CN102925545A - New Bursaphelenchus xylophilus detection kit and detection method thereof - Google Patents
New Bursaphelenchus xylophilus detection kit and detection method thereof Download PDFInfo
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- CN102925545A CN102925545A CN2012102315558A CN201210231555A CN102925545A CN 102925545 A CN102925545 A CN 102925545A CN 2012102315558 A CN2012102315558 A CN 2012102315558A CN 201210231555 A CN201210231555 A CN 201210231555A CN 102925545 A CN102925545 A CN 102925545A
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- wood nematode
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Abstract
The present invention provides a new Bursaphelenchus xylophilus detection kit and a detection method thereof. According to the present invention, special primers having a marker and competitive primers of the Bursaphelenchus xylophilus are cleverly designed, and a PCR amplification and test strip combination detection technology is adopted to achieve rapid and efficient Bursaphelenchus xylophilus detection. The detection method has characteristics of strong specificity, high sensitivity, good stability, Bursaphelenchus xylophilus detection accuracy improvement, detection time shortening, convenience, rapidness, cost and time saving, simple principle and operation, and no requirement of special equipment, and is suitable for application and popularization in inspection and quarantine departments.
Description
Technical field
The present invention relates to the quarantine field of plant pest, a kind of method and test kit thereof that utilizes pcr amplification and nucleic acid test strip associated detection technique rapid detection pine wood nematode is provided, be applicable to Check and Examination of Port quarantine mechanism and use.
Background technology
Pine wood nematode (Bursaphelenchus xylophilus) is the topmost harmful organism that current serious threatened and endangered pine forest safety, and the pine nematode of its initiation is one of our times four large forest-crop diseases.This disease mainly is to propagate by the allocation and transportation of conifer plants and plant product, is the destructive disease of a kind of pine tree, in case occur, difficult anti-refractory can cause huge financial loss.This disease mainly is distributed in a plurality of countries such as the U.S., Canada, Japan.China finds pine wood nematode in nineteen eighty-two first in the Zhongshan Tomb, Nanjing, subsequently, again in succession in Jiangsu, the partial area on the ground such as Anhui, Guangdong and Hong Kong, Taiwan find that successively the forest in China partial area has been caused huge destruction, and it is particularly important therefore to strengthen port quarantine.
Studying efficient, quick, sensitive pine wood nematode inspection technology is effectively to find and control the important means of disease.In order to seek accurately and rapidly detection method, a lot of scholars are furtheing investigate aspect morphology, genetics, immunology, pathology and the biological chemistry of nematode, and worked out such as detection methods such as pH value detection method, cellulase diffusion detection methods, played positive effect for the detection of pine wood nematode, but these methods are subject to the restriction of the aspects such as pine wood nematode growth and development stage, insect population quantity, experiment condition stability, have certain limitation in actual application.
In the last few years, along with the fast development of molecular biology and biotechnology, the method for researchist's applied molecular biology detected pine wood nematode, to overcome the deficiency of bringing owing to traditional method.Although PCR-RAPDL, PCR-RFLP and PCR-SSCP technology equimolecular biological detecting method have improved the accuracy that detects, but these methods are higher to technician and requirement for experiment condition, thereby these methods promote the use of, therefore seek a kind of simply, fast and accurately detection method be still current in production reality urgent problem.
This research is used pcr amplification and nucleic acid test strip associated detection technique by designing dexterously Auele Specific Primer and the competitive primer for the tape label of pine wood nematode, has realized the efficient rapid detection of pine wood nematode.The method high specificity, highly sensitive and good stability is for improving accuracy that nematode detects, shortening detection time and have important effect.Convenient and swift, save cost and time, principle and simple to operate does not need specific apparatus, is fit to inspection and quarantine department utilization and popularization, simultaneously the rapid detection of other nematodes is also had important reference.
Summary of the invention
The technical issues that need to address of the present invention provide for detection of the Auele Specific Primer of pine wood nematode and competitive primer.
Another problem that the present invention need to solve provides the PCR detection kit that comprises above-mentioned primer.
The present invention for detection of the PCR primer sequence of pine wood nematode is:
Upstream primer Bx-F3-bio:5 ' biotin-TCTGCACGTTGTGACAGTC-3 '
Downstream primer Bx-B3-fit:5 ' fitc-TCATCCGAACGTCCCTGAC-3 '
Competition primer Bx-B3-comp:5 '-GTCAGGGACGTTCGGAACT-3 '
Pine wood nematode detection kit of the present invention comprises following composition:
(1) pine wood nematode dna profiling and negative control are intended the pine wood nematode dna profiling
(2) pine wood nematode upstream and downstream primer and competition primer
Upstream primer Bx-F3-bio:5 ' biotin-TCTGCACGTTGTGACAGTC-3 '
Downstream primer Bx-B3-fit:5 ' fitc-TCATCCGAACGTCCCTGAC-3 '
Competition primer Bx-B3-comp:5 '-GTCAGGGACGTTCGGAACT-3 '
(3) 10 * ExTaq PCR Buffer, DDH
2O, 2.5mM dNTP, 5U/ μ l ExTaq enzyme
With aforesaid method testing sample DNA is carried out pcr amplification, PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that then this sample contains pine wood nematode, if present feminine gender, then illustrates and does not contain pine wood nematode in this sample.
