CN108841984A - It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium oxysporum - Google Patents
It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium oxysporum Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the nucleotide sequence group of Fusarium oxysporum, 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 oxysporum 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 oxysporum
Sequence group, kit and method.
Background technique
Fusarium oxysporum (Fusarium oxysporum) is that one kind can not only infect plant but also can survive in the soil simultaneous
Property parasitical fungi.The pathogen endangers plant from root, causes vascular bundle diseases, causes plant withered, in the entire life of plant
Educating the phase can occur.For by fungus-caused plant disease, pervious discrimination method is usually to fall ill to plant by field observation
Strain, plate separation pathogen, immunology detection and identification, not only time-consuming for these methods, and efficiency and sensitivity are lower, Er Qiejing
The property tested is strong.
In recent years, the development of Protocols in Molecular Biology is the accurate detection of microorganism and quantitatively provides convenience, and no longer
Dependent on traditional technologies such as plate cultures.Real-Time Fluorescent Quantitative PCR Technique be it is a kind of easily and fast, accurately non-bacterial culture
Quantitative approach, refer to and fluorophor be added in PCR reaction system, using fluorescence signal accumulate the entire PCR process of real-time monitoring,
Then a kind of method of quantitative analysis is carried out to unknown template by standard curve.
Fusarium oxysporum and other reaping hook category strains have higher homology, such as Fusarium graminearum, fusarium moniliforme, three
Line 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 sharp spore
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 oxysporum.
The third object of the present invention is to provide a kind of method for detecting Fusarium oxysporum.
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 oxysporum, 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 oxysporum, 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 oxysporum
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 oxysporum, include the following steps：
1) standard items of positive plasmid are obtained：Using the DNA of Fusarium oxysporum 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 are followed
Ring.
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 oxysporum, mainly to point
The EF1a gene of the highly conserved specificity of fusarium oxysporum designs the upstream primer as shown in SEQ ID No.2 as rake sequence
With the downstream primer as shown in SEQ ID No.3, the amplification length of the sequence is 194bp, is had to Fusarium oxysporum preferable
Specificity, effectively it can be distinguished with the Fusarium graminearum of same Fusarium, fusarium moniliforme, fusarium tricinctum；
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 oxysporum 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, it is respectively 1.00 × 10 that a, which represents plasmid concentration,8Copies/ μ L, b represent plasmid concentration as 1.00 ×
107Copies/ μ L, c represent plasmid concentration as 1.00 × 106Copies/ μ L, d represent plasmid concentration as 1.00 × 105copies/
μ L, e represent plasmid concentration as 1.00 × 104Copies/ μ L, f represent plasmid concentration as 1.00 × 103Copies/ μ L, g represent matter
Grain concentration is 1.00 × 102Copies/ μ L, h 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 oxysporum genome, and it is respectively standing grain that b representative, which does not occur standard amplification curve,
Paddy sickle-like bacteria, fusarium moniliforme, fusarium tricinctum, negative control；
Fig. 6 is the specific test result figure of embodiment 8；
Wherein, it is right to be followed successively by Fusarium graminearum, Fusarium oxysporum, fusarium moniliforme, fusarium tricinctum, 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, then adopt using highly conserved, special EF1a gene as rake sequence
Fusarium oxysporum EF1a gene is expanded with round pcr, is connected in plasmid vector pMD18-T using gene recombination technology,
Recombinant plasmid pMD18-T-EF1a is constructed, and carries out corresponding PCR identification and sequencing identification, most afterwards through quantitative as wait establish
The standard items of method 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 oxysporum, 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：
5’-GTCGACTCTGGCAAGTCGACCACTGTGAGTACTCTCCTCGACAATGAGCTTATCTGCCATCGTCAA
TCCCGACCAAGACCTGGTGGGGTATTTCTCAAAGTCAACATACTGACATCGTTTCACAGACCGGTCACTTGATCTAC
CAGTGCGGTGGTATCGACAAGCGAACCATCGAGAAGTTCGAGAAGGTTAGTCACTTTCCCTTCGATCGCGCGTCCTT
TGCCCATCGATTTCCCCTACGACTCGAAACGTACCCGCTACCCCGCTCGAGACCAAAAATTTTGCAATATGACCGTA
ATTTTTTTGGTGGGGCACTTACCCCGCCACTTGAGCGACGGGAGCGTTTGCCCTCT
AATGAGTGCGTCGTCACGTGTCAAGCAGTCACTAACCATTCAACAATAGGAAGCCGCTGAGCTCGGTAAGGGTTCCT
TCAAGTACGCCTGGGTTCTTGACAAGCTCAAGGCCGAGCGTGAGCGTGGTATCACCATCGATATTGCTCTCTGGAAG
TTCGAGACTCCTCGCTACTATGTCACCGTCATTGGTATGTTGTCGCTCATACTTCATTCTACTTCTCTTCGTACTAA
CATATCACTCAGACGCTCCCGGTCACCGTGATTTCATCAAGAACATGATCACTGGGT-3’
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.
