CN101878756B - Method for storing monochamus alternatus hope sample and method for quickly detecting bursaphelenchus xylophilus therein - Google Patents
Method for storing monochamus alternatus hope sample and method for quickly detecting bursaphelenchus xylophilus therein Download PDFInfo
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Abstract
The invention discloses a method for storing a monochamus alternatus hope sample and a method for fast detecting bursaphelenchus xylophilus therein. The method comprises the following step of putting the caught monochamus alternatus hope sample into 75% ethyl alcohol or a refrigeratory with -70 DEG C to be stored, thereby furthest preserving the amount of the bursaphelenchus xylophilus carried by the monochamus alternatus hope. The invention creates a production technology of the bursaphelenchus xylophilus DNA template in the monochamus alternatus hope with the storing state, directly freezes and thaws lyses and centrifuges the middle and back thoracic tracheal tissues of the adult monochamus alternatus hop for PCR amplification, has the amplification rate of 100%, fastens the detecting speed, improves the detecting accuracy, and achieves the aim of convenient, fast and accurate detection.
Description
Technical field
The invention belongs to biological technical field, be specifically related to carry the preservation technology of the Monochamus alternatus Hope sample of pine wood nematode, and the method for quick of pine wood nematode in the Monochamus alternatus Hope sample body.
Background technology
Monochamus alternatus Hope (Monochamus alternatus) pine nematode is important quarantine venereal disease evil.From nineteen eighty-two since pine nematode is found in the Nanjing of China Zhongshan Tomb, pine nematode has spread to ground such as provinces and cities such as Jiangsu, Shandong, Anhui, Zhejiang, Fujian, Hubei, Hunan, Guangdong, Guangxi, Yunnan, Guizhou, Sichuan, Hong Kong, Macao and Taiwan and area, and the whole nation area takes place reaches 8.7 ten thousand hm
2Withered pine tree more than 3,500 ten thousand strains of accumulative total, epidemic-stricken area epidemic disease wood not only can not normally utilize, and influence the circulation of other agricultural byproducts and forestry products, cause 2,500,000,000 yuan of direct economic losses, indirect economic loss reaches 25,000,000,000 yuan (Chen Fengmao etc., 2005), ecotope, foreign export and socio-economic development are had a strong impact on, and are listed in internal, external quarantine object by China.
China has 8.5 hundred million mu of (5,710 ten thousand hectares) conifer trees resources approximately, and wherein as about 500,000,000 mu of the pine tree resource of first seeds (3,330 ten thousand hectares), total accumulation reaches 57 billion cubic meters.Pine nematode is as a kind of destructive disease, and the safety of Chinese numerous scenic spots and historical sites and Scenic Spot Area in serious threat.Simultaneously, pine nematode is just becoming technology barriers, has a strong impact on foreign trade.
Monochamus alternatus Hope is the main vector insect (Xu Fuyuan of China pine wood nematode, 1994)), Monochamus alternatus Hope mainly is distributed in most of provinces and regions of Hebei South, comprise provinces and regions such as Hebei, Henan, Shaanxi, Shandong, Jiangsu, Zhejiang, Jiangxi, Hunan, Guangdong, Guangxi, Fujian, Shaanxi, Taiwan, Sichuan, Guizhou, Yunnan, Tibet (Cheng Xin jumps etc., 2005).The host plant of Monochamus alternatus Hope comprises Pinus, abies (Abies Mill.), Picea (Picea Dietr), Hemlock (Tsuga), Pseudotsuga (Pseudotsuga Carr.), Larch (Larix Mill.) and Cedrus (CedrusTrew.), optimum is pine genus plant (Song Hongmin etc., 2006).
