CN106929572A - A kind of specific primer for identifying diseases of mulberry fruits and the method that diseases of mulberry fruits identification is carried out using the primer - Google Patents
A kind of specific primer for identifying diseases of mulberry fruits and the method that diseases of mulberry fruits identification is carried out using the primer Download PDFInfo
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Abstract
The invention discloses a kind of for identifying by the specific primer of the caruncula shape cup microbial diseases of mulberry fruits of disk, it is characterised in that：It is made up of sense primer CC 18S F and anti-sense primer CC 18S R, the nucleotide sequence such as SEQ ID NO of the sense primer CC 18S F：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer CC 18S R：Shown in 2.The invention also discloses a kind of application, the specific primer identifies that, by the method for the caruncula shape cup microbial diseases of mulberry fruits of disk, the method can Rapid identification diseases of mulberry fruits in the morbidity early stage of diseases of mulberry fruits.
Description
Technical field
The invention belongs to sclerotiniose technical field, and in particular to a kind of to be reflected by the caruncula shape cup microbial diseases of mulberry fruits of disk
Fixed specific primer and its application.
Background technology
Diseases of mulberry fruits, also known as gingko disease, is the Major Diseases of mulberry fruit, and the disease is caused by fungi, and germ is when mulberry fruit blooms
Start to invade.It is in aubergine when the mulberry fruit of health is ripe, the mulberry fruit infected by mulberry reality cup cup fungi becomes white or canescence, and perianth shows
Write loose.After sick mulberry landing, perianth is partially disengaged and becomes black sclerotium.With the continuous expansion of mulberry fruit cultivated area, mulberry fruit bacterium
Core disease continues large area outburst, and in spreading trend, even No kernels or seeds are gathered, as in a year of scarcity in serious orchard, greatly influences the economic effect of Planting household
Benefit, threatens the development of mulberry fruit industry.The pathogen of diseases of mulberry fruits mainly has 4 kinds, respectively sclerotinite, mulberry reality cup cup fungi,
Caruncula shape cup cup fungi and core ground cane bacterium.Through research, the diseases of mulberry fruits pathogen of In Guangdong Province is mainly caruncula shape cup cup fungi.
At present, the main method of identification diseases of mulberry fruits is phenotypic evaluation.Phenotypic evaluation is the morbidity by observing mulberry fruit
Phenotypic characteristic judges whether diseases of mulberry fruits occurs.Because plant condition phenotype can just be observed in the morbidity middle and later periods, and it is sick
Opportunistic pathogen is when mulberry fruit blooms i.e. in intrusive body, therefore prevention and control of the phenotypic evaluation method to diseases of mulberry fruits have little significance.
The content of the invention
First purpose of the invention be provide a kind of specific primer for identifying diseases of mulberry fruits, application this draw
Thing can be carried out by the detection of the caruncula shape cup microbial diseases of mulberry fruits of disk.
Second object of the present invention is to provide a kind of identification of application specific primer to be caused by caruncula shape cup cup fungi
Diseases of mulberry fruits method, the method diseases of mulberry fruits morbidity early stage can Rapid identification diseases of mulberry fruits.
Third object of the present invention is that the specific primer for providing above-mentioned diseases of mulberry fruits identification is being prepared with mirror
Determine the application in the medicine of diseases of mulberry fruits function.
First purpose of the invention is achieved through the following technical solutions：
It is a kind of for identify by caruncula shape cup the microbial diseases of mulberry fruits of disk specific primer, it is characterised in that：By
Sense primer CC-18S-F and anti-sense primer CC-18S-R is constituted, the nucleotide sequence such as SEQ of the sense primer CC-18S-F
ID NO：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer CC-18S-R：Shown in 2.
According to 18SrRNA (the GenBank ID of mulberry fruit caruncula shape cup cup fungi in ncbi database：KY449059) sequence is special
Levy, it is specific using PrimerPremier 5.0 and Oligo6.0 designs and the fluorescent PCR for having screened above-mentioned caruncula shape cup cup fungi
Primer (CC-18S-F:GAGTGAGCATAAGCTCACCCCGA, such as SEQ ID NO：Shown in 1；CC-18S-R:
CTCCACCCCCCGAGAGCGGTC, such as SEQ ID NO：Shown in 2).The primer pair theoretical amplification clip size is 344bp, should
Primer can be synthesized by companies such as Hua Da genes.
