CN101712983B - Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof - Google Patents
Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof Download PDFInfo
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- CN101712983B CN101712983B CN2009100446882A CN200910044688A CN101712983B CN 101712983 B CN101712983 B CN 101712983B CN 2009100446882 A CN2009100446882 A CN 2009100446882A CN 200910044688 A CN200910044688 A CN 200910044688A CN 101712983 B CN101712983 B CN 101712983B
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Abstract
The invention relates to the biological detection technology, in particular to primers for detecting citrus canker pathogenic bacteria and a detection method thereof. The invention aims to provide the primers and method for dual PCR-DHPLC detection of the citrus canker pathogenic bacteria. The primers for detecting the citrus canker pathogenic bacteria of the invention have good primer specificity and high detection sensitivity; and the detection method of the invention has high accuracy and high sensitivity and can quickly and simply judge whether a sample has the citrus canker pathogenic bacteria or not, thereby ensuring the safety in import and export.
Description
Technical field
The present invention relates to Measurement for Biotechnique, relate in particular to the c itrus canker germ and detect with primer and detection method.
Background technology
Citrus bacterial canker disease is by the important quarantine venereal disease evil due to citrus ulcer bacteria (the Xanthomonas axonopodis pv.Citri) infection; Be domestic and international Quarantine Objects; Have a strong impact on citrus safety in production and foreign trade, listed in " the People's Republic of China enter the territory Plant Quarantine property harmful organism register " that announced in 2007 by China State General Administration for Quality Supervision.
This germ survives the winter in the scab of leaf, branch and fruit, overflows from sick portion when spring in next year condition is suitable, borrows wind and rain, entomochory, and through host's pore, hole skin and wound are invaded.Make blade, the tender tip and fruit morbidity, also do not have the method for a kind of effective this germ of elimination at present.The most sweet oranges of China and this disease of shaddock class producing region have generation in various degree, and what cause damage maximum economically is oranges and tangerines.
Most at present the master that is viewed as of the quarantine of citrus bacterial canker disease with the plant disease symptom; Can judge with naked eyes fully in the ordinary course of things; But many disease-resistant or non-sensitive kinds lack manifest symptom; Be prone to obscure with other diseases, simultaneously because kind and the mutation of Xanthomonas campestris are various, very numerous and diverse and difficult to the biochemical identification of citrus ulcer bacteria.The detection technique research that is directed against the c itrus canker germ at present both at home and abroad is based upon on the basis of conventional PCR and real-time fluorescence PCR basically; Wang Zhongkang etc. have designed the detection primer of c itrus canker germ and have adopted conventional PCR method that this germ is detected with H.D.Coletta-Filho, and application real time fluorescent PCR methods such as Vessela Mavrodieva have carried out the research of context of detection to the c itrus canker germ.
The double PCR technology can detect to the dna sequence dna of two different zones, improves the precision that dna molecular detects greatly, reaches the purpose that improves recall rate.Sex change performance liquid chromatography (DHPLC) technology is through PCR nucleic acid fragment to be measured is carried the DNA separator column that flows through patent down at damping fluid; Utilize the different graded of damping fluid; Realization is to the separation and the analysis of the big or small nucleic acid fragment of difference, by the separated DNA sample of ultraviolet detection; And under the column temperature condition of partially denaturing, pass through heterozygosis and the different principle of zygoid RT in post, check and analysis DNA site difference and sudden change are carried out more sensitive analysis and comparison to the result.Still the research report that does not adopt double PCR to combine the technology of efficient sex change liquid chromatography (doublx PCR-DHPLC) that citrus bacterial canker disease is quick and precisely detected at present.
Summary of the invention
The object of the invention is to be provided for primer and the method that c itrus canker germ double PCR-DHPLC detects.
