CN102925545B - 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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- CN102925545B CN102925545B CN201210231555.8A CN201210231555A CN102925545B CN 102925545 B CN102925545 B CN 102925545B CN 201210231555 A CN201210231555 A CN 201210231555A CN 102925545 B CN102925545 B CN 102925545B
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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 the application of Check and Examination of Port quarantine mechanism.
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 is mainly to propagate by the allocation and transportation of conifer plants and plant product, is the destructive disease of a kind of pine tree, once occur, difficult anti-refractory, can cause huge financial loss.This disease is mainly 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, Anhui, Deng Di partial area, Guangdong and Hong Kong, Taiwan find that the forest in ，Dui China partial area caused huge destruction successively, therefore strengthens port quarantine particularly important.
Studying efficient, quick, sensitive pine wood nematode inspection technology is effectively to find and control the important means of disease.In order to find detection method accurately and rapidly, a lot of scholars are furtheing investigate aspect morphology, genetics, immunology, pathology and the biological chemistry of nematode, and worked out as detection methods such as pH value detection method, cellulase diffusion detection methods, for the detection of pine wood nematode, played positive effect, 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 due to traditional method.Although PCR-RAPDL, P CR-RFLP and PCR-SSCP technology equimolecular biological detecting method have improved the accuracy detecting, 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 by designing dexterously Auele Specific Primer and the competitive primer for the tape label of pine wood nematode, and application pcr amplification and nucleic acid test strip associated detection technique, realized the efficient rapid detection of pine wood nematode.The method high specificity, highly sensitive and good stability, for improving accuracy that nematode detects, shorten detection time and have important effect.Convenient and swift, the cost-saving and time, principle and simple to operate, does not need specific apparatus, is applicable to inspection and quarantine department and uses and promote, and the rapid detection of other nematodes is also had to important reference simultaneously.
Summary of the invention
The technical issues that need to address of the present invention are to provide for detection of the Auele Specific Primer of pine wood nematode and competitive primer.
Embodiment
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
301 CTGCCCTGAC AAAACATTCA TTTTACATTT ATTTTGTTGG AAAAGAGCTT TAAGTTACTC
361 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, 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 '
Another problem that the present invention need to solve is to provide 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 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 to pcr amplification, PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that this sample contains pine wood nematode, if present feminine gender, illustrates and in this sample, does not contain pine wood nematode.
Compared with prior art, progress of the present invention is: use instrument simple, without 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 applicable laboratories is promoted the use of.
Accompanying drawing explanation
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 is food cap aphelenchoides, and 6 is Bursaphelenchus rainulfi, and 7 belong to nematode (Aphelenchus SP.) for really sliding sword, 8,9 is pine wood nematode, uses as can be seen from Figure 1 this test kit can detect exactly pine wood nematode.
Specimen origin
This experiment test 8 the different line insect populations of originating in Tianjin entry and exit plant in recent years, comprising 2 pines
Line insect population, intends pine wood nematode population for 2,3 umbrella aphelenchoides populations, and 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 in 50uL TE ,-20 ℃ save backup.
Pcr amplification: using sterilized water as blank, using respectively BmSk1, BmSk2, BdSk1, BFSk1, BrSk1, Auk, BxUs1 and BxUs2 nematode gene group DNA as template, carry out pcr amplification.
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 in the sample pad of test strip, then drip 2-3 and drip damping fluid, interpretation record result after 5min, result is as Fig. 1, swimming lane 1:ddH2O; 2:BmSk1; 3:BmSk2; 4:BdSk1; 5:BFSk1; 6:BrSk1; 7:Auk; 8:BxUs1; 9:BxUs2..
Result shows to only have 8,9 pine wood nematodes to occur specificity red stripes at detection line, and all the other water and other respectively contrast nematode and all without band, occur, have shown the good specificity of this detection method.
Claims (3)
1. the Auele Specific Primer and the competitive primer that for pine wood nematode, detect, 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 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, ddH2O, 2.5mM dNTP, 5U/ μ l ExTaq enzyme
(4) vitamin H is that Biotin, fluorescein are the membrane chromatographic test strip of Fitc antigenic mark.
3. a method for quick for pine wood nematode, comprises the steps:
(2) 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
(3) result of determination: PCR product ELISA test strip, if control line is normal, detection line presents the positive, illustrates that this sample contains pine wood nematode, if present feminine gender, illustrates and does not contain pine wood nematode in this sample.
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| CN201210231555.8A CN102925545B (en) | 2012-07-06 | 2012-07-06 | New Bursaphelenchus xylophilus detection kit and detection method thereof |
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| 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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