CN1244703C - Pine wireworm microsatellite amplifying primer, detection method and detection kit - Google Patents
Pine wireworm microsatellite amplifying primer, detection method and detection kit Download PDFInfo
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Abstract
The present invention discloses a specificity micro-satellite amplification primer of pine wood nematode and a method for quickly detecting the molecules of the pine wood nematode, and the method is arranged on the basis of the primer. More specifically, samples which are to be detected are amplified in a PCR mode by the specificity DNA micro-satellite amplification primer of the pine wood nematode, and the pine wood nematode is justified by which amplification strips exist or don't exist after the amplification primer passes through gel electrophoresis. Simultaneously, the present invention also provides a reagent kit for detecting the pine wood nematode. The present invention is characterized in that one nematode can be identified, the DNA of the nematode need not be extracted, the reagent kit is formed by used identification reagents, and the identification work can be completed within 2.5 hours after the nematode is obtained.
Description
Technical field
The invention belongs to technical field of biological, specifically, the present invention relates to little satellite amplimer, detection method and the detection kit of a kind of pine wood nematode.
Background technology
From nineteen eighty-two since finding pine nematode first near the Zhongshan Tomb, Nanjing, " cancer " of this pine tree spread to more than 100 the counties and cities district of provinces and cities surplus Jiangsu, Anhui, Zhejiang, Guangdong, Hubei, Guangxi, Fujian, Shandong, Shanghai, Chongqing, Jiangxi, Hunan, Taiwan, Hong Kong etc. 14, existing more than 70,000 hectare of pine forest catches an illness, 1,600 ten thousand strain pine deaths, and occurrence scope enlarges year by year.In a single day the forest disease and pest that is different from other, pine tree infect pine nematode, long then half a year, short then two months will be dead.This is the main forest of forming seeds with the masson pine forest for China south, its seriousness of the harm that pine nematode causes self-evident (the white star moon, Plant Pathology, 16 (4): 59-62,1993; Wang Qiao, popular and improvement, 69-74, China Forest press, 1995 of Chinese pine wood nematode).Therefore, seek effective pine nematode control method and become the task of top priority (Yang Baojun etc., popular and improvement, China Forest press, 1995 of Chinese pine wood nematode; Xiao Gangrou, Chinese forest insect, China Forest press, 1992).
Pine nematode is a kind of worldwide great forest eqpidemic disease, and the international quarantine object has destructive destruction to pine forest.One of key measure of this disease control is the detection of strengthening cause of disease, diffusion of strict control pine wood nematode and propagation (Zhou Jiansheng, the sick worm communication of forest, 3:13-25,1997).Yet the individuality of pine wood nematode is very little, and general method is difficult to identification, in addition the plan pine wood nematode of another kind of toxicological harmless often and pine wood nematode take place simultaneously, seriously disturbed detection to pine wood nematode.Therefore, be necessary to set up the method that a cover identification of morphology combines with Molecular Identification, pine wood nematode is carried out rapid detection so that the diffusion of control pine nematode spreads.
In case pine wood nematode infects pine tree, they are food with relevant fungi parasitic on trunk or the branch and the epithelial cell of pine tree.But nearest studies show that pine wood nematode to get food be not the direct pathogenesis of loose blight.Real fatal reason is because pine wood nematode breeds, stops up screen casing in a large number, cut off the normal transport passage of water and cause pine tree lack of water withered (Holdeman Q.The pine woodnematode and the associated pine wilt disease in Japan, Technical Report.1980) in pine tree limb.In order to prevent the continuation diffusion of pine wood nematode harm, the source of infection that at first needs to follow the trail of pine wood nematode is cut off its possible route of transmission then early.