The pine wood nematode third-instar larvae was assembled to the pupa chamber and is casted off a skin into decentralized 4 instar larvaes (Warren etc., 1993 when Monochamus alternatus Hope (Monochamus alternatus) was sprouted wings; Maehara etc., 1996; Necibi etc., 1998).4 instar larvaes are a kind of diffused larvas of not getting food, thereby survive (Ishibashi etc., 1977 in uncomfortable environment; Kondo etc., 1978).From the pupa development adult between one-period, a large amount of 4 instar larvaes enter its respiratory system by the valve of Monochamus alternatus Hope promerous at Monochamus alternatus Hope.Longicorn sprout wings fly out after, 4 age the decentralized larva overflow along valve, creep to the longicorn abdomen end, leave longicorn (Enda, 1977) again.Pine wood nematode 4 instar larvaes are laid eggs by longicorn or are got the food wound and invade healthy pine tree, and casting off a skin becomes adult (Mamiya etc., 1972 again; Morimoto etc., 1972).Pine wood nematode breeds in pine tree, and susceptible pine tree then stops gummosis (Hashimoto etc., 1974 in several weeks; Mamiya 1983; Fukuda, 1997).Susceptible pine tree is withered death after 1～2 month.Concentrate on when the Monochamus alternatus Hope that carries 1000～10000 pine wood nematodes and get food on the pine tree of a health and just can propagate enough nematodes pine tree is fallen ill, when the quantity of carrying nematode can not cause pine tree fall ill then (Togashi, 1985) when following at 1000.
The monitoring of pine nematode epidemic situation is the primary link of preventing and controlling pine wilt disease work, only finds epidemic situation in time, and finds out on its basis that harm situation takes place.Just can make the scientific and reasonable scheme of preventing and treating and job design, effectively control epidemic situation (Jiang Liya etc., 2006).At present, mainly carry out from disease tree and two aspects of Monochamus alternatus Hope for the monitoring of pine nematode epidemic situation.With the diseased wood be the object epidemic monitoring mainly by the generaI investigation of artificial ground, task is heavy, landform standing forest situation complexity, the separating difficulty of in addition taking a sample is bigger, initial stage of origination often can not in time be found, in case find that epidemic situation has spread.There is the latent infection phenomenon in woodland simultaneously, is difficult to from the pine tree angle it be monitored (Yang Baojun etc., 2002; Zhang Zhiyu etc., 2003; Futai, 2003).For doubtful epidemic place, Monochamus alternatus Hope density is low, and withered pine tree is few, and pine tree up-sampling separation difficulty utilizes attractant to lure Monochamus alternatus Hope to detect pine wood nematode, can accomplish to find early, for control is raced against time.
Utilize the Monochamus alternatus Hope adult to monitor pine nematode, the most important thing is in time, the pine wood nematode that fast detecting is carried in the Monochamus alternatus Hope body.The nematode detection method that adopts is at present, after the Monochamus alternatus Hope adult of transporting to test point shredded, be immersed in the water 24 hours, after nematode separated, identify with the following the whole bag of tricks of setting up in succession: the morphology authentication method of pine wood nematode, pine wood nematode protein electrophorese method, pine wood nematode enzyme linked immunosorbent assay (ELISA) and the protein imprinted method of pine wood nematode (Westenblots), pine wood nematode DNA hybridization technique, pine wood nematode RAPD technology and pine beam thread insect PCR authentication method.
But there is following defective in existing detection method:
At first, aspect the sample preservation, prior art is used the generation of Monochamus alternatus Hope monitoring woodland pine wood nematode, often needs to detect a large amount of samples, the store method of the Monochamus alternatus Hope sample that uses at present, be that Monochamus alternatus Hope is supported in the insect case of fresh pine branch is housed, like this, Monochamus alternatus Hope is in the process that supplements the nutrients, pine wood nematode will leave the Monochamus alternatus Hope polypide, detected pine wood nematode quantity is significantly reduced, or detect less than, and influence the accuracy of monitoring result.
Secondly, mainly there is following defective in existing detection analytical technology:
1, the separating resulting temperature influence of pine wood nematode is bigger in the Monochamus alternatus Hope body, when the Monochamus alternatus Hope after especially surviving the winter is sprouted wings, because temperature is lower, under the situation of low temperature, may can not separate nematode, thereby influence testing result.
2, pine nematode mainly is according to its cause of disease--the morphological feature of the female adult of pine wood nematode, identify (Feng Zhixin etc., 2001).And this nematode form small (about long 1mm, about wide 30 μ m) need be identified by the professional at microscopically.And pine wood nematode exists with 4 instar larvae forms in the Monochamus alternatus Hope, and turning out the pine wood nematode adult needs the regular hour.These often cause doubtful epidemic place to can not get in time making a definite diagnosis, and have incured loss through delay best control period.