Specific primer of the invention can be used for Standard PCR and fluorescent quantitation qPCR
Second object of the present invention is achieved through the following technical solutions：
A kind of method of the identification for applying above-mentioned specific primer to carry out diseases of mulberry fruits, it is characterised in that including following
Step：
(1) morbidity mulberry fruit and healthy each 10 of mulberry fruit are taken, the DNA of these mulberry fruits is extracted；
(2) it is template with the morbidity mulberry fruit extracted and the DNA of healthy mulberry fruit, using the spy of above-mentioned caruncula shape cup cup fungi
Specific primer carries out fluorescent quantitation qPCR；The Ct values of the qPCR reaction systems of record morbidity mulberry fruit and healthy mulberry fruit DNA；To measure
All described healthy mulberry fruit DNA qPCR reaction systems Ct values in minimum value as judge the mulberry fruit whether have mulberry fruit
The critical value of sclerotiniose；
(4) mulberry fruit to be detected is taken, and extracts the DNA of the mulberry fruit；
(5) with the DNA of mulberry fruit to be detected that extracts in step (4) as template, using the special of the caruncula shape glass cup fungi
Property primer carries out fluorescent quantitation qPCR, and records the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected；
(6) when the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected are less than described critical value, show described
Mulberry fruit to be detected has infected diseases of mulberry fruits；When the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected are more than or wait
When described critical value, show that the mulberry fruit to be detected is healthy mulberry fruit.
In the methods described above, the extraction of mulberry fruit DNA is carried out preferably by DNA extraction kit.
One of the invention preferred embodiment in, the reaction system of above-mentioned fluorescent quantitation qPCR includes：SYBR
The μ L of Premix Ex Taq II (2 ×) 10.0, the μ L of sense primer RS-16S-F 0.8 that concentration is 10 μM, concentration are under 10 μM
The μ L of trip primer RS-16S-R 0.8, the μ L of template 2.0, the μ L of distilled water 6.4.
The amplification program of fluorescent quantitation qPCR is preferably in step (2)：95 DEG C of predegeneration 30s of the first step, 95 DEG C of second step
Denaturation 5s, 55 DEG C of annealing 30s, 72 DEG C of extension 15s, totally 45 circulations.
In a preferred embodiment in accordance with this invention, described critical value is 35.
Third object of the present invention is achieved through the following technical solutions：
The specific primer of above-mentioned diseases of mulberry fruits identification is in the medicine with identification diseases of mulberry fruits function is prepared
Using.
Described medicine is including kit etc..
Beneficial effect：
It is of the invention for identify by caruncula shape cup the microbial diseases of mulberry fruits of disk specific primer it is sensitive, efficient,
Specificity is good.Method by the diseases of mulberry fruits identification obtained by the specific primer combined with fluorescent quantitative PCR can be early in morbidity
Phase Rapid identification diseases of mulberry fruits, for the early prevention and treatment of diseases of mulberry fruits provides reliable foundation.
Brief description of the drawings
Fig. 1 is Ralstonia solanacearum fluorescence PCR primer specific detection in embodiment 1.The template of swimming lane 1 is mulberry fruit caruncula shape cup cup fungi
DNA；The template of swimming lane 2 is trichoderma DNA；The template of swimming lane 3 is sclerotinite DNA；The template of swimming lane 4 is grey mold DNA；The template of swimming lane 5 is black
Aspergillus DNA；The template of swimming lane 6 is flavus dna；The template of swimming lane 7 is cerevisiae dna；M is 2000bp DNALadder；
Fig. 2 is the Ct values of different onset degree mulberry fruit and healthy mulberry fruit sample in embodiment 3.
Specific embodiment
In examples below, the Standard PCR instrument for being used is the ABI-9700 type PCR instruments produced by ABI companies.Institute
The Taq PCRMix (2 ×) that use, DNAiso reagent kit and T-easy carriers are produced by precious biotech firm.qPCR
Experiment is produced using the real-time fluorescence PCR instrument of LightCycler 480 and precious biotech firm of Roche Holding Ag (Roche) production
SYBR Premix Ex TaqTMKit is carried out.