Pine wood nematode is not that produce in the native country, Asia, the country of origin is at first imported Japan (Mamiya Y.In M.J.Wingfield (ed.) .pp 59-65.1987 in 20 beginnings of the century because of the goods from North America imported timber, woodwork and band wooden packing box in North America and Europe; Mamiya Y.J.Nematology20 (2): 219-226,1988).In decades subsequently, pine wood nematode is popular wantonly in Japanese various places.1979 these years only, pine wood nematode the hazard area of Japan account for national pine forest 1/4th (LiLiebhold, A.M., W.L.MacDonald, D.Bergdahl and V.C.Mastro, For.Sci.Monogr., 30:1-49.1995).Nowadays, pine wood nematode is diffused into south east asia such as China's Mainland, Taiwan again, and its route of transmission equally mainly is the import of timber or woodwork.By to day, in, the analysis of the phyletic evolution of U.S. three ground pine wood nematode plastosome rRNA has confirmed that the sample of gathering in various places is to belong to a kind of.In conjunction with the generation history of pine wood nematode in various places, we can conclude that pine wood nematode imports China into along aforesaid passage just.Heavy lesson is told us, and strict timber quarantine and forbid the input of diseased wood is the first line of defence (Yang Baojun, Gao Jianfu, the control of pine wilt disease, China Forest press, 1989) of control pine wood nematode.
Pine wood nematode is the activity by Monochamus alternatus Hope in forest, from disease tree be transmitted to healthy wood (Linit M.J., J.Nematology 20 (2): 227-235,1988; Heisuke S.J.Jpn.For.Soc.69:492-496,1987).The investigation of pine wood nematode and Monochamus alternatus Hope life history their mutual relationships when each etap have been disclosed.Generally, from mid-April, the pine wood nematode dispersion pattern larva in four ages in the sick tree will pour in the valve chamber of the Monochamus alternatus Hope adult that firm emergence comes out.Along with the Monochamus alternatus Hope adult breaks away from sick tree, import on the healthy wood by getting food or laying eggs, cause the loose blight of a new round.Studies have shown that Japan an entrained pine wood nematode of Monochamus alternatus Hope adult quantity up to the hundreds of thousands of bar.And in the U.S. that pine wood nematode does not cause loose blight, the quantity of an entrained pine wood nematode of longicorn adult and carry the ratio of the Monochamus alternatus Hope adult of pine wood nematode, all much lower.As seen, monitor the quantity of the entrained pine wood nematode of somewhere longicorn adult and carry the ratio of the Monochamus alternatus Hope adult of pine wood nematode, can predict the generation scale of this area's pine blight.
To the early diagnosis of pine wood nematode, the meaning of particularly important is arranged.Just like people's disease, diagnosed in early days, be hopeful more to obtain medical treatment.And to those precious gardens pine tree woodss and the protection that the Age-old pine trees that important historical relic is worth are arranged, more need early diagnosis accurately.Need only the existence that detects pine wood nematode at the initial stage of loose blight, just can in time take practical measures, rescue the trees that those face impasse.But, the morbidity shortage early symptom of loose blight, normally the quantity pine wood nematode rolls up so that the screen casing that will set and pitch tube could be found preliminary symptom after stopping up.Therefore, early diagnosis is badly in need of setting up one and is overlapped detection technique fast, accurately, so that just can make a definite diagnosis the existence of pine wood nematode at the initial stage of a disease.
In fact, some researchists have also carried out such trial (Wang Yang, Wang Laifa etc., structure the 14th volume (supplementary issue) of pine wood nematode RAPD standardization reaction system, Yunnan Prov Agriculture University's journal, 1999; Zhang Lihai, Liao Jinling, Feng Zhixin, Plant Pathology.31(1)：84-89，2001)。But the method that they set up still exists many problems, and particularly sensitivity is not high, need carry out extracting to genomic dna, can not identify a nematode.
In sum, set up the Fast Detection Technique of a cover pine wood nematode, be used for the quarantine of timber, the prediction of the condition of a disaster and early diagnosis are the keys that pine wood nematode is quarantined and prevents and treats.At present to the detection of pine wood nematode mainly according to the formalness feature, normally from susceptible tree body or separate nematode in the polypide, then, confirm whether be pine wood nematode according to the morphological specificity of record.Except systematicalian, common people usually can be with the qualification result obfuscation.And, the nature pine wood nematode usually with other nematodes, for example, intend the pine wood nematode coexistence.No matter intend pine wood nematode in form, still all very similar to pine wood nematode in living environment, both forms are difficult to distinguish larva and male imago period especially.But intend pine wood nematode and can not cause the loose blight of pine tree.Therefore, easy, the rapid differentiation pine wood nematode of a kind of energy and other non-morbidity nematodes are very important.