SEQ ID No.1 harrows sequence by the way that such as Fig. 1, SEQ ID No.1 harrow sequence as the result is shown to ncbi database blast
99-100% is reached with the matching degree height of the gene order of a variety of Fusarium oxysporum separation strains strains, similarity.
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 oxysporum.
The nucleotides sequence group includes the probe sequence as shown in SEQ ID No.4：
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 oxysporum reference culture genomic DNA using bacterial genomes extracts kit, uses
Make the template of EF1a gene PCR amplification.Its extracts kit used can be selected from the bacterium base of hundred Imtech of Beijing production
Because of a group extracts kit, specific extracting method is 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 buffer 2.5μL,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 hundred Tyke Power Taq Plus DNA Polymerse of Beijing, and PCR amplification instrument is Hangzhou
The serial 96 grads PCR instrument of Lang Ji 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.Take 5 μ L
Amplified production carries out 2% agarose electrophoresis, detects PCR product size, is then returned using the DNA gel of Shanghai Sangon Biotech Company's production
It receives kits and recycles remaining pcr amplification product, obtain 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 are followed
Ring；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-EF1a is extracted using the Plasmid Preparation kit of hundred Imtech production, is measured dense
Degree and purity, while drawing a part of plasmid purification and supreme marine growth Engineering Co., Ltd is given to be sequenced, determine Insert Fragment
Gene order it is consistent with aim sequence.
D the standard items of positive recombinant plasmid) are obtained：
1) step C is taken) the obtained 100 μ L transferred species of bacillus coli DH 5 alpha containing recombinant plasmid pMD18-T-EF1a is in 5mL
LB liquid medium, 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-EF1a concentration (copy number) calculates
Molecular weight=2885bp × 660 (average molecular weight of each pair of base) of plasmid
Measuring plasmid concentration is 192.17ng/ μ L, plasmid purity A260/A280=1.84, 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=192.17 × 10 plasmid concentration copies/ μ extracted-9g/μL×6.02×1023copies/mol÷
(2885bp × 660g/bpmol)=6.07 × 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 oxysporum
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 of series of concentrations standard items is：
1.00×108copies/μL、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/μL)、
(concentration is 6.07 × 10 to positive control10The positive recombinant plasmid of 3 step 3) of the embodiment preparation of copies/ μ L) 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
Circulation.
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=-
3.2871x+39.619。
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=0.9981, meet the requirement of real-time fluorescence quantitative PCR detection.
Embodiment 6：A method of for detecting Fusarium oxysporum
Gradient standard items (the series of concentrations mark of the positive plasmid obtained respectively with sample to be tested DNA and 3 step 4) of embodiment
The series of concentrations of quasi- product is：1.00×108copies/μL、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, the kit obtained using embodiment 5 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 processed 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 oxysporum that is obtained using embodiment 5, to embodiment 6 obtain for detecting point
The method of fusarium oxysporum carries out sensitivity, specific test
A) sensitivity test
(series of series of concentrations standard items is dense for the gradient standard items of the positive plasmid obtained respectively with 3 step 4) of embodiment
Degree is：1.00×108copies/μL、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
It L is) template, with ddH2O is negative control, carries out 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 are followed
Ring.The fluorescence signal according to detected by instrument handles through software and obtains fluorescence curve, observes the signal of fluorescence curve, as a result joins
According to 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
It is carried out using the reaping hook bacteria microorganism Fusarium graminearum of common clinical infection, fusarium moniliforme, fusarium tricinctum special
Specific assay.
