CN106434999A - Method for rapidly detecting constrictive sclerotinia pathogenic bacteria of mulberry at early stage on basis of nested PCR (polymerase chain reaction) - Google Patents
Method for rapidly detecting constrictive sclerotinia pathogenic bacteria of mulberry at early stage on basis of nested PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention relates to a method for rapidly detecting constrictive sclerotinia pathogenic bacteria of mulberry at the early stage on basis of a nested PCR (polymerase chain reaction). The method comprises steps as follows: (1) tissue sample DNA extracted with a CTAB method is taken as a template, and first round PCR amplification is performed with the adoption of the universal primer ITS1/ITS4; (2) a first round product is taken as a template for second round PCR amplification, a nested primer adopts a specific primer of scleromitrula shiraiana and has the sequences shown in SEQ ID NO.1 and SEQ ID NO.2; (3) constrictive sclerotinia of the mulberry is diagnosed according to the amplification conditions of the step (2). The detection method has excellent accuracy and specificity and greatly improves the detection sensitivity.
Description
Technical field
The invention belongs to field of plant disease control, be related to a kind of detection method and in particular to a kind of based on nest-type PRC
Fructus Mori constrictive sclerotinios pathogen early stage method for quick.
Background technology
Bao Kuo China, the East Asia including India, the Central Asia and South Asia region, mulberry as a kind of important for breeding silkworms
Economic plants are extensively planted, and people mainly utilize its Folium Mori to feed silkworm, and most including Turkey and Greece
Number European countries, people plantation mulberry is primarily used to produce Fructus Mori.Fructus Mori as the potential source of functional food, because having
Numerous biological activitys and pharmacological action and receive much concern.Research show its have good antioxidation, antiinflammatory, protection nerve,
Numerous activity such as anticancer, blood sugar lowering and blood fat reducing.Transition with sericulture there and people numerous health cares, pharmacologically active to Fructus Mori
Understanding, domestic gradually emerging a large amount of fruit Mulberry and seedless mulberry field.Development fruit Mulberry is not only the emerging product that soil increases income
Industry, is also silkworm and mulberry transition and a big approach of upgrading.Fruit Mulberry cultivated area increases considerably year by year, only the fruit leaf of Chongqing City
Dual-purpose Mulberry cultivated area adds up to reach hundreds thousand of mus.But it easily infects fatal sclerotiniose and (is commonly called as Cortex Mori fruit disease, is loose type bacterium
Core disease, constrictive sclerotinios, the general designation of little graininess sclerotiniose), sickness rate up to more than 90% when serious, ruin product total crop failure, such as anti-
Control not in time, year after year repeated infection, make mulberry planter suffer serious financial consequences.This disease becomes the bottle hindering fruit Mulberry industrialized development
Neck.
Diseases of mulberry fruits is the most fatal disease that fruit Mulberry industry is faced at present, and it is in China In Middle And Lower Reaches of Changjiang River and Korea Spro
State's report is more, and its sclerotium can be survived the several years in soil, and Fructus Mori is infected in circulation, and when morbidity is serious, even No kernels or seeds are gathered, as in a year of scarcity.
The detection of diseases of mulberry fruits and diagnosis use traditional method at present, carry out experience according to Fructus Mori disease symptom
Property judges roughly, or collecting sample carries out isolating and purifying of pathogen, is then made a definite diagnosis according to Koch's Postulates.The method
Have the disadvantages that:
1st, could find to catch an illness after need to waiting until to show related symptoms.But it is now " cancer of late stage ", almost without can rescue
Medicine, even if spray preventing and treating, also sheerly " mends the fold after the sheep is lost ", takes effect little.Because this disease is fallen ill from Pathogen Infection to sorosis in a large number,
Course is very short, and patient's condition is violent, once display symptom, in mulberry field, most of disease fruit, to the infection later stage, is that bacterium bag is inedible entirely,
Suffer heavy losses.
2nd, time-consuming, empirical strong.Make a definite diagnosis, need to isolate and purify out pathogen, then carry out morphological observation, routine
The means such as Molecular Identification, reversal connection infection are made a definite diagnosis, and take and are up to tens of skies, or even all longer than the sorosis phase.
