CN108866214A - It is a kind of for detecting RPA primer, probe, kit and the detection method of tobacco ralstonia solanacearum - Google Patents
It is a kind of for detecting RPA primer, probe, kit and the detection method of tobacco ralstonia solanacearum Download PDFInfo
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- CN108866214A CN108866214A CN201810709756.1A CN201810709756A CN108866214A CN 108866214 A CN108866214 A CN 108866214A CN 201810709756 A CN201810709756 A CN 201810709756A CN 108866214 A CN108866214 A CN 108866214A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of for detecting RPA primer, probe, kit and the detection method of tobacco ralstonia solanacearum, belongs to Biosafety Technique field；The RPA primer is designed based on tobacco ralstonia solanacearum Rip TALI-9 gene, RPA primer sequence such as SEQ ID No：Shown in 1 and 2；The PRA probe is designed according to the primer amplified region RPA, and sequence size 45bp-54bp, 5 ' ends are marked with FAM, and 3 ' ends are modified with C3-Spacer, and THF modification is carried out at the 34bp at the sequence medium spacing 5 ' of RPA probe end.The present invention also provides the kits for containing the RPA primer and probe.The present invention further provides the detection methods based on the RPA primer and probe foundation, carry out RPA amplified reaction as template using the DNA extracted in sample；The present invention is for the first time by the Molecular Detection of RPA technical application to tobacco ralstonia solanacearum, it has both the feature that high specificity, high sensitivity, practicability are good, lower to instrument and equipment requirement, provides new method for the diagnosis of tobacco bacterial wilt, disease control and pesticide reduction use.
Description
Technical field
The invention belongs to Biosafety Technique fields, specifically, being related to a kind of for detecting the RPA of tobacco ralstonia solanacearum
Primer, probe, kit and detection method.
Background technique
Tobacco bacterial wilt is the bacterial disease as caused by Ralstonia solanacearum (Ralstonia solanacearum), main
Occur in the higher tropical and subtropical region of temperature, it is extremely serious to the harm of continuous-cropping tobacco, it is the destruction in leaf tobacco production
Venereal disease evil.Tobacco ralstonia solanacearum is soil inhabitant, overwintering in plant invalid body, after the intrusion of cigarette strain root in vascular bundle
Field planting causes withered cigarette strain, death and root system necrosis.High temperature (30 DEG C or more), high humidity (90% or more relative humidity)
It is the essential condition of tobacco bacterial wilt prevalence, it, should since the tobacco bred and chemopreventive effects that lack high resistance to bacterial wilt are bad
Disease causes to seriously endanger to China's main product cigarette district throughout the year.Therefore, tobacco ralstonia solanacearum is accurately detected, observing and predicting and integrating to the disease
Prevention and treatment all has important scientific meaning.
The detection of tobacco ralstonia solanacearum mainly includes traditional Physiology and biochemistry detection method, Serology test and molecule
Detection method.Traditional Physiology and biochemistry detection method mainly passes through the means such as plate streaking or coating, utilizes different culture medium
Combination with antibiotic identifies tobacco ralstonia solanacearum, and this method detection cycle is long, and sensitivity is low, above has biggish limitation in application
Property.Serologic detection technology detects tobacco ralstonia solanacearum based on the principle that antigen and antibody specific combines, and mainly includes
Enzyme linked immunosorbent assay (Elisa), indirect IF staining method (IFAS) and immunity test strip method etc., this method sensitivity
Height, but the deficiency that there are false positive rates is higher, testing cost is excessively high.Molecular detecting method is the most common inspection of tobacco ralstonia solanacearum
Survey means mainly include that polymerase chain reaction (PCR), Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR) and ring mediate
Isothermal amplification technique (LAMP).The characteristics of that PCR and Real-time PCR has is accurate, efficient, high sensitivity, while can realize
To the quantitative detection of tobacco ralstonia solanacearum, but the technology is higher scarce to instrument and equipment, experiment condition, personnel specialty competency profiling
Point.L AMP technology can complete detection reaction in 65 DEG C of constant temperature 1h, lower to instrument and equipment requirement, but its reaction system needs 6
Primer, and easily there is false positive.Therefore, continual exploitation it is accurate to tobacco ralstonia solanacearum, it is efficient, require instrument and equipment low inspection
Survey technology has important more practical value.