3, pine wood nematode morphology authentication method is simple and easy to do, is the conventional method (Ryss A., 2005) that existing line insect types is identified.The evaluation of pine wood nematode mainly relies on the morphological feature of adult.Because variation umbrella aphelenchoides (B.abruptus Giblin-Davis, Mundo-Ocampo, Baldwin, the Norden﹠amp of pine wood nematode group; Batra, 1993), awl tail chute aphelenchoides (B.conicaudatus), pseudo-umbrella aphelenchoides (B.fraudulentus R-ü hm, 1956 (J.B.Goodey, 1960)), Ke Limu umbrella aphelenchoides (B.kolymensisKorentchenko, 1980) and intend pine wood nematode (B.mucronatus (Braasch, 2001; Kanzaki﹠amp; Futai, 2003)) all have the form similar with pine wood nematode, and Wingfield et al (1983) becomes pointed at the female adult tail end of pine wood nematode that the glue fir is separated to, and Fukushige et al (1985) will observe pine wood nematode, and to have the tail point prominent this be not that very the stable morphology feature has been brought difficulty to evaluation.
4, the protein electrophorese analysis is a molecular engineering that early is applied to the nematode means of taxonomic research.After the eighties in 20th century, many scholars have carried out the research of isozyme difference to pine wood nematode and plan pine wood nematode strain system.The sample size that the isoenzyme technique requirement is analyzed is big (being no less than 3000), could obtain electrophoresis pattern clearly, and protein expression is relevant with the growth and development stage of environment and nematode.Up to the present, also not having a kind of isodynamic enzyme to be acknowledged as can distinguish pine wood nematode clearly and intend pine wood nematode.
5, many scholars utilize the DNA hybridization technique to identify pine wood nematode, Abad et al (1991), Tares et al (1992) respectively with the hybridization of allos probe and homologous probe to the pine wood nematode evaluation of classifying, Tares etc. (1994) have been separated to species specificity satellite DNA probe Mspl from Japanese pine wood nematode, can directly squeeze broken nematode to the wall scroll of point on filter screen with this probe identifies, can identify pine wood nematode fast and accurately, but it is bigger that the shortcoming of this technology is technical difficulty, the expense height, and have radiological hazard.
6, the RAPD technology be Williams in nineteen ninety set up with 10 oligonucleotides molecular marking technique that is random primer.It is quick, easy, sensitive and need not labelled with radioisotope.Braasch et al (1995), Zhang, et al (1998), Wang Laifa etc. (2001) and a road equality (2002) adopt the RAPD-PCR technology to pine wood nematode and intend studying with intraspecies variation and their genetic diversity between the pine wood nematode kind.Contain much information although the RAPD technology is contained, because its collection of illustrative plates more complicated, repeatability and comparativity are poor, make the RAPD technology identify that there is certain limitation in pine wood nematode.
7, the application of round pcr has broken through dna molecular and has identified suffered DNA quantitative limitation, has improved the sensitivity that detects greatly.Hoyer et al (1998) has set up the ITS-PCR-RFLP technology of distinguishing 5 kinds of nematodes.This technology of utilizing Iwahori et al (2000) has not only successfully detected pine wood nematode and has intended pine wood nematode, and has only set up quick, sensitive pine wood nematode diagnostic method with the wall scroll living nematode.The PCR-SSCP technology that Zhang Lihai (2001) sets up, (2005) such as (2005), Zhang Weidongs such as Cao etc. (2005), Wang Mingxu are utilized PCR in real time to detect respectively and have been identified pine wood nematode.From China's present circumstances, Auele Specific Primer PCR method (especially SCAR mark detection method) is short detection time with real time quantitative PCR method, and the accuracy of detection height can satisfy the requirement of quick test quarantine, and begun large-scale application (Tang Jian etc., 2008) in production.But existing P CR The Application of Technology technology after all needing nematode separated from wood or vector insect, is identified.