Therefore, develop new fast and accurate diseases of mulberry fruits detection of pathogens side on the basis of forefathers' research
Method has important practical significance, and it can not only carry out early prediction forecast, has the more fully time to take prophylactico-therapeutic measuress, simultaneously
Can also be quarantined in new mulberry field and introduction seedling etc..
Content of the invention
In view of this, it is an object of the invention to provide a kind of Fructus Mori constrictive sclerotinios pathogen based on nest-type PRC
Early stage method for quick.
For reaching above-mentioned purpose, the present invention provides following technical scheme:
Based on the Fructus Mori constrictive sclerotinios pathogen early stage method for quick of nest-type PRC, comprise the following steps:
(1) using the tissue sample DNA of CTAB method extraction as template, the first round is carried out using universal primer ITS1/ITS4
PCR expands;
(2) the second wheel PCR amplification is carried out for template with first round product, nested primer is drawn using the specificity of core ground cane bacterium
Thing, sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2;
(3) amplified band according to step (2) diagnoses Fructus Mori constrictive sclerotinios.
Preferably, the sample in step (1) is Fructus Mori.
Preferably, in step (1), the condition of first round PCR amplification is as follows:95 DEG C of denaturations 3min;94 DEG C of degeneration 30s,
55 DEG C of annealing 50s, 72 DEG C of extension 50s, 32 circulations, 72 DEG C of extension 10min, 4 DEG C, ∞.
Preferably, in step (1), the sequence such as SEQ ID NO.3 of universal primer ITS1/ITS4 and SEQ ID NO.4 institute
Show.
Preferably, in step (2), the second wheel amplification condition is similar to the first round, and annealing temperature is arranged to 56,58,60,62
Or the different gradients such as 64 DEG C, every kind of primer optimal condition is found with this.
It is further preferred that the annealing temperature of the second wheel amplification is 56,58,60 or 62 DEG C.
A pair of core ground cane bacterium primer, sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The screening technique of above-mentioned a pair of core ground cane bacterium primer, be with Mulberry reality cup cup fungi, caruncula shape cup cup fungi, sclerotinite and
Mulberry Phoma sp is comparison, and the corresponding pathogenic bacteria ITS sequence in NCBI carries out Multiple Sequence Alignment, ESPript with ClustalX 2
3.0 carry out variation analyses, and Primer 5.0 carries out the design of primer and assessment and obtains.
The beneficial effects of the present invention is:
In the gene of fungal gene group encoding ribosomal, transcriptional units are formed by 18S, 5.8S and 28S rDNA, its
In spacer be internal transcribed spacer region (Internal Transcribed Spacer, ITS).ITS region include ITS1 and
Two regions of ITS2, wherein ITS1 is located between 18S and 5.8S, and ITS2 is located between 5.8S and 28S.Because ITS1 and ITS2 enters
Change relatively rapid, there is conservative in certain inter-species specificity and kind, have become as funguses (especially filamentous fungis) classification
The research emphasis of identification.Present invention employs nest-type PRC, its have quickly, high specific the features such as, using ITS as the first round
Amplimer, specificity nested primer carries out diseases of mulberry fruits cause of disease quick detection as the second wheel amplimer, and method is feasible,
And there is wider annealing temperature, and early prediction forecast can be carried out, have the more fully time to take prophylactico-therapeutic measuress, simultaneously can also be right
Quarantine in new mulberry field and introduction seedling etc..Empirical tests, the detection method of the present invention has splendid accuracy and specificity, and
Substantially increase detection sensitivity.
