CN102051415A - LAMP primer group for rapidly detecting phytophthora. medicaginis at normal temperature - Google Patents
LAMP primer group for rapidly detecting phytophthora. medicaginis at normal temperature Download PDFInfo
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- CN102051415A CN102051415A CN2010105665766A CN201010566576A CN102051415A CN 102051415 A CN102051415 A CN 102051415A CN 2010105665766 A CN2010105665766 A CN 2010105665766A CN 201010566576 A CN201010566576 A CN 201010566576A CN 102051415 A CN102051415 A CN 102051415A
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- phytophthora
- clover
- root rot
- primer
- normal temperature
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Abstract
The invention relates to an amplifying nucleic acid primer for rapidly detecting phytophthora. medicaginis at normal temperature. The LAMP amplifying nucleic acid primer comprises an FIP primer, an F3 primer, a BIP primer, a B3 primer, an LF primer and an LB primer. The LAMP detecting system of the invention has favorable specificity and can rapidly, conveniently and efficiently detect phytophthora. medicaginis under the condition of 63 DEG C and meet the rapid detecting requirement of a port on the phytophthora. medicaginis.
Description
Technical field
The present invention relates to the plant protection field, and the normal temperature method for quick at clover phytophthora root rot bacterium is provided, and is applicable to that Check and Examination of Port quarantine mechanism uses.
Background technology
Alfalfa is pulse family, Medicago per nnial herb, is to plant maximum herbage varieties in the world. owing to advantages such as its wide adaptability, output height, quality betters, have the laudatory title of " king of herbage ".The clover root rot is one of main disease that causes the alfalfa production loss, causing this sick pathogen is clover pine root fungus (Phytophthora medicaginis E.M.Hans.etD.P.Maxwell), belong to algae thing circle, the oomycetes door, Oomycete, rotten mould order, pythiaceae, phytophthora, the parasitic clover of energy, garbanzo, Kiwifruit, the multiple important cash crop of Radix Dauci Sativae or the like, alfalfa wherein, garbanzo is its main host, clover phytophthora root rot bacterium all has distribution in each continent, scope comprises: Japan, Pakistan, Turkey, Greece, Canada, U.S. Argentina, Australia, South Africa etc., the clover root rot does not also have the report in China's generation at present.
China in 2007 issue and " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register " that implements are increased to 435 kinds with quarantine harmful organisms by original 84 kinds, and clover phytophthora root rot bacterium is exactly one of newly-increased disease, up to the present, each port also is in the state that does not have according to complying with to the quarantine to clover phytophthora root rot bacterium, therefore need set up detection method and standard as early as possible, prevent that this danger pathogenic bacteria from importing China into.
Compare the regular-PCR amplification method, the LAMP isothermal amplification technology has the following advantages: (1) is special strong, article 4, primer is to the identification in 6 distinguished sequence districts of target sequence, guaranteed the high degree of specificity of LAMP amplification, (2) susceptibility height, its waterfall type amplification has improved amplification efficiency greatly, is much higher than conventional P CR method, (3) amplification homo(io)thermism, need not accurate temperature controlling, the LAMP method has realized increasing under the isothermal condition, need not the control of frequent heating and cooling and precise dose, and simple water-bath can be realized amplification, thereby greatly reduce the input of instrument, (4) easy and simple to handle, need not complicated programdesign, greatly reduce the technical requirements of nucleic acid amplification.
The present invention uses the rapid detection that LAMP primer isothermal amplification technology applies to clover phytophthora root rot bacterium, and the common alternating temperature amplification of specificity, remolding sensitivity PCR method is higher, can exempt high instrument input simultaneously, is convenient to basic unit and promotes the use of.
Summary of the invention
The technical issues that need to address of the present invention provide the LAMP primer sets that is used to detect clover phytophthora root rot bacterium.
The LAMP primer sequence that the present invention is used to detect clover phytophthora root rot bacterium is:
F3：5’-AAACCTATCAGCGAAGGC-3’
B3 5’-ATATTGGACATAACGAGCCT-3’
FIP?5’-TCACCATGCATGCGTACTTAGTGGATTTCCTAAGTTCGCACCA-3’
BIP?5’-GTGCAGCGTTGACGTGTTAAACGGAACTTTCGTTTTGGAGGA-3’
Carry out the constant temperature expansion with the above-mentioned primer sets testing sample DNA of team, if the result produces scalariform DNA band, then this sample contains clover phytophthora root rot bacterium, if band does not appear in amplification, does not then contain clover phytophthora root rot bacterium in this sample.
Compared with prior art, progress of the present invention is: detecting instrument is simple, need not expensive PCR instrument, only needs simple water-bath or other temperature control devices to get final product, and has reduced the laboratory input, is fit to the basic unit laboratory and promotes.