8, the pine wood nematode that directly detects Monochamus alternatus Hope is the problem that people are studying always.Wang Xinrong etc. (2009) utilize one couple of PCR primers, and the pine wood nematode that the Monochamus alternatus Hope of living is carried detects.The method that catches the Monochamus alternatus Hope adult is to hang trapper between pine forest, is transported to the laboratory behind the trapping Monochamus alternatus Hope adult and detects.But as previously mentioned, the Monochamus alternatus Hope sample is being transported to the way in laboratory from doubtful epidemic place, death can take place in Monochamus alternatus Hope, existing detection technique can only detect on the Monochamus alternatus Hope alive with pine wood nematode alive, dead sample does not meet the detection requirement; Perhaps pine wood nematode has left Monochamus alternatus Hope in the way of transporting, and influences the accuracy of testing result.
Summary of the invention
An object of the present invention is to overcome the deficiency of sample preservation technology in the existing pine wood nematode detection technique, the store method of a kind of Monochamus alternatus Hope (Monochamus alternatus) sample is provided, solve to be used for the transportation that Monochamus alternatus Hope sample that pine wood nematode detects runs into or to preserve in the Monochamus alternatus Hope of living pine wood nematode quantity and lose, testing result can not the accurate response Monochamus alternatus Hope be carried the pine wood nematode actual quantity at woodland problem.
Another object of the present invention is the deficiency that overcomes pine wood nematode detection technique in the existing Monochamus alternatus Hope body, effectively, sensitive, accurately, in the Monochamus alternatus Hope body, detect pine wood nematode easily, overcome in the traditional detection method that nematode separates, incubation time is long, pine wood nematode in the large sample Monochamus alternatus Hope body is difficult to technical problems such as efficient preservation, Protocols in Molecular Biology is applied in the Monochamus alternatus Hope pine wood nematode to be gone in detecting, accelerate the speed of detection, improve the accuracy that detects.
Purpose of the present invention is achieved by following technical proposals:
A kind of store method of Monochamus alternatus Hope sample is provided, and is that the Monochamus alternatus Hope sample that will capture is put in 75% alcohol or-70 ℃ of refrigerators and preserves.
The present invention provides the method for quick of pine wood nematode in a kind of Monochamus alternatus Hope sample body simultaneously, and the Monochamus alternatus Hope sample of preserving is according to the method described above carried out the detection of following steps:
(1) preparation PCR primer: by the synthetic a pair of specific pcr amplification primer of gene Synesis Company, this primer draws from Wang Xinrong (2009) (Xinrong Wang Xiaowei Zhu HuanhuaHuang et al.A PCR-based Method for Detect ing the Pine WoodNematodes-Bursaphelenchus xylophilus from Monochamus alternatus. forest-science 2009 (45) 7:70-74):
P155：5’-CTACGTGCTGTTGTTGAGTTGGC-3’；
P538：5’-TGGTGCCTAACATTGCGCGA-3’ 。
The pcr amplification primer can place-20 ℃ of refrigerators to preserve.
(2) catching Monochamus alternatus Hope adopts said method to preserve;
(3) preparation of pine wood nematode DNA masterplate in the 2mg Monochamus alternatus Hope: with metathorax tracheal tissue in the 2mg Monochamus alternatus Hope, put into the centrifuge tube of the 1.5ml of sterilization, do in the liquid nitrogen slightly and grind, the back adds 50 μ l～88 μ l WLB liquid (2.5mmolL
-1Dithiothreitol (DTT) (DTT), 1.125% (mass percent) Tween-20 (Tween 20), 0.025% (mass percent) gelatin (Gelatin), 125mmolL
-1KCl, 25mmolL
-1Tris-HCl (pH 8.0), 3.75mmolL
-1MgCl
2), finish temperature cycles then in water-bath: 95 ℃ of 15min, 65 ℃ of 2min repeat once, add 8 μ l～10 μ l Proteinase K (20mgmL
-1) back is in 65 ℃ of 1h, 95 ℃ of 15min cracking, the DNA extract after the cracking through 15,294g, after 3min is centrifugal, is drawn 2 μ l and is done the pcr amplification template.