Positive recombinant plasmid with the preparation of 3 step 3) of embodiment is positive template, with Fusarium graminearum, fusarium moniliforme,
Fusarium tricinctum 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 are followed
Ring.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
It is anisotropic.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 oxysporum 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 graminearum, fusarium moniliforme,
Fusarium tricinctum 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 oxysporum 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 graminearum, sharp spore
Sickle-like bacteria, fusarium moniliforme, fusarium tricinctum, negative control, DL2000 Marker.
As the result is shown：Only Fusarium oxysporum 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 oxysporum
<130> 0000
<141> 2018-06-29
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 660
<212> DNA
<213>It harrows sequence ()
<400> 1
gtcgactctg gcaagtcgac cactgtgagt actctcctcg acaatgagct tatctgccat 60
cgtcaatccc gaccaagacc tggtggggta tttctcaaag tcaacatact gacatcgttt 120
cacagaccgg tcacttgatc taccagtgcg gtggtatcga caagcgaacc atcgagaagt 180
tcgagaaggt tagtcacttt cccttcgatc gcgcgtcctt tgcccatcga tttcccctac 240
gactcgaaac gtacccgcta ccccgctcga gaccaaaaat tttgcaatat gaccgtaatt 300
tttttggtgg ggcacttacc ccgccacttg agcgacggga gcgtttgccc tcttaaccat 360
tctcacaacc tcaatgagtg cgtcgtcacg tgtcaagcag tcactaacca ttcaacaata 420
ggaagccgct gagctcggta agggttcctt caagtacgcc tgggttcttg acaagctcaa 480
ggccgagcgt gagcgtggta tcaccatcga tattgctctc tggaagttcg agactcctcg 540
ctactatgtc accgtcattg gtatgttgtc gctcatactt cattctactt ctcttcgtac 600
taacatatca ctcagacgct cccggtcacc gtgatttcat caagaacatg atcactgggt 660
<210> 2
<211> 18
<212> DNA
<213>Upstream primer ()
<400> 2
agttcgagaa ggttagtc 18
<210> 3
<211> 18
<212> DNA
<213>Downstream primer ()
<400> 3
aggttgtgag aatggtta 18
<210> 4
<211> 26
<212> DNA
<213>Probe ()
<400> 4
ctttcccttc gatcgcgcgt cctttg 26
<210> 5
<211> 194
<212> DNA
<213>Target sequence ()
<400> 5
agttcgagaa ggttagtcac tttcccttcg atcgcgcgtc ctttgcccat cgatttcccc 60
tacgactcga aacgtacccg ctaccccgct cgagaccaaa aattttgcaa tatgaccgta 120
atttttttgg tggggcactt accccgccac ttgagcgacg ggagcgtttg ccctcttaac 180
cattctcaca acct 194
Claims (10)
1. a kind of for detecting the nucleotide sequence group of Fusarium oxysporum, 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 oxysporum, 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 oxysporum, 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 oxysporum, 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 oxysporum 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 oxysporum 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 oxysporum 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 oxysporum, which is characterized in that include the following steps：
1) standard items of positive plasmid are obtained：Using the DNA of Fusarium oxysporum 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 oxysporum, 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 oxysporum, 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 oxysporum, 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.
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| CN108841985A (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 solani |
| TWI716094B (en) * | 2019-09-02 | 2021-01-11 | 國立屏東科技大學 | Kit and method for detection of fusarium oxysporum f. sp. melonis |
| CN114058729A (en) * | 2021-11-22 | 2022-02-18 | 河北省农林科学院植物保护研究所 | Primer and probe for detecting fusarium oxysporum cucumber specialization type and application thereof |
| CN117683933A (en) * | 2024-01-02 | 2024-03-12 | 吉林农业科技学院 | Primer group, kit and method for detecting fusarium oxysporum on basis of RPA isothermal amplification technology |
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