Embodiment 1
The design and checking of mulberry fruit caruncula shape cup cup fungi special primer
1. according to 18SrRNA (the GenBank ID of mulberry fruit caruncula shape cup cup fungi in ncbi database：KY449059) sequence
Feature, designs and has screened the PCR specific primers of caruncula shape cup cup fungi using Primer Premier 5.0 and Oligo6.0
(CC-18S-F:GAGTGAGCATAAGCTCACCCCGA/CC-18S-R:CTCCACCCCCCGAGAGCGGTC).The primer pair is managed
It is 344bp by amplified fragments size, the primer is synthesized by Huada gene company.Before primer dilution first under the conditions of 4 DEG C, with 1 ×
104Rpm is centrifuged 2min, be subsequently adding deionized water be made into 10 μM of storage liquid be stored in -20 DEG C it is stand-by；
2. it is control, all experimental strains with 6 kinds of fungies such as trichoderma, sclerotinite, grey mold, aspergillus niger, aspergillus flavus and yeast
It is inoculated in PDA culture medium, with the rotating speed of 200rpm in constant temperature gas bath shaking table, 24h is cultivated under the conditions of 28 DEG C.Using
DNAiso reagent kit extract the DNA of each bacterial strain, stand-by under the conditions of being stored in -20 DEG C；
3., with caruncula shape cup cup fungi and each control strain DNA as template, drawn using caruncula shape cup cup fungi fluorescent PCR specificity
Thing CC-18S-F and CC-18S-R carry out standard PCR amplification, and PCR reaction systems are as follows：12.5 μ are included in the reaction system of 25 μ L
L Taq PCR Mix (2 ×), 8.5 μ L distilled waters, 2 μ L templates, 1 μ L sense primers CC-18S-F, 1 μ L anti-sense primers CC-18S-
R.Amplification reaction condition is：94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 20s, 30 circulations；
72 DEG C of extension 5min, 4 DEG C of preservations.After the completion of PCR, product is detected through 1.2% agarose gel electrophoresis；
4. simultaneously by electrophoretic band gel extraction, it is connected on T-easy carriers, and converts into E.coli, obtains positive
Bacterium colony is simultaneously sequenced.Sequencing result is compared on NCBI, its Pseudomonas type is determined.
As a result
It is control with 6 kinds of fungies such as trichoderma, sclerotinite, grey mold, aspergillus niger, aspergillus flavus and yeast, using caruncula shape cup disk
Bacterium fluorescent PCR specific primer CC-18S-F and CC-18S-R carry out standard PCR amplification, and electrophoretic analysis result is as shown in Figure 1：Only
Caruncula shape cup cup fungi can amplify band, and 6 kinds of control strain trichoderma, sclerotinite, grey mold, aspergillus niger, aspergillus flavus and yeast etc.
Fungi does not amplify band.
Electrophoretic band is reclaimed after converting and being sequenced, by sequencing result on NCBI known caruncula shape cup cup fungi
18SrRNA sequences (KY449059) are compared, and comparison result shows that the sequence is the 18SrRNA feature sequences of caruncula shape cup cup fungi
Row.
In sum, caruncula shape cup cup fungi primer is specific good, and the fluorescence that can be used for follow-up caruncula shape cup cup fungi is determined
Amount analysis experiment.
Embodiment 2
The method of early stage Rapid identification diseases of mulberry fruits
The determination of Ct values
1. morbidity mulberry fruit and healthy each 10 of mulberry fruit are taken, these mulberry fruits are extracted using DNAiso reagent kit kits
DNA；
2. the DNA for being extracted with step 1 is template, using the specific primer CC-18S-F/ of above-mentioned caruncula shape cup cup fungi
CC-18S-R carries out fluorescent quantitation qPCR experiments.20 μ L qPCR reaction systems include：SYBRPremix Ex Taq Ⅱ(2×)
10.0uL, the μ L of sense primer CC-18S-F (10 μM) 0.8, the μ L of anti-sense primer CC-18S-R (10 μM) 0.8, the μ L of template 2.0, double steamings
The μ L of water 6.4.The amplification program of fluorescent quantitation qPCR is：95 DEG C of predegeneration 30s of the first step, 95 DEG C of denaturation 5s of second step, 55 DEG C are moved back
Fiery 30s, 72 DEG C of extension 15s, totally 45 circulations.PCR carries out solubility curve analysis immediately after terminating, verify the specificity of amplification.