The present invention has designed through the c itrus canker germ Xanthomonas axonopodis pv.citri OPM-12 SCAR fragment regional sequence of having reported (gb|AF312370.1) and has been used for primer of the present invention to SEQ ID NO.1 and 2; And combine primer SEQ ID NO.3 and 4 pairs of report such as Coletta-Filho to carry out double PCR-DHPLC method detection c itrus canker germ; Described two pairs of primers of being made up of forward and reverse primer are right, and its nucleotide sequence is respectively
SEQ?ID?NO.1：5’-ACGCTCGATCTGCACCTATT-3’；
SEQ?ID?NO.2：5’-CCAACGAGTCTCAAGCATCA-3’；
SEQ?ID?NO.3：5’-CGCCATCCCCACCACCACCACGAC-3’；
SEQ?ID?NO.4：5’-AACCGCTCAATGCCATCCACTTCA-3’。
The present invention gives double PCR-DHPLC method of using above-mentioned primer, and it is template with the DNA of bacteria, carries out the double PCR amplification, and the PCR product after the amplification directly carries out DHPLC and analyzes.
Its reaction system is: 10 * PCR buffer, 2.5 μ L; DNTPs (10mmol/L) 0.5 μ L, MgCl2 (25mmol/L) 2 μ L, each 1.0 μ L of 10 μ mol/L primers; Taq DNA polymerase (2U/ μ L) 1.0 μ L; 1ng/ μ L～10ng/ μ L, dna profiling 5 μ L, the sterilization ultrapure water complements to 25 μ L.
Wherein, above-mentioned reaction conditions can be: 96 ℃ of preparatory sex change 5min; 96 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30s, circulating reaction 30 times; 72 ℃ are extended 5min.
The DHPLC analysis condition is set at: sample size 5 μ L, 50 ℃ of column temperatures select full fragment trace routine to detect flow velocity 0.9mL/min.
The present invention has also set up further double PCR-DHPLC analyzed and has obtained DHPLC Molecular Identification system and the molecule sample that male PCR product is as a result identified.Promptly with the PCR product of designed primer SEQ ID NO.1 and 2 amplification citrus bacterial canker disease bacteria strain DNA affinity tag as the PCR-DHPLC authentication method.Unknown amplified production can carry out the evaluation of c itrus canker germ to unknown bacterium through carrying out half sex change DHPLC analysis with this affinity tag.The DHPLC testing conditions is: the setting clip size is 236bp, each sample introduction 5 μ L, and detected temperatures: Tm=65 ℃, 0.9mL/min analyzes with flow velocity.
For double PCR-DHPLC, selecting suitable primer is the guarantee of sensitivity of analytical method and accuracy.
The present invention also provides the test kit that contains said primer.
Further, the nucleic acid molecule that also has primer SEQ ID NO.1 or 2 to combine and amplify with c itrus canker germ DNA amplifying nucleic acid specificity site in the test kit provided by the invention.Those skilled in the art know, SEQ ID NO.1 or/and in 2 the variation of indivedual bases can tolerate.
It is good that the present invention is designed for the primer specificity that detects the c itrus canker germ; Detection sensitivity is high; Detection method accuracy of the present invention is high, highly sensitive, its can be fast simply judgement sample whether the c itrus canker germ is arranged, for imports and exports safety provides assurance.
Description of drawings
Fig. 1 double PCR-DHPLC detects c itrus canker germ DNA result;
Fig. 2 the inventive method sensitivity detected result;
Fig. 3 primer SEQ ID NO.1 and 2 amplification c itrus canker germ dna fragmentation DHPLC, half sex change detect.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting scope of the present invention.
Embodiment 1
Primer design is with synthetic
C itrus canker germ Xanthomonas axonopodis pv.citri OPM-12 SCARfragment regional sequence (gb|AF312370.1) according to report, designed primer sequence and be:
SEQ ID NO.1 (forward): 5 '-ACGCTCGATCTGCACCTATT-3 ';
SEQ ID NO.2 (oppositely): 5 '-CCAACGAGTCTCAAGCATCA-3 '.