Brief description
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below to carry out
Explanation:
Fig. 1 is various pathogenic bacteria ITS sequence comparison result;
Fig. 2 a and Fig. 2 b is the nest-type PRC result of different annealing temperature, and 6 swimming lanes therein are respectively M-marker, 1-
56 DEG C, 2-58 DEG C, 3-60 DEG C, 4-62 DEG C, 5-64 DEG C;Fig. 2 a is the result of sample1, and Fig. 2 b is the result of sample2;
Fig. 3 a and Fig. 3 b is the application result of method for quick.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Material and reagent
Material:Adopt in the disease in It In Beibei, Chongqing West seed station mulberry field and Can Ye scientific and technical research institute of Chongqing City mulberry field
Sorosis, is shown in Table 1.
Table 1. two parts of materials used
Reagent:Main agents are as shown in table 2.
Table 2. main agents
The configuration of related reagent:
The configuration of 1M Tris-HCl:Accurate weighing 121.1g Tris is placed in 1L beaker.Add the deionization of about 800mL
Water, is sufficiently stirred for dissolving.About 42mL concentrated hydrochloric acid is added to adjust pH to 8.0.By solution constant volume to 1L.After autoclave sterilization, room
Temperature preserves.
The configuration of 0.5M EDTA (pH 8.0):90.05g bis- water disodiumedetate is added in 400mL water
(EDTANa2 2H2O), stirring and dissolving, adjust pH to 8.0 (about 10g NaOH granule) with NaOH, be settled to 500mL, high pressure goes out
Bacterium.
4×CTAB:CTAB 4g, NaCl 16.364g, 1M Tris-HCl 20mL (PH8.0), 0.5M EDTA 8mL.First
Use 70mL ddH2O dissolves, and is settled to 100mL autoclaving.Using front plus 1mL beta -mercaptoethanol.
Instrument and equipment:
Used by test, key instrument equipment is as shown in table 3
Table 3. key instrument equipment
Embodiment 1:
The design of nested primer:
From NCBI (National Center for Biotechnology Information, US National biotechnology
Information centre, http://www.ncbi.nlm.nih.gov/nuccore/) upper three kinds of (hypertrophy (Mulberry reality cup disks of download tradition
Bacterium), little graininess (caruncula shape cup cup fungi), contractility (core ground cane bacterium)) diseases of mulberry fruits pathogen ITS sequence (sclerotinite and Mulberry
Phoma sp is comparison), Multiple Sequence Alignment is carried out with ClustalX 2, ESPript 3.0 carries out variation analyses, and Primer 5.0 enters
The design of row primer and assessment, obtain the specific primer of every kind of diseases of mulberry fruits pathogen, and its comparison result is as shown in Figure 1.
Downloaded correlated serieses are as shown in table 4.
The ITS sequence accession number of table 4. diseases of mulberry fruits pathogen
Primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The foundation of diseases of mulberry fruits method for quick:
Using nest-type PRC as the method for quick detection diseases of mulberry fruits pathogen, with CTAB method (Liu Li, Zhang Yongjun, perhaps long
Levy, Luo Feng, a kind of CTAB method of improvement is extracted and produced polysaccharide fungal DNA, Chinese biological engineering magazine, 2014,34 (5):75-79)
The tissue sample DNA extracting, as template, carries out the amplification of first round PCR, primer sequence (this using universal primer ITS1/ITS4
Primer sequence listed by literary composition is all defaulted as 5 ' 3 ' directions) be:(TCCGTAGGTGAACCTGCGG, as SEQ ID NO.3 institute for ITS1
Show) and ITS4 (TCCTCCGCTTATTGATATGC, as shown in SEQ ID NO.4) (purchased from Hua Da gene), with first round product
Carry out the second wheel PCR amplification for template, nested primer adopts aforesaid corresponding specific primer, and amplification system is all using 25 μ L bodies
System, concrete consumption is as shown in table 5.
The concrete consumption of table 5.PCR amplification system
First round PCR amplification condition is as shown in table 6.
6. first round of table PCR Amplification
Second wheel amplification condition is similar to the first round, and annealing temperature is arranged to the different gradients such as 56,58,60,62 or 64 DEG C,
Every kind of primer optimal condition is found with this.
Embodiment 2:
The checking of method for quick
The theory testing of primer specificity:
The primer of design in embodiment 1 is brought in common mulberry fungal disease ITS sequence and compares, to its specificity
Carry out preliminary test.