Little satellite (Simple Sequence Repeats, abbreviating SSR as) research of dna polymorphism is restriction fragment length multiformity (the Restriction Fragment Length Polymorphism that continues, abbreviate RFLP as), random amplification DNA multiformity (Random Amplified polymorphic DNA, abbreviate RAPD as) afterwards, the s-generation molecular genetic marker technique that development in recent years is got up, be meant the section of DNA that short sequence that a class is made up of 1-6 Nucleotide joins end to end and repeats repeatedly to constitute, be distributed widely in the eukaryotic gene group.SSR label information content height covers whole genome, is codominant inheritance; Can utilize PCR to analyze; Compare with other dna molecular marker, SSR mark good reproducibility, high specificity, and also ssr analysis is not high to DNA quantity and purity requirement; Working method is simple, promptly with the specific short nucleotide sequence design primer in SSR both sides in the genome, genomic dna is carried out pcr amplification, and amplified production carries out its polymorphism of electrophoresis detection.At present, the SSR labeling technique has been widely used in (Lane L in the researchs such as cultivar identification and detection, group structure and genetic diversity, germ plasm resource and breeding, genomic mapping, Bargelloni L, and Patranello T.Molecular Ecology:11:1-16,2002).
Summary of the invention
Purpose of the present invention just is the SSR labeled analysis method of utilizing pine wood nematode special, thereby the little satellite amplimer of specificity of pine wood nematode is provided.
Another object of the present invention is to provide the molecule method for quick of a kind of pine wood nematode.
Another object of the present invention is to set up the molecule quick detection kit of a kind of pine wood nematode.
For achieving the above object, the present invention is according to following principle: the efficient of (1) DNA cloning; (2) be easy to distinguish with dimer; (3) three Tm temperature that different amplification lengths is close with (4) have designed the little satellite amplimers of specificity of following three pairs of pine wood nematodes, and expanding fragment length is between 110-190bp.
(1) forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
(2) forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
(3) forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 ';
Pine wood nematode molecule method for quick of the present invention may further comprise the steps:
1) utilize the little satellite amplimer of following three pairs of pine wood nematode specificitys that testing sample is increased:
(1) forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
(2) forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
(3) forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 ';
2) amplified production carries out gel electrophoresis, judges whether to be pine wood nematode according to having or not of amplified band between the 110-190bp.
The present invention has also prepared the quick detection kit of pine wood nematode molecule according to the pine wood nematode specificity amplification primer of above-mentioned design, wherein contain three pairs of little satellite amplimers of specificity.
Pine wood nematode quick detection kit of the present invention can be finished the evaluation quick and precisely of pine wood nematode within 2.5 hours, tolerance range is more than 99%.Simultaneously, the present invention also will play a positive role to the rapid detection of other alien species.
The molecule Fast Detection Technique of pine wood nematode of the present invention has the following advantages:
The first, can utilize micro-satellite primers that pine wood nematode is increased and overcome additive method such as the defective of RAPD and AFLP equistability difference; Still the report that does not have microsatellite marker to be used to detect before this;
The second, the primer that filters out is the Auele Specific Primer of pine wood nematode, does not have amplified band to intending pine wood nematode, and two kinds of nematodes are identified simultaneously only needs to judge having or not of band;
The 3rd, need not carry out the DNA extracting to template, saved the required time of detecting greatly, therefore can within 2.5 hours, finish detection, and all methods before this need the time more than 10 hours at least;
The four, the three pair of primer increases simultaneously to a nematode can guarantee the accuracy that detects.Overcome like this in the traditional method many nematodes have been carried out the DNA extracting, may sneak into the deficiency of other nematodes.
