CN105087800A - PCR (polymerase chain reaction) detection primer and reagent kit for camellia root-knot nematode - Google Patents
PCR (polymerase chain reaction) detection primer and reagent kit for camellia root-knot nematode Download PDFInfo
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- CN105087800A CN105087800A CN201510526631.1A CN201510526631A CN105087800A CN 105087800 A CN105087800 A CN 105087800A CN 201510526631 A CN201510526631 A CN 201510526631A CN 105087800 A CN105087800 A CN 105087800A
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- pcr
- knot nematode
- primer
- reagent kit
- root knot
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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
Abstract
The invention discloses a PCR (polymerase chain reaction) detection primer and a reagent kit for camellia root-knot nematode. The PCR detection primer mainly comprises a pair of specific primers. Nucleotide sequences of upstream primers are TTCGAGAAAC TTGGGGACCG, and nucleotide sequences of downstream primers are ATGTCCTCAA ACGCGTCACT. The PCR detection primer and the reagent kit have the advantages that the specific primers are specific and sensitive to the camellia root-knot nematode, the sizes of amplified specific fragments are 361bp, and the specific primers are free of specific bands and fragments for other root-knot nematode; detection can be completed within 4 hours by the aid of the specific primers and the reagent kit, and accordingly the PCR detection primer and the reagent kit are high in specificity, sensitivity and practicality and low in cost.
Description
Technical field
The present invention relates to the detection technique that root knot belongs to nematode, the PCR being specifically related to a kind of camellia root knot nematode detects primer and test kit.
Background technology
Root knot belong to nematode (
meloidogynegoeldi, 1887) be root system of plant obligate endoparasitism nematode, be most species in plant pathogeny line insect, distribution the widest, one of the most serious monoid of causing harm, there is very important economic implications.Root knot nematode (non-China seed) was listed in 2007 by China " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register ", implemented strict quarantine at port, to protect China's agricultural and ecological safety.It is 98 kinds that root knot known at present belongs to nematode, and wherein great majority not yet find in China, are important quarantine nematodes.Camellia root knot nematode
meloidogynecamelliaeGolden1978 intercepted and captured in 2012 first for Ningbo Port from from the camellia of Japan and tea plum, had done quarantine disinfection subsequently to this batch sample.At present to the detection of camellia root knot nematode except Morphological Identification, publication number is the LAMP method that the application for a patent for invention of CN103924001 discloses rapid detection camellia root knot nematode, although special, sensitive, testing cost is higher.
Summary of the invention
Technical problem to be solved by this invention is to provide that a species specificity is comparatively strong, the PCR of higher, the lower-cost camellia root knot nematode of sensitivity detects primer and test kit.
The rrna ITS gene (InternalTranscribedSpacer) with reference to camellia root knot nematode in GenBank is selected by system of the present invention, use PrimerExplorerV4(http: //primerexplorer.jp) devise Auele Specific Primer, through a large amount of reaction conditionss preferably, simultaneous test and proof test, and through the detection application evaluation of a large amount of actual sample (with the DNA of camellia root knot nematode for template, Meloidogyne incognita is negative control, water is blank, utilizes primer to carry out PCR detection.By observing the conservative property of amplified production and specificity, the suitableeest primer of screening), screening obtains amplification efficiency and good a pair Auele Specific Primer of specificity.Primer synthesizes by Shanghai Ying Weijie base Bioisystech Co., Ltd.
The PCR of a kind of camellia root knot nematode of the present invention detects primer, and the nucleotide sequence that this PCR detects primer is as follows:
Upstream primer: 5 '-TTCGAGAAACTTGGGGACCG-3 '
Downstream primer: 5 '-ATGTCCTCAAACGCGTCACT-3 '.
The PCR detection kit of camellia root knot nematode, its composition is as follows: without Mg
2+10 × PCR damping fluid, concentration be the MgCl of 25mM
2solution, concentration are the dNTP solution of 0.1mM, the Taq enzyme solution of concentration 5U/ μ L, concentration are the upstream primer solution of 10 μMs and concentration is the downstream primer solution of 10 μMs.