Description of drawings
Accompanying drawing .LAMP primer sets constant-temperature amplification result.No. 2 swimming lane is a clover phytophthora root rot bacterium, can amplify scalariform DNA band, and all the other swimming lanes except the genomic dna band, all do not amplify the scalariform band for contrast.
M:DL2000Marker, 1:H2O, 2, clover phytophthora root rot bacterium, 3: soybean phytophthora, 4: ramie mould, 5: Phytophthora cactorum, 6: the red rotten epidemic disease of potato is mould, and 7: Phytophthora capsici, 8: phytophthora infestans, 9: soybean phytophthora, 10: Phytophthora nicotianae
Embodiment
The LAMP design of primers:
1 TTCGAGCCAG CAGAATGAGA AAGCGAGGAC AAGAAGGATC AAGTCATGAG
AAGACAAGAG
61 AACGACAACC AGACAATCTA CCGGTAGTCG TCGATAAGAG ACCAAATAAA
TGTAGACTCG
121 TCTGCGGCAC GTGTTACTGA TACACCGAAA AAAATGCAGT TTTACAGCTG
TCTATTCAGA
181 AAAAACTGTA GTTCAAGTGA AACATATGTT TCTTTCGCTA TGTACATACG
TAATTAAATG
241 AAATTATAGT TTACGTTTTT AGCATCTGCC TCCATATCTA CAGAATTCAA
CTTTATTCAA
301 ACGACCAAGT?TTTGTAGGTA ATATCACATG ATCCAAATCG ATTCACATTC
GGTATTGCAA
F3
361 TGTAAACCTT CGGTATCATT TGGTGTATGC AACGGGGCCC CACGAGAGCA
ATTTCCATAA
F2 LF
421 TCCAGTACCA ATGTGCTGGA?TTTTGAAACC?TATCAGCGAA GGCTGTCCCA
AAACCAGTAG
F1 B1 LB
481 GGATTTCCTA AGTTCGCACC AATTAATAGT CAGTACTTTA ACTAAGTACG
CATGCATGGT
B2 B3
541 GATTAATACT CGGTGTTGCG AGGACGTGCA?GCGTTGACGT GTTAAAACTT
CTTCGATGGG
601 CTAAACGCGC ACTTCCTCCA?AAACGAAAGT TCCGATGGAA?GGCTCGTTAT
GTCCAATATT
661 AACTCCATTT AGAGCTTTGC ATCAATTTGT GGCGATGAAA TGGGTACTAA
CTTTATGTCT
F3：CGGTATTGCAATGTAAACCT
B3：ATCACCATGCATGCGTAC
FIP：TCCAGCACATTGGTACTGGATTATATCATTTGGTGTATGCAACGG
BIP：AACCTATCAGCGAAGGCTGTCCTGACTATTAATTGGTGCGAAC
LF：TGGAAATTGCTCTCGTG
LB：AAACCAGTAGGGATTTCC
Reagent and equipment
Water-bath, primer is synthetic by the handsome Bioisystech Co., Ltd in Shanghai, and the Bst archaeal dna polymerase is available from NEB, and trimethyl-glycine (Betaine), DNTPmix purchase Sigma,,
The sample source
The present invention says that the red rotten epidemic disease of clover phytophthora root rot bacterium, soybean phytophthora, potato of employing is mould etc. available from ATCC, and Phytophthora cactorum, Phytophthora nicotianae, ramie mould, Phytophthora capsici, potato late blight are mould etc. from the isolate of China different areas, see the following form:
Coding kind of name source
DNA extraction: 50mg left and right sides mycelia, extract DNA with Qiagen Dneasy Plant Mini Kit test kit.
Reaction system: the mixed solution cumulative volume of every reaction is 25 μ l
10*ThermoPol?Buffer?2.5μl
10mM?DNTPmix 1μl
5M?Betaine 1μl
10μMFIP 2μl
10μMBIP 2μl
10μMF3 0.5μl
10μMB3 0.5μl
10μMLF 1μl
10μM?LB 1μl
8U/ μ lBst enzyme 1 μ l
DDH2O 12μl
Template DNA 0.5 μ l
25μl
Response procedures: 65 ℃ of reaction 1h.
The result: No. 2 swimming lanes are clover phytophthora root rot bacterium, the scalariform specific band appears, all the other swimming lanes are except the genomic dna band, the scalariform band does not all appear, the result shows the high specificity that primer of the present invention is right, to other belong to that other fungi such as soybean phytophthora, ramie mould, Phytophthora cactorum, the red rotten epidemic disease of potato are mould together, Phytophthora capsici, potato late blight are mould, Phytophthora nicotianae or the like does not have non-specific band to occur.