Recombinase polymeric enzymatic amplification technology (Recombinase polymerase amplification, RPA) is in recent years
Carry out a kind of emerging isothermal amplification technology, reaction process includes three kinds of core enzymes：Recombinase (Recombinase), single-stranded knot
Hop protein (S SB) and archaeal dna polymerase (Ploymerase).RPA technology reaction principle is：1) recombinase is in the presence of ATP
RPA primer, which combines, forms nucleoprotein microfilament, and nucleoprotein microfilament can be mobile to template DNA, and primer and template DNA sequence are compared
It is right；2) when base pair complementarity occurs for primer and template DNA, recombining reaction occurs for nucleoprotein microfilament and template DNA, and in list
Under the action of chain binding protein, template DNA unwinding；3) under the action of archaeal dna polymerase, the exponential amplification of nucleic acid, shape occurs
The DNA cloning product of Cheng Xin.RPA technology only needs the primer of a pair of of 30bp-35bp, in 37 DEG C of -42 DEG C of isothermal reaction 20min
Complete amplified reaction.RPA amplified production combination lateral flow chromatographs examination technology, and detection reaction can be completed in 10min, that is, is utilized
RPA technology can complete the Visual retrieval to tobacco ralstonia solanacearum in 30min.Currently, RPA technology is widely used to disease
Poison, bacterium, mycoplasma and animal parasitic nematodes quick detection in, but there is not yet its tobacco ralstonia solanacearum context of detection report
Road.
Summary of the invention
1, it to solve the problems, such as
Aiming at the problem that tobacco ralstonia solanacearum there is no effectively progress RPA technology detection, the present invention provides a kind of for detecting
RPA primer, probe, kit and the detection method of tobacco ralstonia solanacearum, the special of tobacco ralstonia solanacearum detection can be improved in it
Property and sensitivity.
2, technical solution
To solve the above problems, the present invention adopts the following technical scheme that.
It is a kind of for detecting the RPA primer and RPA probe of tobacco ralstonia solanacearum,
The RPA primer is based on tobacco ralstonia solanacearum Rip TALI-9 gene (GenBank No.：LN874053.1 it) sets
Meter,
The upstream primer of the RPA primer such as SEQ ID No：Shown in 1,
The downstream primer of the RPA primer such as SEQ ID No：Shown in 2；
The PRA probe is designed according to the primer amplified region RPA,
The sequence size of the RPA probe is 45bp-54bp, and 5 ' ends are marked with FAM, and 3 ' ends are repaired with C3-Spacer
Decorations, and THF modification is carried out at the 34bp at the sequence medium spacing 5 ' of RPA probe end.
Preferably, the downstream primer of the RPA primer includes such as SEQ ID No：Sequence shown in 2, and its 5 ' end is used
Biotin modification；The RPA probe includes such as SEQ ID No：Sequence shown in 3,5 ' ends are marked with FAM, and C3- is used at 3 ' ends
Spacer modification, and THF modification is carried out at the 34bp at the sequence medium spacing 5 ' of RPA probe end.
A kind of kit for being used to detect tobacco ralstonia solanacearum containing RPA primer and RPA probe described above.
Preferably, the kit further include RPA reaction tube, negative control template, in positive control template at least
It is a kind of.
It is a kind of for detecting the detection method of tobacco ralstonia solanacearum,
Include the following steps：
(1) DNA in sample is extracted；
(2) it using the DNA that step (1) is extracted as DNA profiling to be detected, is visited using RPA primer described above and RPA
Needle carries out RPA amplified reaction in RPA reaction tube；
(3) RPA amplified production is analyzed.
Preferably, sample described in step (1) is pathogen to be checked, diseased plant tissue or morbidity soil.
Preferably, sample described in step (1) is pathogen to be checked, the detection limit of the RPA primer and RPA probe
For 1pg/ μ L；Sample described in step (1) is diseased plant tissue, and the detection of the RPA primer and RPA probe is limited to
103CFU/g disease sense plant tissue；Sample described in step (1) is morbidity soil, the inspection of the RPA primer and RPA probe
Rising limit is 104CFU/g soil.
Preferably, the reaction system of RPA amplified reaction is calculated as in step (2) with 50 μ L：
Preferably, the reaction condition of RPA amplified reaction is in step (2)：37 DEG C -45 DEG C (when concrete application, can choose
37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C, 45 DEG C) under react 20 minutes, then terminate reaction on ice.