Compared with prior art the invention has the advantages that the PCR of a kind of camellia root knot nematode detects primer and test kit, mainly comprise a pair Auele Specific Primer, the nucleotide sequence of upstream primer: TTCGAGAAACTTGGGGACCG, the nucleotide sequence of downstream primer: ATGTCCTCAAACGCGTCACT.This Auele Specific Primer is special, sensitive to camellia root knot nematode, and the specific fragment size of amplification is 361bp, to other root knot nematode without specific band fragment; Apply Auele Specific Primer of the present invention and test kit, can complete detection in 4 hours, its specificity is comparatively strong, and sensitivity is higher, and practicality is comparatively strong, and cost is lower.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
The PCR detection kit of embodiment 1, camellia root knot nematode
100 times or 200 secondary response systems, 50 μ L reaction systems comprise: without Mg
2+10 × PCR damping fluid 5.0 μ L, concentration is the MgCl of 25mM
2solution 5.0 μ L, concentration is the Taq enzyme solution 0.6 μ L of the dNTP solution 4.0 μ L of 0.1mM, concentration 5U/ μ L, and concentration is all upstream primer solution and each 3.0 μ L of downstream primer solution of 10 μMs.Taq enzyme liquid, dNTP liquid is purchased from precious biotechnology (Dalian) company limited, and wherein upstream primer solution contains the primer that nucleotides sequence is classified as TTCGAGAAACTTGGGGACCG, and downstream primer solution contains nucleotides sequence and is classified as ATGTCCTCAAACGCGTCACT primer.
Embodiment 2, DNA extraction method
Picking wall scroll nematode is put into 200 μ LPCR pipes and [has been added with 10 μ L distilled waters and 5 μ L10 × PCR damping fluids (without Mg
2+)], place in liquid nitrogen 1min(or-70 degree place 0.5h) more than, after taking-up immediately 85 DEG C heating 2min.Then in PCR pipe, add 1 μ L1mg/mL Proteinase K, 56 DEG C of heating more than 30min, 95 DEG C of heating 10min, obtain DNA profiling.
Embodiment 3, PCR detection method
Detect by the PCR detection kit of camellia root knot nematode, PCR reaction system is: without Mg
2+10 × PCR damping fluid 5.0 μ L, concentration is the MgCl of 25mM
2solution 5.0 μ L, concentration is the Taq enzyme solution 0.6 μ L of the dNTP solution 4.0 μ L of 0.1mM, concentration 5U/ μ L, and concentration is all upstream primer solution and each 3.0 μ L of downstream primer solution of 10 μMs, DNA profiling 1.0 ~ 3.0 μ L, ddH
2o polishing 50 μ L.PCR reaction conditions: 94 DEG C of 4min; 94 DEG C of 40sec, 52 DEG C of 1min, 72 DEG C of 1.5min, 36 circulations; 72 DEG C of 8min.Electrophoresis: by 5 μ LPCR product 1.0%(weight/volume) agarose gel electrophoresis separation, video picture under ultraviolet gel imaging system after DNA dyeing, occurring that specific fragment size is 361bp, is then camellia root knot nematode.
The specificity of embodiment 4, camellia root knot nematode and sensitivity test
With the extracting method of embodiment 2, the PCR detection method of embodiment 3, the each root knot nematode respectively the present inventor gathered in Ningbo: camellia root knot nematode (picking up from the camellia of Japanese import), apple root knot nematode (picking up from the Acer palmatum of Japanese import), javanese root knot nematode (picking up from the tobacco of Foochow, Fujian), Meloidogyne incognita (picking up from the tobacco in Lunan, yunnan county), peanut root-knot nematode (picking up from the tomato that is produced from Xuzhou), M hapla (picking up from the tree peony that Heze City, Shandong Province produces), Meloidogyne enterolobii (picking up from the piscidia that is produced from Danzhou, Hainan) and Meloidogyne hispanica (picking up from the papaya that is produced from Danzhou, Hainan) detect, camellia root knot nematode is only had effectively to increase, there is band, and its specific fragment size is 361bp, illustrate that pair of primers of the present invention has good specificity.10 are carried out to the DNA profiling of camellia root knot nematode
0, 10
﹣ 1, 10
﹣ 2, 10
﹣ 3, 10
﹣ 4, 10
﹣ 5dilution, then carry out PCR detection, result display PCR detects and is limited to stoste dilution 10
-2doubly.