Description of drawings
Fig. 1 is three kinds of micro-satellite primers D, e and the f pcr amplification collection of illustrative plates to the single head pine wood nematode; Wherein M is Marker, and swimming lane 1,4 is the amplified production of primer D to the single head pine wood nematode, and 2,5 is the amplified production of primer e to the single head pine wood nematode, and 3,6 is the amplified production of primer f to the single head pine wood nematode.Each primer shows reproducible results to the amplification of two nematode.
Fig. 2 is that the e primer is to pine wood nematode and the specific PCR amplification collection of illustrative plates of intending pine wood nematode; Wherein M is Marker; First to the 4th swimming lane is a pine wood nematode; The the 5th to the 8th swimming lane is for intending pine wood nematode; The the 9th to the 12 swimming lane is a pine wood nematode; The 13 to the 16 swimming lane is a pine wood nematode; The 17 swimming lane is that the PCR product of the gene pool DNA of pine wood nematode T4 (Japanese strain system) is used for contrast.
Embodiment
Adopt the little satellite amplimer of following three pairs of pine wood nematode specificitys, on PCR instrument (FlexigeneThermal Cycler), increase:
Primer is right:
PW-f forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
PW-e forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
PW-D forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 '
Prepare PCR reaction system (10 μ l) according to following ratio:
10 * damping fluid, 1.0 μ l
Primer is to 0.4 μ l
10mM?dNTPs 0.3μl
dd?H
2O 2.1μl
Taq enzyme (5U/ μ l) 0.2 μ l
1 nematode DNA diluent 6.0 μ l
Wherein:
10 * PCR reaction buffer composed as follows:
200mM?Tris-HCl；
100mM?(NH
4)
2SO
4；
100mM?KCl；
1％Triton?X-100；
20mM?MgCl
2；
pH?9.0；
The PCR reaction is undertaken by following condition:
94 ℃ of pre-sex change 5 minutes enter circulation then; 94 ℃ of sex change 30 seconds, 51 ℃ of annealing 30 seconds, 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 5 minutes again.
The pcr amplification product that obtains is electrophoresis detection on the 1.5%Agarose gel.With EB dyeing, observe under UV-light and photograph after 30 minutes, its structure as shown in Figure 1.
As can be seen from Figure 1 three kinds of primers all can detect pine wood nematode.The D primer only amplifies a band, then demonstrates two bands on the electrophoresis picture of e primer and f primer.The amplification of D primer is an ideal results, is easy for determining of result.Though e primer and f primer have two bands, a following band is shorter than 110bp, belongs to dimer, can not influence result's judgement.The length of these bands is all within the interval of design primer.See between the individuality of pine wood nematode having some differences on the amplified band degree of depth in Fig. 1, this mainly is because the pine wood nematode individuality is minimum, has some deviations in the template amount of carrying out obtaining when template is handled and causes.But these differences can not have influence on the judgement to amplification yet.In addition, because three pairs of primers all can detect pine wood nematode, so optional wherein a pair of or two pairs also can reach same effect.
The comparison and detection of embodiment 2, pine wood nematode and plan pine wood nematode
Adopt pcr amplification system and the reaction conditions of embodiment 2 respectively pine wood nematode and plan pine wood nematode to be increased, amplified production is electrophoresis detection on the 1.5%Agarose gel.With EB dyeing, observe under UV-light and photograph after 30 minutes, its result as shown in Figure 2.
As shown in Figure 2, amplified band occurred between 110bp and 147bp, this is consistent with the length that design is considered during primer.The nethermost band of amplified production is a dimer, and their length is less than 60bp, and this is for the not influence of judgement of amplified production.As shown in the figure, the e primer has obtained the band of expection in the amplification of single head pine wood nematode, and intending pine wood nematode (5-8 swimming lane) does not then have amplified band.In other the repeatedly repeat amplification protcol (9-16 swimming lane),, can not influence judgement to amplification though the band depth exists difference.The difference of the band depth is likely because the difference of the interior DNA extracting amount of nematode body causes.