Compared with prior art, progress of the present invention is: use instrument simple, need not agarose gel electrophoresis, ultraviolet gel imaging, thereby avoided the pollution of ethidium bromide, saved time, high specificity, highly sensitive, good stability has reduced the input in laboratory, and suitable laboratories is promoted the use of.
Description of drawings
Fig. 1 is pine wood nematode test kit specificity test result, 1 negative contrast, 2,3 for intending pine wood nematode, 4 is Bursaphelenchus doui, 5 are food cap aphelenchoides, and 6 is Bursaphelenchus rainulfi, and 7 is really to slide sword to belong to a kind of of nematode (Aphelenchus SP.), 8,9 is pine wood nematode, can detect exactly pine wood nematode by this test kit of output from Fig. 1
Embodiment
The PCR design of primers:
1 CGTAACAAGG TAGCTGTAGG TGAACCTTCG GCTGGATCAT TACCGATCCT ATGACACATT
61 TATTCGTGCT CGTCACGATG ATGCGATTGG TGACTTCGGT TGCCGCGCAT GATGGCGGTT
121 CGATTCGCGT CGTTCCGCCT ACTTATGGTT CGCATGGAAG CCGAGAGGCG ACCGTGCAAC
181 GGTGAAGTCT GGGTTTCTAC GTGCTGTTGT TGAGTTGGCG TTTTACCGTG CCGACAGATG
241 AGACCAGCCA GCTGCTTGCC GATTCGTTCT GGCGAGCGTA GGATTGAAAA GCCCGAGAGG
3O1 CTGCCCTGAC AAAACATTCA TTTTACATTT ATTTTGTTGG AAAAGAGCTT TAAGTTACTC
36l CGGTGGATCA CTTGGCTCGC GGGTCGATGA AGAACGCAGT GAATTGCGAT AATAAGTACG
421 AATTACAGAT ATTATGAGTA CCATGTTTTT GAATGCATAT TGCGCTCTTG GGCTTTGCTC
481 TTGAGCATAT TCGATTCAGG GTGTGTTTTT AAACTCGAGC AGAAACGCCG ACTTGTTTTT
541 TTCAAGTT
TC TGCACGTTGT GACAGTCGTC TCGCATTGTT CACGCAATGT TAGGCACCAT
F3
601 CTGTTTTACG CGGTTTGTTC CGCGACCAAT ATCTTCTACG CACTGTTTGT TCGTGCGGCG
661 AGAGGGCTTC GTGCTCGATT GTCGTGCGGC TAAACCGTTT GGTGATGTTG TTTCAACGGC
721 GCGGCC
GTCA GGGACGTTCG GATGAGAATG TTTGGAGTCC TGGCTGCGGT TTGTTGAGCT
B3
781 TCGTCGTGAA GCCTTGCGGG CAGTGTTGTC GGAATTGGTT GAAACCACCT GAGTTGGGTA
841 TGACTACCTG CTGAACTTAA GCATATCAGT AAGCAGAGGA AAAGAAACAA ACATGGATTC
901 CCTTAGTAAC GGCGAGTGAA A
The dna sequence dna of the pine wood nematode of announcing according to Genbank, utilize primer-design software to design one group of primer, clip size is 197bp, 5 of upstream and downstream primer ' end uses respectively vitamin H (Biotin) and fluorescein (Fitc) to modify, and is synthetic by Invitrogen (the handsome Bioisystech Co., Ltd in Shanghai) company:
Upstream primer Bx-F3-bio:5 ' Biotin-TCTGCACGTTGTGACAGTC-3 '
Downstream primer Bx-B3-fit:5 ' Fitc-TCATCCGAACGTCCCTGAC-3 '
Competition primer Bx-B3-comp:5 '-GTCAGGGACGTTCGGAACT-3 '
Specimen origin
This experiment test 8 different line insect populations of source in the Tianjin entry and exit plant in recent years, comprising 2 pine wood nematode populations, intend the pine wood nematode populations for 2,3 umbrella aphelenchoides populations, 1 very sliding sword belongs to nematode, sees the following form:
The detection of embodiment 1, pine wood nematode
DNA extraction: operate according to TIANamp Genomic DNA Kit tissue gene group DNA extraction test kit specification sheets, extract nematode gene group DNA and be dissolved among the 50uL TE ,-20 ℃ save backup.