<110> Ningbo Institute of Inspection and Quarantine Science Technology
The PCR of a <120> camellia root knot nematode detects primer and test kit
<160>2
<170>PatentInversion3.5
<210>1
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of the gene design of camellia root knot nematode
<400>1
TTCGAGAAACTTGGGGACCG20
<210>2
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of the gene design of camellia root knot nematode
<400>2
ATGTCCTCAAACGCGTCACT20
Claims (2)
1. the PCR of camellia root knot nematode detects a primer, it is characterized in that the nucleotide sequence of this PCR detection primer is as follows:
Upstream primer: TTCGAGAAACTTGGGGACCG
Downstream primer: ATGTCCTCAAACGCGTCACT.
2. the PCR detection kit be made up of the PCR detection primer of a kind of camellia root knot nematode according to claim 1, is characterized in that composition is as follows: without Mg
2+10 × PCR damping fluid, concentration be the MgCl of 25mM
2solution, concentration are the dNTP solution of 0.1mM, the Taq enzyme solution of concentration 5U/ μ L, concentration are the upstream primer solution of 10 μMs and concentration is the upstream primer solution of 10 μMs.
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| CN201510526631.1A CN105087800A (en) | 2015-08-25 | 2015-08-25 | PCR (polymerase chain reaction) detection primer and reagent kit for camellia root-knot nematode |
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| CN201510526631.1A Pending CN105087800A (en) | 2015-08-25 | 2015-08-25 | PCR (polymerase chain reaction) detection primer and reagent kit for camellia root-knot nematode |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355396A (en) * | 2018-11-02 | 2019-02-19 | 宁波检验检疫科学技术研究院 | The PCR of China fir and root-knot nematode detection specific primer and kit |
| CN109486960A (en) * | 2018-11-02 | 2019-03-19 | 宁波检验检疫科学技术研究院 | A kind of method, RPA primer and kit using RPA technology detection China fir and root-knot nematode |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586461A (en) * | 2012-03-16 | 2012-07-18 | 中国农业科学院植物保护研究所 | Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method |
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2015
- 2015-08-25 CN CN201510526631.1A patent/CN105087800A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586461A (en) * | 2012-03-16 | 2012-07-18 | 中国农业科学院植物保护研究所 | Loop mediated isothermal amplification (LAMP) detection method for meloidogyne hapla and application of method |
Non-Patent Citations (2)
| Title |
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| 何洁 等: "6种根结线虫的形态和分子检测技术研究", 《植物检疫》 * |
| 王江岭 等: "日本苗木中山茶根结线虫的鉴定", 《植物检疫》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355396A (en) * | 2018-11-02 | 2019-02-19 | 宁波检验检疫科学技术研究院 | The PCR of China fir and root-knot nematode detection specific primer and kit |
| CN109486960A (en) * | 2018-11-02 | 2019-03-19 | 宁波检验检疫科学技术研究院 | A kind of method, RPA primer and kit using RPA technology detection China fir and root-knot nematode |
| CN109486960B (en) * | 2018-11-02 | 2021-11-16 | 宁波检验检疫科学技术研究院 | Method for detecting Meloidogyne incognita by applying RPA technology, RPA primer and kit |
| CN109355396B (en) * | 2018-11-02 | 2021-11-16 | 宁波检验检疫科学技术研究院 | PCR detection specific primer and kit for Meloidogyne haplocalyx |
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Application publication date: 20151125 |