Preferably, the method for analysis RPA amplified production includes the following steps in step (3)：Take RPA amplified production and buffering
Liquid mixing prepares mixed liquor, and test strips loading area is placed in mixed liquor, result is observed and recorded after 4min-5min；As a result
Determine as follows：If band (as detection band) occurs in the detection line position of test strips, and there is band (as in Quality Control line position
Quality control band), then show to contain tobacco ralstonia solanacearum in the sample, if only band (as Quality Control occurs to test strips in Quality Control line position
Band), then show in the sample without containing tobacco ralstonia solanacearum.
3, beneficial effect
Compared with the prior art, beneficial effects of the present invention are：
RPA primer, probe, kit and detection method of the present invention provide new technology for the detection of tobacco ralstonia solanacearum,
For the first time by the Molecular Detection of RPA technical application to tobacco ralstonia solanacearum, it is good, right to have both high specificity, high sensitivity, practicability
Instrument and equipment requires lower feature, provides new method for the diagnosis of tobacco bacterial wilt, disease control and pesticide reduction use；Its
Design of the middle RPA primer and probe based on tobacco ralstonia solanacearum Rip TALI-9 gene, can stablize amplification and specificity is higher, real
The Detection accuracy of existing tobacco ralstonia solanacearum is high；Wherein kit and detection method detection sensitivity with higher, while can
The constant-temperature amplification and Visual retrieval to tobacco ralstonia solanacearum are completed at 38 DEG C and in 30min, are not necessarily to any PCR instrument, gel
Electrophoresis and imaging system, detection efficiency solve existing much higher than more traditional Physiology and biochemistry detection method even PCR detection method
There are phenomenons longer the time required to detection method, high according to lazyness height, testing result false positive to instrument and equipment for routine techniques.By
This does not need cumbersome electrophoresis and purple as it can be seen that the tobacco ralstonia solanacearum RPA detection method established is easy to operate and result easily determines
Outer observation provides strong technical support for the on-site test or quick detection of tobacco ralstonia solanacearum.
Detailed description of the invention
Fig. 1 is the testing result figure of the foundation of 2 tobacco ralstonia solanacearum RPA detection method of embodiment；
Fig. 2 is the specific detection result figure of 3 tobacco ralstonia solanacearum RPA primer and probe of embodiment；
Fig. 3 is the sensitivity technique result figure of 4 tobacco ralstonia solanacearum RPA primer and probe of embodiment；
Fig. 4 is the testing result figure of 5 Wan Nan cigarette district tobacco ralstonia solanacearum incidence of embodiment.
Specific embodiment
The present invention is further described below combined with specific embodiments below.
RPA reaction tube and reaction buffer used in the following embodiment are purchased from TwistDX company of Britain, and commodity are compiled
Number T ANFO02KIT；Wherein, recombinase, single strand binding protein and archaeal dna polymerase are present in RPA reaction with RPA freeze-dried powder state
Guan Zhong, in use, being dissolved with reaction buffer, entire RPA amplified reaction carries out in RPA reaction tube.