The practice test of primer specificity:
With two parts of materials (referring to table 1), the method set up in embodiment 1 is tested, the material in table 1 is expanded
Increase.
Product after nested PCR amplification is linked to pMD 19-T carrier, connector after the recovery of glue reclaim test kit
System is as shown in table 7.
Table 7. linked system
After fully mixing, 16 DEG C of connection 4h.
Connection product transformed competence colibacillus escherichia coli are enterprising after Amp (ampicillin, ampicillin) resistant panel
Row screening, after choosing speckle amplification, carries out the detection of bacterium solution PCR, and positive colony bacterium solution is delivered to Hua Da gene sequencing.
The Preliminary Applications of method for quick:
Spring in 2016 does not show disease in the collection of It In Beibei, Chongqing different regions mulberry field diseases of mulberry fruits field occurred frequently
And immature Fructus Mori, it is allocated as 14 parts altogether, concrete condition is shown in Table 8, detected using said method.
The immaturity Fructus Mori that table 8. is gathered
Embodiment 3:
The experimental result of embodiment 2 and analysis:
Different sclerotiniose pathogen ITS sequence comparison results:
Find after being compared using ClustalX 2, same pathogen different strains ITS sequence indifference, therefore final choosing
Mulberry reality cup cup fungi, caruncula shape cup cup fungi, core ground cane bacterium, sclerotinite and five groups of Mulberry Phoma sp is taken to compare, its result is such as
Shown in Fig. 1, the primer being combined using Primer 5.0 designed by Fig. 1 is as shown in table 9.
The nested primer sequence going out designed by table 9.
The foundation of sclerotiniose method for quick:
Carry out grads PCR mensure, primer SW-SX2F/2R (core ground cane using the bi-material in the primer pair table 1 in table 9
Bacterium) expected clip size be 325bp, measurement result as shown in Figure 2 a and 2 b, it can be seen that temperature from Fig. 2 a and Fig. 2 b
On three on primer impact less, in addition to when 64 DEG C, expanding effect is not so good, 56~62 DEG C.But in Fig. 2 a and Fig. 2 b according to
There is a faint miscellaneous band, it may be mulberry ITS sequence near dilute visible 700bp;500bp about have one faint miscellaneous band it may be possible to
Template used (first round amplified production) excessive concentration causes.And amplified production concentration is slightly higher, there are some conditions of streakings.Always
For body, the method has certain feasibility, and has wider annealing temperature.
The result of diseases of mulberry fruits method for quick is shown in Table 10.
The theoretical validation of table 10. primer specificity
As shown in table 10, as can be seen from Table 10, designed primer can only be specific for the theoretical validation of specific primer
Detect in pathogen, other mulberry fungal disease no same clip, there is certain specificity in theory.
Shown by sequencing, amplified production fits like a glove (as shown in SEQ ID NO.5) with expected fragment, shows the method
There is accuracy and specificity.
SW-SX2F/SW-SX2R 325bp
TTGCGCCTCCGGGTGCTTCAGGGCTTGCTCGCCCGCCGAAGGATATTTAAACTCTGTTTATTATTGTCGTCTGAGTA
CTATATAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAG
TAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATG
CCTGTTCGAGCGTCATTTCAACCCTCAAGCTTTGCTTGGTATTGGGCCTCGCCAGTAAAATGGCGGGCCTTAAAATC
AGTGTGCGGTGCCGTTG, as shown in SEQ ID NO.5.
The application result of method for quick:
Do not show symptom using the method for quick that the present invention sets up to 14 parts and immaturity Fructus Mori detects, result
As shown in Figure 3 a and Figure 3 b shows, after first round ITS1/ITS4 primer amplification, this 4 parts of samples of only a1-a4 have amplified pathogenic fungi
ITS sequence (size 500bp about), also amplified in 8 parts of samples such as c1, c2 a 700bp about band, it may
For mulberry ITS sequence.Although the ITS1/ITS4 primer of the first round only amplifies 4 parts in 14 parts of samples, the second wheel nido draws
After thing amplification, most sample standard deviations can amplify respective strap, shows that the method can greatly improve the sensitivity of detection, can examine
Go out the more lower bound degree that conventional single PCR can not detect, simultaneously the explanation main mulberry field in Beibei district infection diseases of mulberry fruits situation
More serious.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and various changes are made to it, without departing from claims of the present invention limited range in details.