According to the primer of report, primer sequence is:
SEQ ID NO.3 (forward): 5 '-CGCCATCCCCACCACCACCACGAC-3 ';
SEQ ID NO.4 (oppositely): 5 '-AACCGCTCAATGCCATCCACTTCA-3 '.
Embodiment 2
The collection of thalline
The bacterium colony of growth on the flat board: at least 5 of direct picking colonies of transfering loop add the 1mL aqua sterilisa and fully vibrate the centrifugal 3min of 8000g to the 1.5mL centrifuge tube; Abandon supernatant, in pipe, add 1mL aqua sterilisa, triplicate again; Get deposition and extract DNA, carry out double PCR-DHPLC and detect.
Oranges and tangerines blade or material: beat on the oranges and tangerines blade with the 6mm punch tool and to get 50 of apertures; There is the scab material to beat and gets the scab part; Or got the about 1g of illness organization material and add the washing of fully vibrating of 1mL sterile purified water, get the centrifugal 5min of washings 8000g, supernatant discarded; Get deposition and extract DNA, carry out double PCR-DHPLC and detect.
Embodiment 3
The extraction of DNA of bacteria
1) in deposition, add TE damping fluid 600 μ L, 10%SDS solution 30 μ L, 20mg/mL Proteinase K 15 μ L, mixing, 1h is hatched in 37 ℃ of water-baths.
2) add isopyknic trichloromethane-primary isoamyl alcohol (24: 1), mixing.The centrifugal 5min of 10000g moves to supernatant in the new centrifuge tube.
3) add equal-volume phenol-trichloromethane-primary isoamyl alcohol (25: 24: 1), mixing, the centrifugal 5min of 10000g moves to a new centrifuge tube with supernatant.
4) add the Virahol of 0.6 times of volume, mixing gently ,-20 ℃ of deposition 1h, the centrifugal 5min of 10000g abandons supernatant.
5) add 1mL70% washing with alcohol deposition, 8000g is centrifugal, abandons supernatant, dries.
6) add 50 μ LTE damping fluid dissolving DNAs deposition ,-20 ℃ of prolonged preservation are subsequent use.
Embodiment 4
The foundation of double PCR-DHPLC detection method
Present method has selected the citrus bacterial canker disease bacteria strain of 5 strain different sourcess as positive control, has adopted separated portions mikrobe in the oranges and tangerines blade, several frequently seen and compare analysis with the phytopathogen of the nearly edge of c itrus canker germ and other phytopathogen of part as the bacterial strain of participating in the experiment simultaneously.Strains tested particular case such as table 1.
Table 1 supplies examination bacterium and source thereof
After above-mentioned bacterial strains carried out DNA extraction, use reactions system and condition and carry out double PCR and detect and study.The PCR instrument that uses is the Biometra Company products.
Reaction system: 10 * PCR buffer, 2.5 μ L, dNTPs (10mmol/L) 0.5 μ L, MgCl
2(25mmol/L) 2 μ L, each 1.0 μ L of 10 μ mol/L primers, Taq DNA polymerase (2U/ μ L) 1.0 μ L, 1ng/ μ L～10ng/ μ L, dna profiling 5 μ L, the sterilization ultrapure water complements to 25 μ L.
Reaction conditions: 96 ℃ of preparatory sex change 5min; 96 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30s, circulating reaction 30 times; 72 ℃ are extended 5min.
The DHPLC analysis condition is set at: sample size 5 μ L, 50 ℃ of column temperatures select full fragment trace routine to detect flow velocity 0.9mL/min.
The PCR product is put into the DHPLC Sample Room, and elutriant is made up of buffer A (0.1mol/L triethylamine acetyl salt) and buffer B (0.1mol/L triethylamine acetyl salt and 25% acetonitrile), and sample size 5 μ L are set; 50 ℃ of column temperatures select full fragment trace routine to detect flow velocity 0.9mL/min; System is automatically according to the mode of linear increment; Flow velocity with flow velocity 0.9mL/min elutes the dna molecular on the DNASep post, and wavelength 260nm reads light absorption value in the place, after system handles automatically; Form DHPLC peak type collection of illustrative plates, supply Analysis and Identification.