Embodiment 1
Tobacco ralstonia solanacearum is used for the screening of RPA primer and probe.It is with tobacco ralstonia solanacearum Rip TALI-9 gene
Target gene；It is required according to TwistDX operation manual, design can be used for primer and the progress of RPA kit (TwistAmp nfo)
Screening obtains the primer pair that amplification efficiency is high, sensitivity and specificity are best.Screen obtained best primer (as RPA primer
Upstream and downstream primer) sequence such as SEQ ID No：1 and SEQ ID No：Shown in 2.Then above-mentioned primer is modified, in RPA
Biotin (Biotin) marker site is added in the end of downstream primer 5 ' of primer, in addition, being designed according to RPA primer amplification segment
5 ' ends of the probe that one size is 45bp-54bp, probe are marked with FAM, and 3 ' ends carry out C3-Spacer modification, and in probe
Medium spacing 5 ', which is held, carries out THF modification at 34bp.Finally according to requiring screening to obtain high sensitivity and can specific amplification target gene
, for detecting the RPA primer and probe of tobacco ralstonia solanacearum, their sequence is as follows：
Upstream primer Rs-RPA-F (SEQ ID No.1)：5'-GGCTATTCCCGTGAGCAGATCCGGAAGCT CAAG-
3 ',
Downstream primer Rs-RPA-R (SEQ ID No.2)：5'-Biotin-GCCGAGCGATGTCCACGATGTGGG
CCCTGGTCA-3'；
Probe Rs-LF-Probe (SEQ ID No.3)：5'-FAM-TGCCGGATCAGCCGCAGACGGCAGTC
GCTCCGG-(THF)-TGGTCGCCAGGAACTA-(C3-Spacer)-3’。
Embodiment 2
The foundation of tobacco ralstonia solanacearum RPA detection method
The detection method for being used to detect tobacco ralstonia solanacearum of the present embodiment, includes the following steps：
(1) DNA in sample is extracted；
(2) DNA extracted using step (1) as DNA profiling to be detected, using RPA primer described in embodiment 1 and
RPA probe carries out RPA amplified reaction in RPA reaction tube；
The reaction condition of RPA amplified reaction is in step (2)：It is reacted at 38 DEG C 20 minutes, then terminates reaction on ice；
The reaction system of RPA amplified reaction is calculated as in step (2) with 50 μ L：
(3) RPA amplified production is analyzed；It takes RPA amplified production and buffer mixing to prepare mixed liquor and (takes above-mentioned 1 μ L RP A
Amplified production is added in 49 μ L PBST buffers and mixes, wherein containing the P BS of 0.1% Tween-20 in the PBST buffer
Buffer), by test strips (Milenia Genline Hybridetect-1, German Milenia Biotec company) loading area
It is placed in mixed liquor, result is observed and recorded after 4min-5min；As a result judgement is as follows：If the detection line position of test strips goes out
Existing band, and band occurs in Quality Control line position, then shows to contain tobacco ralstonia solanacearum in the sample, if test strips only nature controlling line
There is band in position, then shows in the sample without containing tobacco ralstonia solanacearum.
As shown in Figure 1, carrying out the test of negative control template and positive control template respectively；Wherein positive control template
DNA profiling to be detected in test replaces with the DNA of tobacco ralstonia solanacearum, and other content is with above-mentioned for detecting tobacco bacterial wilt
The detection method of bacterium, the label " 1 " in Fig. 1 refer to the testing result figure of positive control template, it is seen that the detection line of test strips
There is band in position, and band occurs in Quality Control line position, then shows to contain tobacco ralstonia solanacearum in the sample；It is wherein negative right
DdH is replaced with according to the DNA profiling to be detected in the test of template2O, other content is with above-mentioned for detecting tobacco ralstonia solanacearum
Detection method, the label " 2 " in Fig. 1 refer to the testing result figure of negative control template, it is seen that test strips only Quality Control line position
There is band, then shows in the sample without containing tobacco ralstonia solanacearum.
The testing result figure of above-mentioned Fig. 1 shows that RPA primer and probe of the present invention can stablize amplification.
Embodiment 3
The specific detection of tobacco ralstonia solanacearum RPA primer and probe
With embodiment 2, difference is the detection method for detecting tobacco ralstonia solanacearum of the present embodiment：
The sample is respectively tobacco ralstonia solanacearum, Flavobacterium, Arthrobacter, Escherichia coli, pseudomonad, saprophytic
Arthrobacterium, bacillus amyloliquefaciens, bacillus genus, bacillus subtilis, serratia marcescens, bacterial leaf streak of rice
Bacterium, Phytophthora nicotianae and black root of tobacco bacterium.
As shown in Fig. 2, the label " 1 " in Fig. 2 refers to the testing result of tobacco ralstonia solanacearum, the label " 2 " in Fig. 2 refers to
Be Flavobacterium testing result, the label " 3 " in Fig. 2 refers to the testing result of Arthrobacter, the label " 4 " in Fig. 2
Refer to the testing result of Escherichia coli, the label " 5 " in Fig. 2 refers to the testing result of pseudomonad, the label in Fig. 2
" 6 " refer to the testing result of saprophytic arthrobacterium, and the label " 7 " in Fig. 2 refers to the testing result of bacillus amyloliquefaciens, figure
Label " 8 " in 2 refers to the testing result of bacillus genus, and the label " 9 " in Fig. 2 refers to the inspection of bacillus subtilis
It surveys as a result, the label " 10 " in Fig. 2 refers to the testing result of serratia marcescens, the label " 11 " in Fig. 2 refers to rice
The testing result of Xanthomonas campestris PV.oryzicola, the label " 12 " in Fig. 2 refer to the testing result of Phytophthora nicotianae, the label in Fig. 2
" 13 " refer to the testing result of black root of tobacco bacterium；
The testing result figure of above-mentioned Fig. 2 shows the detection line position and nature controlling line of the only testing result of tobacco ralstonia solanacearum
There is band simultaneously in position, and only band occurs to the testing result of other germs in Quality Control line position, this shows RPA primer of the present invention
It is higher with probe specificity.