Sequence table
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<400>1
CGGTATTGCAATGTAAACCT?20
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<400>2
ATCACCATGCATGCGTAC?18
<210>1
<211>45
<212>DNA
＜213〉artificial sequence
<400>3
TCCAGCACATTGGTACTGGATTATATCATTTGGTGTATGCAACGG?45
<210>1
<211>43
<212>DNA
＜213〉artificial sequence
<400>4
AACCTATCAGCGAAGGCTGTCCTGACTATTAATTGGTGCGAAC?43
<210>1
<211>17
<212>DNA
＜213〉artificial sequence
<400>5
TGGAAATTGCTCTCGTG?17
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<400>6
AAACCAGTAGGGATTTCC?18
Claims (3)
1. LAMP primer sets that is used for clover phytophthora root rot bacterium normal temperature rapid detection is characterized in that sequence is:
F3：5’-CGGTATTGCAATGTAAACCT-3’
B3：5’-ATCACCATGCATGCGTAC-3’
FIP：5’-TCCAGCACATTGGTACTGGATTATATCATTTGGTGTATGCAACGG-3’
BIP：5’-AACCTATCAGCGAAGGCTGTCCTGACTATTAATTGGTGCGAAC-3’
LF：5’-TGGAAATTGCTCTCGTG-3’
LB：5’-AAACCAGTAGGGATTTCC-3’
2. one kind is used for clover phytophthora root rot bacterium normal temperature method for quick, it is characterized in that, with the LAMP primer sets in the claim 1 testing sample is carried out DNA cloning, amplification condition: 63 ℃ 1 hour, if electrophoresis result shows special ladder-like spectrum belt, then this sample contains clover phytophthora root rot bacterium, if special ladder-like spectrum belt does not appear in the result, does not then contain clover phytophthora root rot bacterium in the sample.
3. be used for clover phytophthora root rot bacterium normal temperature quick detection kit based on claim 1 and claim 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586469A (en) * | 2012-03-31 | 2012-07-18 | 中国科学院微生物研究所 | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers |
| CN103773865A (en) * | 2014-01-15 | 2014-05-07 | 福建省农业科学院植物保护研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof |
| CN104498589A (en) * | 2014-08-29 | 2015-04-08 | 兰州大学 | Pair of specific molecular detection primers for Embellisia astragali and detection method |
| CN106434990A (en) * | 2016-11-30 | 2017-02-22 | 福建省农业科学院植物保护研究所 | Nested PCR (Polymerase Chain Reaction) detection method for phytophthora of alfalfa |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705294A (en) * | 2009-12-04 | 2010-05-12 | 天津出入境检验检疫局动植物与食品检测中心 | Primer pair for LAMP for detecting Phytophthora medicaginis |
-
2010
- 2010-11-30 CN CN2010105665766A patent/CN102051415A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705294A (en) * | 2009-12-04 | 2010-05-12 | 天津出入境检验检疫局动植物与食品检测中心 | Primer pair for LAMP for detecting Phytophthora medicaginis |
Non-Patent Citations (2)
| Title |
|---|
| KENTARO NAGAMINE等: "Loop-mediated Isothermal Amplification Reaction Using a Nondenatured Template", 《CLINICAL CHEMISTRY》 * |
| 张裕君等: "基于LAMP方法的苜蓿疫霉根腐病菌分子检测研究", 《中国植保导刊》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586469A (en) * | 2012-03-31 | 2012-07-18 | 中国科学院微生物研究所 | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers |
| CN102586469B (en) * | 2012-03-31 | 2013-10-23 | 中国科学院微生物研究所 | Loop-mediated isothermal amplification (LAMP) primers for detecting Phytophthora capsici and kit comprising primers |
| CN103773865A (en) * | 2014-01-15 | 2014-05-07 | 福建省农业科学院植物保护研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof |
| CN103773865B (en) * | 2014-01-15 | 2015-05-20 | 福建省农业科学院植物保护研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof |
| CN104498589A (en) * | 2014-08-29 | 2015-04-08 | 兰州大学 | Pair of specific molecular detection primers for Embellisia astragali and detection method |
| CN104498589B (en) * | 2014-08-29 | 2016-06-29 | 兰州大学 | A pair specific molecular detection primer of prairie milk vetch yellow dwarf's root rot and detection method |
| CN106434990A (en) * | 2016-11-30 | 2017-02-22 | 福建省农业科学院植物保护研究所 | Nested PCR (Polymerase Chain Reaction) detection method for phytophthora of alfalfa |
| CN106434990B (en) * | 2016-11-30 | 2019-06-25 | 福建省农业科学院植物保护研究所 | A kind of clover phytophthora nested PCR detection method |
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Application publication date: 20110511 |