Specific detection
The c itrus canker germ positive strain DNA that adopts extraction to obtain detects with other bacterial strain DNA that participates in the experiment, and water increases as contrast.
Test-results:
5 strain c itrus canker germs all amplify tangible positive peak, see Fig. 1.Other bacterial strain DNA that participates in the experiment does not all have positive signal.
Sensitivity detects
Inoculation c itrus canker germ is in 100ml PSA liquid nutrient medium, and 24h is cultivated in 28 ℃ of concussions, with colony counting method each bacteria concentration is measured, and nutrient solution is processed 2 * 10 with saline water
5, 2 * 10
4, 2 * 10
3, 2 * 10
2, 2 * 10
16 dilution gradients.Extract bacterium liquid DNA, the performing PCR of going forward side by side-DHPLC analyzes.
Test-results such as Fig. 2 utilize present method can be effectively from containing 2 * 10
2Detect this pathogenic bacteria in the bacterium liquid of the c itrus canker germ of CFU/mL.
Embodiment 5
The foundation of PCR-DHPLC molecular criteria system
Whether the homology that is tested and appraised 5 strain c itrus canker germ DNA cloning product sequences is consistent, in the hope of setting up unified molecule sample and molecular criteria system.
Having carried out sequence half sex change between the PCR product that adopts DHPLC that designed primer is voluntarily obtained amplification detects: with per two kinds with the PCR product of order of magnitude concentration each other according to 1: 1 mixed; Mix products and single PCR product are carried out following denaturation renaturation process on the PCR appearance: 95 ℃ of initial sex change 5min; 94.5 ℃ sex change 20s; Circulation of later every 20s reduces by 0.5 ℃, slowly cools to 25 ℃, to form homology or heteroduplex DNA molecule mixture.PCR product after the cooling processing is put into the DHPLC Sample Room, import fragment sequence to be detected, set each sample introduction 5 μ L.Detected temperatures is reference with the melting temperature(Tm) (Tm=64.9 ℃) of this sequence fragment, and selecting column temperature respectively is 65 ℃, sets clip size 236bp, with flow velocity 0.9mL/min, carries out DHPLC and analyzes.
PCR mix products through sex change and slow renaturation processing; Carrying out half sex change DHPLC analytical results is: a tangible absorption peak all only appears in the PCR mix products of primer SEQ ID NO.1 and 2 amplification c itrus canker germ A1-A5 DNA about 4.6min (Fig. 3), and the single PCR product result after result and the same treatment is consistent.Evidence: after carrying out pcr amplification through primer SEQ ID NO.1 and 2, the PCR product fragment sequence of each c itrus canker germ DNA is consistent, and homology is high, no mutational site.
Because of the PCR product sequence of primer SEQ ID NO.1 and 2 amplifications, 5 strain bacterial strains is all consistent, can sets up with primer SEQ ID NO.1 and 2 and increase the PCR product of any strain bacterial strain DNA among the A1-A5 as the affinity tag of PCR-DHPLC authentication method.PCR product that the clip size of the unknown is consistent or close and molecule sample carry out DHPLC half sex change analysis after carrying out sex change and slow renaturation processing.DHPLC identifies that condition is: the setting clip size is 236bp, each sample introduction 5 μ L.Detected temperatures: Tm=65 ℃,, carry out DHPLC and analyze with flow velocity 0.9mL/min.The result occurs can being judged as the c itrus canker germ under single peak type and other peak type normal circumstances.