Pcr amplification: with sterilized water as blank, respectively with BmSk1, BmSk2, BdSk1, BFSk1, BrSk1, Auk, BxUs1 and BxUs2 nematode gene group DNA as template, carry out pcr amplification.
The PCR reaction conditions: 94 ℃ of denaturation 1min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min.
ELISA test strip: get 4 μ L PCR products and be added on the sample pad of test strip, then drip 2-3 and drip damping fluid, interpretation and record the result, result such as Fig. 1, swimming lane 1:ddH2O behind the 5min; 2:BmSk1; 3:BmSk2; 4:BdSk1; 5:BFSk1; 6:BrSk1; 7:Auk; 8:BxUs1; 9:BxUs2..
The result shows to only have 8,9 pine wood nematodes the specificity red stripes to occur at detection line, and all the other water and other respectively contrast nematode and all occur without band, have shown the good specificity of this detection method.
Claims (3)
1. one kind is used for Auele Specific Primer and the competitive primer that pine wood nematode detects, and it is characterized in that sequence is:
Upstream primer Bx-F3-bio:5 ' Biotin-TCTGCACGTTGTGACAGTC-3 '
Downstream primer Bx-B3-fit:5 ' Fitc-TCATCCGAACGTCCCTGAC-3 '
Competition primer Bx-B3-comp:5 '-GTCAGGGACGTTCGGAACT-3 '
2. the test kit for detection of pine wood nematode is characterized in that, comprises following composition:
(1) positive control pine wood nematode dna profiling and negative control are intended the pine wood nematode dna profiling
(2) pine wood nematode upstream and downstream primer and competition primer
Upstream primer Bx-F3-bio:5 ' Biotin-TCTGCACGTTGTGACAGTC-3 '
Downstream primer Bx-B3-fit:5 ' Fitc-TCATCCGAACGTCCCTGAC-3 '
Competition primer Bx-B3-comp:5 '-GTCAGGGACGTTCGGAACT-3 '
(3) 10 * ExTaq PCR Buffer, ddH
2O, 2.5mM dNTP, 5U/ μ l ExTaq enzyme
(4) the membrane chromatographic test strip of vitamin H (Biotin), fluorescein (Fitc) antigenic mark.
3. the method for quick of a pine wood nematode comprises the steps:
(3) PCR reaction conditions: 94 ℃ of denaturation 1min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 5min.
(4) result of determination: PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that then this sample contains pine wood nematode, if present feminine gender, then illustrates and does not contain pine wood nematode in this sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278098A (en) * | 2014-10-13 | 2015-01-14 | 天津市畜牧兽医研究所 | Kit and method for fast detecting enterohemorrhagic escherichia coli 0157:H7 |
| CN104278099A (en) * | 2014-10-13 | 2015-01-14 | 天津市畜牧兽医研究所 | Streptococcus suis type 2 LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit and detection method thereof |
| CN112322751A (en) * | 2020-11-19 | 2021-02-05 | 南京耐德高科技有限公司 | Fluorescence and colorimetric two-in-one kit for rapidly detecting pine wood nematode nucleic acid and detection method |
| CN112481386A (en) * | 2020-11-19 | 2021-03-12 | 南京耐德高科技有限公司 | Normal-temperature rapid PCR (polymerase chain reaction) kit for detecting nucleic acid of pine wood nematode and detection method |
-
2012
- 2012-07-06 CN CN201210231555.8A patent/CN102925545B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| 杨贤等: "高灵敏可视化核酸试纸条法快速检测 HBV DNA", 《现代生物医学进展》 * |
| 王明旭等: "松材线虫rDNA-ITS2的TaqMan探针实时荧光PCR检测", 《林业科学》 * |
| 赵立荣等: "松材线虫和拟松材线虫的PCR快速检测", 《华南农业大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278098A (en) * | 2014-10-13 | 2015-01-14 | 天津市畜牧兽医研究所 | Kit and method for fast detecting enterohemorrhagic escherichia coli 0157:H7 |
| CN104278099A (en) * | 2014-10-13 | 2015-01-14 | 天津市畜牧兽医研究所 | Streptococcus suis type 2 LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection kit and detection method thereof |
| CN112322751A (en) * | 2020-11-19 | 2021-02-05 | 南京耐德高科技有限公司 | Fluorescence and colorimetric two-in-one kit for rapidly detecting pine wood nematode nucleic acid and detection method |
| CN112481386A (en) * | 2020-11-19 | 2021-03-12 | 南京耐德高科技有限公司 | Normal-temperature rapid PCR (polymerase chain reaction) kit for detecting nucleic acid of pine wood nematode and detection method |
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