(4) preparation pcr amplification technical system
The pcr amplification reaction system is as follows: 10 * PCR buffer, 2.5 μ l (do not contain Mg
2+), dNTP (2.5mmolL
-1) 2 μ l, Mg
2+(25mmolL
-1) 2.5 μ l～3.5 μ l, each 1 μ l of primer P1 (P155) 10 μ M and P2 (P538) 10 μ M, Taq archaeal dna polymerase 5UuL
-10.125 μ l, template DNA 2 μ l, the sterilization distilled water is supplemented to cumulative volume 25 μ l.
(5) carry out pcr amplification
The PCR reaction condition is as follows: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 48 ℃～58 ℃ annealing 30s, 72 ℃ are extended 1min, and after 40 circulations, last 72 ℃ are extended 10min.In 4 ℃ of refrigerators, preserve.
(6) conventional agarose gel electrophoresis detects
Preparation 1.0～1.2% Ago-Gels when treating that gel is chilled to about 60 ℃, add GoldView, and making the GoldView final concentration is 0.5 μ g/ml.Get sample on 5 μ l pcr amplification products and an amount of DNA load sample buffer solution (containing 0.2% bromjophenol blue, the 0.5g/ml sucrose) mixing, with the Bio-Rad PowerPac300 of company type voltage-stabilized power supply, in 0.5 * tbe buffer liquid with the about 30min of 5v/cm voltage electrophoresis.Observe under the uviol lamp, take a picture with the gel images analytical system.
The present invention is directed to the deficiency of existing pine wood nematode detection technique, comprise the transportation that runs in the preservation of Monochamus alternatus Hope sample or preserve back Monochamus alternatus Hope death and can not use prior art and detect, perhaps the interior pine wood nematode quantity of the Monochamus alternatus Hope body of Huoing is lost and is caused testing result not carry problems such as pine wood nematode actual quantity at woodland by the accurate response Monochamus alternatus Hope, overcome nematode separation in the traditional detection method, incubation time is long, pine wood nematode in the large sample Monochamus alternatus Hope body is difficult to technical barriers such as efficient preservation, simple and easy to do sample store method is provided, in conjunction with pine wood nematode DNA masterplate technology of preparing in the effective and easy Monochamus alternatus Hope body, sensitive pine wood nematode specific PCR amplimer, High Efficiency PC R amplification system and the pcr amplification reaction condition that is fit to, Protocols in Molecular Biology is successfully applied in the Monochamus alternatus Hope during pine wood nematode detects, accelerate the speed of detection, improve the accuracy that detects, determine the kainogenesis epidemic place as early as possible, for the prevention and control of pine nematode contribute.The beneficial effect that the present invention gives prominence to is summarized as follows:
(1) provides a kind of new Monochamus alternatus Hope sample preservation technology.The present invention is put into the Monochamus alternatus Hope sample that captures in 75% alcohol or-70 ℃ of refrigerators and preserves.75% alcohol or-70 ℃ of pine wood nematodes that refrigerator can kill Monochamus alternatus Hope simultaneously and carry have at any time preserved the quantity of the pine wood nematode that Monochamus alternatus Hope carries to greatest extent, make PCR testing result subsequently more realistic.And the preservation technology of prior art is with the Monochamus alternatus Hope adult, raises in dependent insect cage, and the pine wood nematode that the Monochamus alternatus Hope adult is carried might leave Monochamus alternatus Hope, influences the accuracy of testing result.
(2) the present invention has created pine wood nematode dna profiling technology of preparing in the Monochamus alternatus Hope of above-mentioned preservation state.The preparation of pine wood nematode dna profiling in the Monochamus alternatus Hope body of preserving, employing be directly the middle metathorax tracheal tissue of Monochamus alternatus Hope adult is carried out freeze thawing, cracking, centrifugal after, be used for pcr amplification, amplification efficiency reaches 100%.This technology need not be separated pine wood nematode earlier from the longicorn body, improved pine wood nematode detection efficiency in the Monochamus alternatus Hope body.