SEQUENCE LISTING
<110>Southwest University
<120>Fructus Mori constrictive sclerotinios pathogen early stage method for quick based on nest-type PRC
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Core ground cane bacterium SW-SX2F
<400> 1
ttgcgcctcc gggtgcttca 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Core ground cane bacterium SW-SX2R
<400> 2
caacggcacc gcacactgat t 21
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223> ITS1
<400> 3
tccgtaggtg aacctgcgg 19
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223> ITS4
<400> 4
tcctccgctt attgatatgc 20
<210> 5
<211> 325
<212> DNA
<213>Artificial sequence
<220>
<223> SW-SX2F/ SW-SX2R
<400> 5
ttgcgcctcc gggtgcttca gggcttgctc gcccgccgaa ggatatttaa actctgttta 60
ttattgtcgt ctgagtacta tataatagtt aaaactttca acaacggatc tcttggttct 120
ggcatcgatg aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa 180
tcatcgaatc tttgaacgca cattgcgccc cttggtattc cggggggcat gcctgttcga 240
gcgtcatttc aaccctcaag ctttgcttgg tattgggcct cgccagtaaa atggcgggcc 300
ttaaaatcag tgtgcggtgc cgttg 325
Claims (6)
1. the Fructus Mori constrictive sclerotinios pathogen early stage method for quick based on nest-type PRC is it is characterised in that include following
Step:
(1) using the tissue sample DNA of CTAB method extraction as template, the expansion of first round PCR is carried out using universal primer ITS1/ITS4
Increase;
(2) the second wheel PCR amplification is carried out for template with first round product, nested primer adopts the specific primer of core ground cane bacterium,
Sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2;
(3) amplified band according to step (2) diagnoses Fructus Mori constrictive sclerotinios.
2. detection method according to claim 1 is it is characterised in that in step (1), the condition of first round PCR amplification is such as
Under:95 DEG C of denaturations 3min;94 DEG C of degeneration 30s, 55 DEG C of annealing 50s, 72 DEG C of extension 50s, 32 circulations, 72 DEG C of extension 10min,
4 DEG C, ∞.
3. detection method according to claim 1 is it is characterised in that in step (1), the sequence of universal primer ITS1/ITS4
Row are as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. detection method according to claim 1 is it is characterised in that in step (2), the second wheel amplification condition is similar to first
Wheel, annealing temperature is arranged to 56,58,60,62 or 64 DEG C of different gradients, finds every kind of primer optimal condition with this.
5. a pair of core ground cane bacterium primer is it is characterised in that sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
6. the screening technique of a pair of core ground cane bacterium primer described in claim 5 is it is characterised in that be with Mulberry reality cup cup fungi, meat
Abundant shape cup cup fungi, sclerotinite and Mulberry Phoma sp are comparison, by the traditional Fructus Mori constrictive sclerotinios pathogen ITS sequence in NCBI
Row, carry out Multiple Sequence Alignment with ClustalX 2, ESPript 3.0 carries out variation analyses, Primer 5.0 carries out setting of primer
Count and assess and obtain.
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104513860A (en) * | 2015-01-12 | 2015-04-15 | 西南大学 | Method for rapidly diagnosing shrunken-fruitsclerotiniose of mulberry |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN104513860A (en) * | 2015-01-12 | 2015-04-15 | 西南大学 | Method for rapidly diagnosing shrunken-fruitsclerotiniose of mulberry |
Non-Patent Citations (2)
Title |
---|
胡君欢等: "宁波桑果基地菌核病菌的多样性与ITS 初步分析", 《宁波大学学报( 理工版)》 * |
苏正川: "桑葚缩小型菌核病病原学及其分子检测体系", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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