Embodiment 4
The sensitivity technique of tobacco ralstonia solanacearum RPA primer and probe
With embodiment 2, difference is the detection method for detecting tobacco ralstonia solanacearum of the present embodiment：
(1) sample described in is pathogen to be checked
Wherein pathogen to be checked is tobacco ralstonia solanacearum, takes tobacco ralstonia solanacearum single bacterium to fall in 10mL LB solution and expands
The extraction that the bacterium solution in exponential phase of growth is used for genomic DNA is collected in culture afterwards for 24 hours；
By genomic DNA obtained above by 10 times of gradients be successively diluted to 100ng/ μ L, 10ng/ μ L, 1ng/ μ L,
100pg/μL、10pg/μL、1pg/μL、100fg/μL、10fg/μL、1fg/μL、0fg/μL。
As shown in (A) in Fig. 3, label " 1 ", " 2 ", " 3 ", " 4 ", " 5 ", " 6 ", " 7 ", " 8 ", " 9 ", " 10 " are respectively referred to
Be various concentration (100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L,
1fg/ μ L, 0fg/ μ L) genomic DNA testing result.
The testing result figure of (A) shows that the detection of tobacco ralstonia solanacearum DNA is limited to 1pg/ μ L in above-mentioned Fig. 3.
(2) sample described in is diseased plant tissue
Wherein diseased plant tissue is susceptible tobacco pathogenesis stem tissue, specially intercepts the susceptible cigarette after surface sterilization respectively
Grass morbidity stem tissue 1g, is added the bacteria suspension of the tobacco ralstonia solanacearum of fresh configuration, until tobacco bacterial wilt after being pulverized with grinding pestle
The final concentration of bacterium is respectively 109CFU/mL、108CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL、
103CFU/mL、102CFU/mL、101CFU/mL、100CFU/mL simulates the susceptible tobacco of nature；Then plus 0.01% Tween-20 is fixed
Hold to 1mL, is placed at room temperature for 10min after vortex 3min, takes 50 μ L supernatants in 95 DEG C of denaturation treatment 10min, i.e. genomic DNA.
As shown in (B) in Fig. 3, label " 1 ", " 2 ", " 3 ", " 4 ", " 5 ", " 6 ", " 7 ", " 8 ", " 9 ", " 10 " are respectively referred to
It is that the susceptible tobacco pathogenesis stem group of 1g is woven in difference and takes under bacterium amount (109CFU/mL、108CFU/mL、107CFU/mL、106CFU/mL、
105CFU/mL、104CFU/mL、103CFU/mL、102CFU/mL、101CFU/mL、100CFU/m L) genomic DNA detection
As a result.
The testing result figure of (B) shows that the detection of tobacco ralstonia solanacearum DNA in diseased plant tissue is limited in above-mentioned Fig. 3
103CFU/g diseased plant tissue.
(3) sample described in is morbidity soil
Wherein morbidity soil is that soil carries tobacco ralstonia solanacearum, specially by OD600 ≈ 1 (about 1 × 109CFU/mL)
Tobacco ralstonia solanacearum presses 10 times of gradient dilutions, is configured to 109CFU/mL、108CFU/mL、107CFU/mL、106CFU/mL、
105CFU/mL、104CFU/mL、103CFU/mL、102CFU/mL、101CFU/mL、100The bacteria suspension of CFU/mL；Then by 1g soil
The ratio of 1mL bacteria suspension is added in earth, and the bacteria suspension of various concentration is added separately in the healthy soil of equivalent, simulation nature hair
Sick soil earth；It is mentioned with soil genomic DNA rapidly extracting kit (DP4002 is purchased from hundred Tyke Biotechnology Co., Ltd of Beijing)
The genomic DNA of tobacco ralstonia solanacearum in morbidity soil is taken, specific experiment process is referring to the kit specification.
