 | 2023-05-02 | 福建省水产研究所（福建水产病害防治中心） | Primer group and kit for detecting fusarium and application of primer group and kit |
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| CN111549165A (en) * | 2020-05-06 | 2020-08-18 | 兰州百源基因技术有限公司 | Primer, probe, kit and method for RT-QPCR (reverse transcription-quantitative polymerase chain reaction) detection of fusarium solani |
| CN113999926A (en) * | 2021-02-24 | 2022-02-01 | 四川省农业科学院植物保护研究所 | Primer group, kit and application of kit in rapid identification of xanthoxylum |
| CN113005218A (en) * | 2021-04-02 | 2021-06-22 | 山西农业大学 | LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani |
| CN116042900A (en) * | 2022-12-29 | 2023-05-02 | 福建省水产研究所（福建水产病害防治中心） | Primer group and kit for detecting fusarium and application of primer group and kit |
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