(3) the present invention has created and has utilized round pcr, from the dead Monochamus alternatus Hope of preserving, directly amplify the technology of the specific fragment of pine wood nematode, the Monochamus alternatus Hope sample is kept in the suitable preservation liquid, carry out pine beam thread insect PCR amplification in the Monochamus alternatus Hope then, can preserve the state of pine wood nematode in the Monochamus alternatus Hope sample at any time, utilize Protocols in Molecular Biology, accelerate the speed of detection, improve the accuracy that detects, the preparation of pine wood nematode dna profiling in the Monochamus alternatus Hope body, only need 5 hours just to obtain the result, reached the purpose of fast detecting.Detection method in the past can't utilize round pcr to detect dead Monochamus alternatus Hope dead wire worm on one's body, also lacks suitable Monochamus alternatus Hope store method, guarantees that the pine wood nematode testing result conforms to actual in the Monochamus alternatus Hope body.
Description of drawings
Fig. 1 pine beam thread insect PCR specific amplified result
Fig. 2 50 μ lWLB+8 μ l Proteinase K (20mgml
-1) lysate makes up the pcr amplification result after 2mg Monochamus alternatus Hope tissue (the adding the wall scroll pine wood nematode) cracking
Fig. 3 88 μ lWLB+10 μ l Proteinase K (20mgml
-1) lysate makes up the pcr amplification result after 2mg Monochamus alternatus Hope tissue (the adding the wall scroll pine wood nematode) cracking
The pcr amplification result of wall scroll pine wood nematode worm alive under Fig. 4 different annealing temperature
The pcr amplification result of Fig. 5 wall scroll pine wood nematode worm alive
The Monochamus alternatus Hope that carries pine wood nematode of Fig. 6-70 ℃ preservation is organized the pcr amplification result
The pcr amplification result of Monochamus alternatus Hope tracheal tissue who carries pine wood nematode that Fig. 7 75% alcohol is preserved
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.
From amusement park, South Lake, Guangzhou, adopt the Monochamus alternatus Hope sample, preserve and identify after 5 days.Obtained expected results, finished the detection task, testing result is seen accompanying drawing 6 and accompanying drawing 7.Concrete steps are as follows:
(1) preparation PCR primer:
Carry the last week than the sampling time, bio-engineering corporation synthesizes a pair of specific pcr amplification primer by match Parkson, Beijing:
P155：5’-CTACGTGCTGTTGTTGAGTTGGC-3’；
P538：5’-TGGTGCCTAACATTGCGCGA-3’。
The pcr amplification primer places-20 ℃ of refrigerators to preserve.
The pine beam thread insect PCR specific amplified the results are shown in shown in the accompanying drawing 1, wherein, and M representation DNA MarkDL2000; 1 represents the pine wood nematode genomic DNA; The pine wood nematode genomic DNA is intended in 2 representatives; 3 represent pine wood nematode and Monochamus alternatus Hope genomic DNA mixture; 4 represent the Monochamus alternatus Hope genomic DNA.
(2) from amusement park, South Lake, Guangzhou, adopt the Monochamus alternatus Hope sample, with the Monochamus alternatus Hope sample that captures, be put in 75% alcohol or-70 ℃ of refrigerators and preserve, carry out detection of the present invention in preferred 5 days.
(3) preparation of pine wood nematode dna profiling in the 2mg Monochamus alternatus Hope:
With the 2mg tracheal tissue of metathorax in the Monochamus alternatus Hope of preserving, put into the centrifuge tube of the 1.5ml of sterilization, do in the liquid nitrogen and grind back adding 50 μ l～88 μ l WLB liquid (2.5mmolL slightly
-1DTT, 1.125% Tween 20,0.025%Gelatin, 125mmolL
-1KCl, 25mmolL
-1Tris-HCl (pH 8.0), 3.75mmolL
-1MgCl
2), in water-bath, finish temperature cycles then: 95 ℃ of 15min, 65 ℃ of 2min repeat once; Add 8 μ l～10 μ l Proteinase K (20mgmL
-1) after carry out the cracking of 65 ℃ of 1h, 95 ℃ of 15min; DNA extract after the cracking after 15294g, 3min are centrifugal, are drawn 2 μ l and is done the pcr amplification template.