As shown in (C) in Fig. 3, label " 1 ", " 2 ", " 3 ", " 4 ", " 5 ", " 6 ", " 7 ", " 8 ", " 9 ", " 10 " are respectively referred to
It is 1g soil in the case where difference takes bacterium amount (109CFU/mL、108CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、
104CFU/mL、103CFU/mL、102CFU/mL、101CFU/mL、100CFU/mL the testing result of genome D NA).
The testing result figure of (C) shows that the detection of tobacco ralstonia solanacearum DNA in morbidity soil is limited in above-mentioned Fig. 3
104CFU/g soil.
By embodiment 4 and Fig. 3 it is found that of the invention (kit and) method detection sensitivity with higher.
Embodiment 5
Wan Nan cigarette district tobacco ralstonia solanacearum incidence
In order to verify tobacco ralstonia solanacearum RPA detection technique practicability, to acquisition from 8 parts of tobacco blueness of Wan Nan cigarette district
The diseased plant tissue and morbidity soil of blight bacterium morbidity field carry out the development of RPA detection technique respectively；Tobacco ralstonia solanacearum
RPA detection method is the same as embodiment 2.
In Fig. 4 (A) be diseased plant tissue testing result figure, label " 1 ", " 2 ", " 3 ", " 4 ", " 5 ", " 6 ", " 7 ",
" 8 " refer respectively to the susceptible tobacco pathogenesis stem tissue of 8 parts of tobacco ralstonia solanacearum morbidity fields, and label " 9 ", " 10 " respectively refer to
It is health tobacco tissue；
(B) is the testing result figure of morbidity soil, label " 1 ", " 2 ", " 3 ", " 4 ", " 5 ", " 6 ", " 7 ", " 8 " point in Fig. 4
Do not refer to that the soil of 8 parts of tobacco ralstonia solanacearum morbidity fields carries tobacco ralstonia solanacearum, label " 9 ", " 10 " refer respectively to
Healthy soil；
The testing result figure of above-mentioned Fig. 4 shows the diseased plant tissue and morbidity soil of 8 parts of tobacco ralstonia solanacearum morbidity fields
Earth can detect tobacco ralstonia solanacearum, and fail to detect tobacco bacterial wilt in 2 parts of health tobacco tissues and healthy soil
Bacterium, this shows that tobacco ralstonia solanacearum RPA primer and probe of the present invention has stronger practicability.
In conclusion by embodiment 1-4 as it can be seen that RPA primer, probe, kit and detection method of the present invention, are that tobacco is green
The detection of blight bacterium provides new technology, for the first time by the Molecular Detection of RPA technical application to tobacco ralstonia solanacearum, has both special
Property it is strong, high sensitivity, practicability are good, require instrument and equipment lower feature, be the diagnosis of tobacco bacterial wilt, disease control and
Pesticide reduction uses offer new method；Wherein design of the RPA primer and probe based on tobacco ralstonia solanacearum Rip TALI-9 gene,
Amplification can be stablized and specificity is higher, realize that the Detection accuracy of tobacco ralstonia solanacearum is high；Wherein kit and detection method tool
There is a higher detection sensitivity, while can be completed at 38 DEG C and in 30min to the constant-temperature amplification of tobacco ralstonia solanacearum and visual
Change detection, be not necessarily to any PCR instrument, gel electrophoresis and imaging system, detection efficiency is much higher than more traditional Physiology and biochemistry detection method
Even PCR detection method, solve existing conventional techniques there are it is longer the time required to detection method, to instrument and equipment according to lazyness
Phenomenon high, testing result false positive is high；The tobacco ralstonia solanacearum RPA detection method that the present invention establishes is easy to operate and result is easy
Determine, do not need cumbersome electrophoresis and ultraviolet visualization, provides strong skill for the on-site test or quick detection of tobacco ralstonia solanacearum
Art is supported.
The above content is specific embodiment is combined, invention is further described in detail, and it cannot be said that the present invention is specific
Implementation is only limited to these instructions, and for those of ordinary skill in the art to which the present invention belongs, is not departing from the present invention
Design under the premise of, several simple deduction or replace can also be made, all shall be regarded as belonging to the power submitted of the present invention
The protection scope that sharp claim determines.