The quick detection kit of preparation pine wood nematode molecule wherein contains following pine wood nematode specificity amplification primer, can be a pair of arbitrarily, two pairs or whole three pairs:
Primer is right:
PW-f forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
PW-e forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
PW-D forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 '
Utilize the little satellite amplimer of pine wood nematode specificity in the test kit that testing sample is increased, amplified production after gel electrophoresis, according to having or not of amplified band between the 110-190bp can judgement sample whether be pine wood nematode.
Claims (8)
1, the little satellite amplimer of the specificity of a kind of pine wood nematode is characterized in that, can be selected from following any a pair of primer sequence:
(1) forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
(2) forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
(3) forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 '.
2, the molecule method for quick of a kind of pine wood nematode is characterized in that may further comprise the steps:
A, utilize the little satellite amplimer of following three pairs of pine wood nematode specificitys that testing sample is carried out pcr amplification:
(1) forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
(2) forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
(3) forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 ';
B, amplified production carry out gel electrophoresis, judge whether to be pine wood nematode according to having or not of amplified band between the 110-190bp.
3, detection method as claimed in claim 2 is characterized in that, the system of the pcr amplification of described steps A is:
10 * damping fluid, 1.0 μ l
Primer is to 0.4 μ l
10mM?dNTPs 0.3μl
dd?H
2O 2.1μl
Taq enzyme (5U/ μ l) 0.2 μ l
1 nematode DNA diluent 6.0 μ l.
4, detection method as claimed in claim 2 is characterized in that, the condition of the pcr amplification of described steps A is:
94 ℃ of pre-sex change 5 minutes enter circulation then; 94 ℃ of sex change 30 seconds, 51 ℃ of annealing 30 seconds, 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 5 minutes again.
5, detection method as claimed in claim 2 is characterized in that, the 1.5%Agarose gel electrophoresis is adopted in the gel electrophoresis of described step B.
As each described detection method of claim 2-5, it is characterized in that 6, the primer of pcr amplification also can be optional wherein a pair of or two pairs.
7, the molecule quick detection kit of a kind of pine wood nematode is characterized in that containing the little satellite amplimer of following three pairs of pine wood nematode specificitys:
(1) forward primer 5 '-TTT CTT CTG GCA TGG CAA AG-3 '
Reverse primer 5 '-AAG ATA TTG AAC GAT CTT TGT GGC-3 '
(2) forward primer 5 '-CTC AAG ACA TTC CGA ATA CC-3 '
Reverse primer 5 '-TCT GGT ACC ACG CAC GTT TTC-3 '
(3) forward primer 5 '-AAC GGA AAA GAG TCC TCA CG-3 '
Reverse primer 5 '-TAG GCC CTC CTT GAC AAA AGC-3 '.
8, detection kit as claimed in claim 7 is characterized in that also can only containing any a pair of in three pairs of primers or two pairs.
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| CN 200310109374 CN1244703C (en) | 2003-12-12 | 2003-12-12 | Pine wireworm microsatellite amplifying primer, detection method and detection kit |
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| CN 200310109374 CN1244703C (en) | 2003-12-12 | 2003-12-12 | Pine wireworm microsatellite amplifying primer, detection method and detection kit |
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| CN1244703C true CN1244703C (en) | 2006-03-08 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100485045C (en) * | 2004-12-14 | 2009-05-06 | 中国科学院微生物研究所 | Method and kit for detecting pine wood nematode, and special-purpose primer and probe for same |
| CN100392100C (en) * | 2005-06-13 | 2008-06-04 | 上海市林业病虫防治检疫站 | Detection kit for pine wood nematode and detection method therefor |
| CN100463947C (en) * | 2005-09-22 | 2009-02-25 | 长江水利委员会长江科学院 | Nano composite aqueous epoxide resin coating material and preparation method |
| CN106755554A (en) * | 2017-03-21 | 2017-05-31 | 四川农业大学 | Pine wood nematode SSR label primer is used for the purposes of pine wood nematode detection and the PCR detection method of pine wood nematode |
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