88 μ lWLB+10 μ l Proteinase K (20mgml
-1) lysate combination the results are shown in shown in the accompanying drawing 3 pcr amplification after 2mg Monochamus alternatus Hope tissue (the adding the wall scroll pine wood nematode) cracking, wherein, M:DNA Marker DL2000; Swimming lane 1～3: wall scroll pine wood nematode; Swimming lane 4～6:2mg Monochamus alternatus Hope tissue (adding the wall scroll pine wood nematode); Swimming lane 7: sterilization deionized water contrast; Swimming lane 8: pine wood nematode genomic DNA.
The preferred above-mentioned 50 μ lWLB+8 μ l systems of the present invention.50 μ lWLB+8 μ l Proteinase K (20mgml
-1) lysate combination the results are shown in shown in the accompanying drawing 2 pcr amplification after 2mg Monochamus alternatus Hope tissue (the adding the wall scroll pine wood nematode) cracking, M:DNA Marker DL2000 wherein; Swimming lane 1～3: wall scroll pine wood nematode; Swimming lane 4～6:2mg Monochamus alternatus Hope tissue (adding the wall scroll pine wood nematode); Swimming lane 7: sterilization deionized water contrast; Swimming lane 8: pine wood nematode genomic DNA.
(4) preparation pcr amplification technical system
The pcr amplification reaction system is as follows: 10 * PCR buffer, 2.5 μ l (Mg
2+Free), dNTP (2.5mmolL
-1) 2 μ l, Mg
2+(25mmolL
-1) 3 μ l, each 1 μ l of primer P1 (10 μ M) and P2 (10 μ M), Taq archaeal dna polymerase 5UuL
-10.125 μ l, template DNA 2 μ l, the sterilization distilled water is supplemented to cumulative volume 25 μ l.
(5) pcr amplification
The PCR reaction condition is as follows: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, and after 40 circulations, last 72 ℃ are extended 10min.In 4 ℃ of refrigerators, preserve.
The pcr amplification of wall scroll pine wood nematode worm alive the results are shown in shown in the accompanying drawing 4 swimming lane M:DL 2000 DNA marker among the figure under the different annealing temperature; Swimming lane 1～7: annealing temperature is respectively 48.0 ℃, 49.0 ℃, 50.0 ℃, 52.4 ℃, 54.8 ℃, 56.0 ℃, 58.0 ℃ pcr amplification product; Swimming lane 8: deionized water contrast.In the temperature of these 7 tests, can both amplify purpose band clearly, see the pcr amplification result of the worm alive of wall scroll pine wood nematode shown in the accompanying drawing 5, wherein swimming lane M:DL2000DNA marker; Swimming lane 1～6, the pcr amplification product of wall scroll pine wood nematode worm alive; Swimming lane 7: deionized water contrast.
(6), agarose gel electrophoresis testing result.
Preparation 1.0～1.2% Ago-Gels (get in 1.0～1.2% the value range arbitrary point value all can), when treating that gel is chilled to about 60 ℃, add GoldView, making the GoldView final concentration is 0.5 μ g/ml.The pcr amplification product of getting 5 μ l above-mentioned steps (5) (contains 0.2% bromjophenol blue with an amount of DNA load sample buffer solution, 0.5g/ml sample on mixing sucrose), with the PowerPac300 of Bio-Rad company type voltage-stabilized power supply, in 0.5 * tbe buffer liquid with the about 30min of 5v/cm voltage electrophoresis.Observe under the uviol lamp, take a picture with the gel images analytical system.Adopt above general electrophoresis method, detect.Can see that the PCR detection technique system of utilizing the inventive method to set up can detect the interior pine wood nematode of preservation Monochamus alternatus Hope sample body and see accompanying drawing 6 and accompanying drawing 7.Illustrate when adopting the inventive method to preserve the PCR specific amplification of pine wood nematode in the Monochamus alternatus Hope sample body, can improve efficient and the precision of detection.
Accompanying drawing 6 is organized the pcr amplification testing result for the Monochamus alternatus Hope that carries pine wood nematode of-70 ℃ of preservations, swimming lane M:DL2000 DNA marker; Swimming lane 1～4: the Monochamus alternatus Hope pcr amplification product that carries pine wood nematode; Swimming lane 5: the Monochamus alternatus Hope pcr amplification product that does not carry pine wood nematode; Swimming lane 6: pine wood nematode genomic DNA pcr amplification product.