Each qPCR reactions are repeated 3 times.The Ct values of qPCR reaction systems are recorded, is recorded in table 1；
3. as shown in the data in table 1,10 Ct values of healthy mulberry fruit are between 35-38, and 10 Ct of morbidity mulberry fruit
Value is between 11-29.Take the Ct values of the qPCR reaction systems of institute unsoundness mulberry fruit DNA, i.e., 35, whether have as detection mulberry fruit
The critical value of diseases of mulberry fruits, i.e., when the Ct values of mulberry fruit sample qPCR detections are less than 35, show there be depositing for caruncula shape cup cup fungi
It is morbidity mulberry fruit in, the sample；When the Ct values of mulberry fruit sample qPCR detections are more than 35, show no mulberry fruit caruncula shape cup cup fungi
Presence, the sample is healthy mulberry fruit.
The identification of diseases of mulberry fruits
1. it is 1 phase (only the small achene of mulberry fruit does not expand, slight internal infects), 2 phases to take in diseases of mulberry fruits occurring degree
(the small achene of mulberry fruit expands and severe infections), 3 phases (the small achene of mulberry fruit is infected to nigrescence, and outside perianth is infected in part) and 4 phases
The mulberry fruit sample and healthy mulberry fruit sample of (the small achene of mulberry fruit and perianth integrally infect concurrent black), rinse dry by it with running water
Only after, with 70% alcohol surface sterilization 1min, with aseptic water washing 1 time, liquid nitrogen flash freezer, be stored in -80 DEG C it is standby；
2., with 1g mulberry fruit samples as material, the DNA of all mulberry fruit samples, agar are extracted using DNAiso reagent kit
It is stand-by under the conditions of being stored in -20 DEG C after sugared detected through gel electrophoresis；
3., for each mulberry fruit sample, the mulberry fruit sample DNA of 1 μ L is taken for template, using the primer CC- in embodiment 1
18S-F/CC-18S-R carries out fluorescent quantitation qPCR experiments.QPCR uses the real-time fluorescence PCR instrument of LightCycler 480 and SYBR
Premix Ex TaqTMKit is carried out.20 μ L qPCR reaction systems include：SYBR Premix Ex Taq Ⅱ(2×)
10.0uL, the μ L of sense primer CC-18S-F (10 μM) 0.8, the μ L of anti-sense primer CC-18S-R (10 μM) 0.8, the μ L of template 1.0, double steamings
The μ L of water 6.4.Amplification program is as follows：95 DEG C of predegeneration 30s of the first step, second step 95 DEG C of denaturation 5s, 55 DEG C of annealing 30s, 72 DEG C are prolonged
15s is stretched, 45 circulations, PCR carries out solubility curve analysis, verifies the specificity of amplification immediately after terminating.Each qPCR reaction weights
It is multiple 3 times；
4. the Ct values of each sample qPCR reaction systems are recorded, and experimental result is summarized in fig. 2.
As seen from Figure 2：
(1) health mulberry fruit sample Ct values are 36.5, more than critical value 35.
(2) the Ct values of the mulberry fruit sample of 1,2,3,4 phases of morbidity respectively 25.4,18.3,14.2,12.2, respectively less than critical
Value 35.
Therefore this method can be used for the identification of diseases of mulberry fruits.
Table 1
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, protection scope of the present invention is included in.
Sequence table
<110>Guangdong Provincial Academy Of Agricultural Sciences Institute Of Sericulture And Farm Product Processing
<120>A kind of specific primer for identifying diseases of mulberry fruits and carry out diseases of mulberry fruits identification using the primer
Method
<160> 2
<170> BiSSAP 1.3.6
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>The nucleotide sequence of sense primer CC-18S-F
<400> 1
gagtgagcat aagctcaccc cga 23
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223> CC-18S-R
<400> 2
ctccaccccc cgagagcggt c 21
Claims (7)
1. a kind of for identifying by the specific primer of the caruncula shape cup microbial diseases of mulberry fruits of disk, it is characterised in that：By upper
Trip primer CC-18S-F and anti-sense primer CC-18S-R compositions, the nucleotide sequence such as SEQ ID of the sense primer CC-18S-F
NO：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer CC-18S-R：Shown in 2.