Sequence table
< 110>Inspection & Quarantine Technology Center of Hu'nan Entry-Exit Inspection & Quarantine Bureau
< 120>primer that is used for the detection of c itrus canker germ is to reaching detection method
<130>PLH091016
<140>
<141>
<160>4
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
< 213>artificial sequence
<400>1
acgctcgatc?tgcacctatt 20
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<400>2
ccaacgagtc?tcaagcatca 20
<210>3
<211>24
<212>DNA
< 213>artificial sequence
<400>3
cgccatcccc?accaccacca?cgac 24
<210>4
<211>24
<212>DNA
< 213>artificial sequence
<400>4
aaccgctcaa?tgccatccac?ttca 24
Claims (8)
1. be used for the primer that the c itrus canker germ detects, its nucleotides sequence is classified as:
SEQ?ID?NO.1：5’-ACGCTCGATCTGCACCTATT-3’；
SEQ?ID?NO.2：5’-CCAACGAGTCTCAAGCATCA-3’；
SEQ?ID?NO.3：5’-CGCCATCCCCACCACCACCACGAC-3’；
SEQ?ID?NO.4：5’-AACCGCTCAATGCCATCCACTTCA-3’。
2. one kind with SEQ ID NO.1 or 2 nucleic acid molecule that to be primer combine and amplify with c itrus canker germ DNA amplifying nucleic acid specificity site.
3. method that detects the c itrus canker germ; This method with the c itrus canker germ extract DNA be masterplate; Utilize the described primer of claim 1 to carry out the double PCR amplification; Reaction finishes the back uses DHPLC double PCR product sequence is carried out check and analysis, and whether set up with the PCR product of primer SEQ ID NO.1 or 2 amplification c itrus canker germ DNA is that the c itrus canker germ is judged as the standards system of molecule sample to unknown bacterium.
4. method as claimed in claim 3 is characterized in that, the annealing temperature in the reaction process of double PCR is 63 ℃.
5. method as claimed in claim 3 is characterized in that, in the DHPLC testing process, detecting fragment setting size is 236bp.
6. method as claimed in claim 3 is characterized in that, in the DHPLC decision process, the nucleic acid molecule in the application rights requirement 2 is as the molecule sample and set up the judgement that the molecular criteria system is carried out the c itrus canker germ, and detecting column temperature is 50 ℃.
7. the test kit that contains the said primer of claim 1.
8. test kit as claimed in claim 7, it also comprises the described nucleic acid molecule of claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102634603B (en) * | 2012-05-10 | 2013-04-10 | 湖南农业大学 | PCR detection method for citrus canker |
| CN103114135A (en) * | 2013-01-25 | 2013-05-22 | 郑媛 | Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for citrus canker pathogenic bacteria |
| CN111690759B (en) * | 2020-08-04 | 2023-10-17 | 西南大学 | Specific primer, kit and method for detecting RPA of citrus canker pathogen |
| CN112280900A (en) * | 2020-11-06 | 2021-01-29 | 宁波市农业科学研究院 | Molecular marker primer combination and method for rapidly and synchronously identifying citrus huanglongbing disease, canker disease, recession disease, leaf shattering disease and split skin disease |
| CN113186318A (en) * | 2021-06-07 | 2021-07-30 | 中国海关科学技术研究中心 | Primer group, kit and detection method for PCR detection of citrus canker pathogen |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404689C (en) * | 2005-12-29 | 2008-07-23 | 重庆大学 | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404689C (en) * | 2005-12-29 | 2008-07-23 | 重庆大学 | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof |
Non-Patent Citations (3)
| Title |
|---|
| Coletta F.Primers based on the rpf gene region provide improved detection of xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants..《journal of applied microbiology》.2006,第100卷(第2期),279-285. * |
| 殷幼平.柑桔溃疡病菌实时荧光定量PCR检测与应用.《植物保护学报》.2007,第34卷(第6期),607-613. * |
| 陈福如.柑橘溃疡病菌PCR快速检测技术研究初报.《福建农业学报》.2005,第20卷(第1期),46-48. * |
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