Accompanying drawing 7 is the Monochamus alternatus Hope tracheal tissue pcr amplification testing result of carrying pine wood nematode that 75% alcohol is preserved, swimming lane M:DL2000 DNA marker; Swimming lane 1～4: the Monochamus alternatus Hope pcr amplification product that carries pine wood nematode; Swimming lane 5: the Monochamus alternatus Hope pcr amplification product that does not carry pine wood nematode.
SEQUENCE LISTING
＜110〉Agricultural University Of South China
＜120〉method for quick of pine wood nematode in the store method of Monochamus alternatus Hope sample and the body thereof
<130>
<160>2
<170>PatentIn version 3.5
<210>1
<211>23
<212>DNA
＜213〉artificial primer sequence P155
<400>1
ctacgtgctg ttgttgagtt ggc 23
<210>2
<211>20
<212>DNA
＜213〉artificial primer sequence P538
<400>2
tggtgcctaa cattgcgcga 20
Claims (3)
1. the method for quick of pine wood nematode in the Monochamus alternatus Hope sample body is characterized in that may further comprise the steps:
(1) synthetic pcr amplification primer;
(2) catch the Monochamus alternatus Hope adult, the Monochamus alternatus Hope adult sample that captures is put in 75% alcohol or-70 ℃ of refrigerators preserves;
(3) with pine wood nematode DNA masterplate in the freeze thawing of middle metathorax tracheal tissue, cracking and the centrifugal back preparation Monochamus alternatus Hope of Monochamus alternatus Hope adult sample;
(4) preparation pcr amplification system is carried out pcr amplification;
(5) conventional agarose gel electrophoresis detects;
Wherein, the described primer of step (1) is:
P155:5’-CTACGTGCTGTTGTTGAGTTGGC-3’；
P538:5’-TGGTGCCTAACATTGCGCGA-3’；
Step (3) is with metathorax tracheal tissue in the 2mg Monochamus alternatus Hope, put into the centrifuge tube of the 1.5ml of sterilization, finish temperature cycles after adding 50 μ l～88 μ l WLB liquid, obtain the DNA extract after adding 8 μ l～10 μ l Proteinase K cracking, the DNA extract obtains the pcr amplification template after 15294g, 3min are centrifugal; Described temperature cycles is 95 ℃ of water-bath 15min and 65 ℃ of water-bath 2min, repeats once; Described cracking is cracking 15min under cracking 1h under 65 ℃ of temperature, 95 ℃ of temperature;
The described pcr amplification reaction system of step (4) is: 10 * PCR buffer2.5 μ l, 2.5mmolL
-1DNTP2 μ l, 25mmolL
-1Mg
2+2.5 μ l～3.5 μ l, each 1 μ l of 10 μ M primer P155 and 10 μ M P538, Taq archaeal dna polymerase 5UuL
-10.125 μ l, template DNA 2 μ l, the sterilization distilled water is supplemented to cumulative volume 25 μ l;
The described PCR reaction condition of step (4) is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 48 ℃～58 ℃ annealing 30s, 72 ℃ are extended 1min, and after 40 circulations, 72 ℃ are extended 10min.
2. method for quick according to claim 1 is characterized in that described WLB liquid consumption is 50 μ l, and the Proteinase K consumption is 8 μ l.
3. method for quick according to claim 1, it is characterized in that step (5) is preparation 1.0～1.2% Ago-Gels, when treating that gel is chilled to about 60 ℃, add GoldView, making the GoldView final concentration is 0.5 μ g/ml, get sample on 5 μ l pcr amplification products and an amount of DNA load sample buffer solution mixing, electrophoresis observation.
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| CN112305101A (en) * | 2020-10-19 | 2021-02-02 | 江西省林业科学院 | Method for determining nitrogen fixation activity of monochamus alternatus living body |
| CN114480376A (en) * | 2022-01-18 | 2022-05-13 | 北京林业大学 | Rapid molecular detection method for pine wood nematode invading forest brown-stem longicorn |
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