2. the method that the specific primer described in a kind of application claim 1 carries out diseases of mulberry fruits identification, it is characterised in that bag
Include following steps：
(1) morbidity mulberry fruit and healthy each 10 of mulberry fruit are taken, the DNA of these mulberry fruits is extracted；
(2) it is template with the morbidity mulberry fruit extracted and the DNA of healthy mulberry fruit, using the specificity of above-mentioned caruncula shape cup cup fungi
Primer carries out fluorescent quantitation qPCR；The Ct values of the qPCR reaction systems of record morbidity mulberry fruit and healthy mulberry fruit DNA；The institute that will be measured
Have the healthy mulberry fruit DNA qPCR reaction systems Ct values in minimum value as judge the mulberry fruit whether have mulberry fruit sclerotium
The critical value of disease；
(4) mulberry fruit to be detected is taken, and extracts the DNA of the mulberry fruit；
(5) DNA with the mulberry fruit to be detected of extraction in step (4) as template, draw by the specificity using caruncula shape cup cup fungi
Thing carries out fluorescent quantitation qPCR, and records the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected；
(6) when the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected are less than described critical value, show described to be checked
The mulberry fruit of survey has infected diseases of mulberry fruits；When the Ct values of the qPCR reaction systems of the mulberry fruit DNA to be detected are more than or equal to institute
During the critical value stated, show that the mulberry fruit to be detected is healthy mulberry fruit.
3. the method that application specific primer according to claim 2 carries out diseases of mulberry fruits identification, it is characterised in that：Adopt
The extraction of mulberry fruit DNA is carried out with DNA extraction kit.
4. the method that application specific primer according to claim 2 carries out diseases of mulberry fruits identification, it is characterised in that：Institute
The reaction system of the fluorescent quantitation qPCR for stating includes：SYBR Premix Ex Taq II (2 ×) 10.0 μ L, concentration are 10 μM
The μ L of sense primer RS-16S-F 0.8, the μ L of anti-sense primer RS-16S-R 0.8 that concentration is 10 μM, the μ L of template 2.0, distilled water 6.4
μL。
5. the method that application specific primer according to claim 2 carries out diseases of mulberry fruits identification, it is characterised in that：Institute
The amplification program of fluorescent quantitation qPCR is in the step of stating (2)：95 DEG C of predegeneration 30s of the first step, 95 DEG C of second step is denatured 5s, 55
DEG C annealing 30s, 72 DEG C extension 15s, totally 45 circulation.
6. the method that application specific primer according to claim 2 carries out diseases of mulberry fruits identification, it is characterised in that：Institute
The critical value stated is 35.
7. described in claim 1 for identify by caruncula shape cup the microbial diseases of mulberry fruits of disk specific primer prepare
Application in medicine with identification diseases of mulberry fruits function.
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| CN201710085720.6A CN106929572B (en) | 2017-02-17 | 2017-02-17 | Specific primer for identifying mulberry sclerotinia sclerotiorum and method for identifying mulberry sclerotinia sclerotiorum by using specific primer |
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| CN110592252A (en) * | 2019-09-05 | 2019-12-20 | 湖北省农业科学院经济作物研究所 | Primer group and kit for mulberry granule sclerotinia sclerotiorum qPCR detection and application of primer group and kit |
| CN110592252B (en) * | 2019-09-05 | 2022-10-14 | 湖北省农业科学院经济作物研究所 | Primer group and kit for mulberry granule sclerotinia sclerotiorum qPCR detection and application of primer group and kit |
| CN110724757A (en) * | 2019-11-26 | 2020-01-24 | 湖北省农业科学院经济作物研究所 | Primer for mulberry fertilize large sclerotinia sclerotiorum detection and application thereof |
| CN110724757B (en) * | 2019-11-26 | 2023-02-28 | 湖北省农业科学院经济作物研究所 | Primer for mulberry fruit fat large sclerotinia sclerotiorum detection and application thereof |
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