Sequence table
<110>Agricultural University Of Anhui
<120>It is a kind of for detecting RPA primer, probe, kit and the detection method of tobacco ralstonia solanacearum
<141> 2018-07-02
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213>Ralstonia solanacearum（Ralstonia solanacearum）(Ralstonia solanacearum)
<400> 1
ggctattccc gtgagcagat ccggaagctc aag 33
<210> 2
<211> 33
<212> DNA
<213>Ralstonia solanacearum（Ralstonia solanacearum）(Ralstonia solanacearum)
<400> 2
gccgagcgat gtccacgatg tgggccctgg tca 33
<210> 3
<211> 49
<212> DNA
<213>Ralstonia solanacearum（Ralstonia solanacearum）(Ralstonia solanacearum)
<400> 3
tgccggatca gccgcagacg gcagtcgctc cggtggtcgc caggaacta 49
Claims (10)
1. a kind of for detecting the RPA primer and RPA probe of tobacco ralstonia solanacearum, it is characterised in that：
The RPA primer is designed based on tobacco ralstonia solanacearum Rip TALI-9 gene,
The upstream primer of the RPA primer such as SEQ ID No：Shown in 1,
The downstream primer of the RPA primer such as SEQ ID No：Shown in 2；
The PRA probe is designed according to the primer amplified region RPA,
The sequence size of the RPA probe is 45bp-54bp, and 5 ' ends are marked with FAM, and 3 ' ends are modified with C3-Spacer, and
THF modification is carried out at the 34bp that the sequence medium spacing 5 ' of RPA probe is held.
2. according to claim 1 for detecting the RPA primer and RPA probe of tobacco ralstonia solanacearum, it is characterised in that：
The downstream primer of the RPA primer includes such as SEQ ID No：Sequence shown in 2, and its 5 ' end is modified with Biotin；
The RPA probe includes such as SEQ ID No：Sequence shown in 3,5 ' ends are marked with FAM, and C3-Spacer is used at 3 ' ends
Modification, and THF modification is carried out at the 34bp at the sequence medium spacing 5 ' of RPA probe end.
3. a kind of reagent for being used to detect tobacco ralstonia solanacearum containing RPA primer of any of claims 1 or 2 and RPA probe
Box.
4. kit according to claim 3, it is characterised in that：The kit further includes RPA reaction tube, negative right
According at least one of template, positive control template.
5. a kind of for detecting the detection method of tobacco ralstonia solanacearum, it is characterised in that：
Include the following steps：
(1) DNA in sample is extracted；
(2) using the DNA of step (1) extraction as DNA profiling to be detected, using RPA primer of any of claims 1 or 2 and RPA
Probe carries out RPA amplified reaction in RPA reaction tube；
(3) RPA amplified production is analyzed.
6. according to claim 5 for detecting the detection method of tobacco ralstonia solanacearum, it is characterised in that：In step (1)
The sample is pathogen to be checked, diseased plant tissue or morbidity soil.
7. according to claim 6 for detecting the detection method of tobacco ralstonia solanacearum, it is characterised in that：In step (1)
The sample is pathogen to be checked, and the detection of the RPA primer and RPA probe is limited to 1pg/ μ L；Described in step (1)
Sample is diseased plant tissue, and the detection of the RPA primer and RPA probe is limited to 103CFU/g disease sense plant tissue；Step
(1) sample described in is morbidity soil, and the detection of the RPA primer and RPA probe is limited to 104CFU/g soil.
8. according to claim 5 for detecting the detection method of tobacco ralstonia solanacearum, it is characterised in that：In step (2)
The reaction system of RPA amplified reaction is calculated as with 50 μ L：
9. according to claim 5 for detecting the detection method of tobacco ralstonia solanacearum, it is characterised in that：In step (2)
The reaction condition of RPA amplified reaction is：It is reacted at 37 DEG C -45 DEG C 20 minutes, then terminates reaction on ice.
10. for detecting the detection method of tobacco ralstonia solanacearum according to claim 5-9 any one, feature exists
In：The method of analysis RPA amplified production includes the following steps in step (3)：RPA amplified production and buffer is taken to mix preparation mixed
Liquid is closed, test strips loading area is placed in mixed liquor, result is observed and recorded after 4min-5min；As a result judgement is as follows：If examination
There is band in the detection line position of paper slip, and band occurs in Quality Control line position, then shows to contain tobacco bacterial wilt in the sample
Bacterium shows in the sample if band occurs in the only Quality Control line position of test strips without containing tobacco ralstonia solanacearum.
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