CN103740857B

CN103740857B - Rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and application of rapid PCR molecular detection method

Info

Publication number: CN103740857B
Application number: CN201410050457.3A
Authority: CN
Inventors: 彭德良; 彭焕; 贺文婷
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2014-02-13
Filing date: 2014-02-13
Publication date: 2015-04-08
Anticipated expiration: 2034-02-13
Also published as: CN103740857A

Abstract

The invention discloses a rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and the application of the rapid PCR molecular detection method, and belongs to the technical field of molecular detection of plant nematodes. A PCR system is involved in the rapid PCR molecular detection method for the ditylenchus destructor thorne. The method is characterized in that the PCR system comprises a group of specific primers, wherein the specific primers are DdF1 and DdR1, and can specifically amplify fragments with lengths of 495bp from A-type and B-type ditylenchus destructor thorne, thereby realizing the rapid molecular detection of the ditylenchus destructor thorne. The method is high in sensitivity and specificity, rapid and accurate, and has high actual application value in the aspects of early diagnosis of ditylenchus destructor thorne, field monitoring and early warning and the like.

Description

Ditylenchus destructor fast PCR molecular detecting method and application thereof

Technical field

The invention belongs to Plant nematode field of molecular detection, relate to Ditylenchus destructor fast PCR molecular detecting method and application thereof.

Background technology

Ditylenchus destructor (Ditylenchus destructor Thorne) be cause potato and sweet potato one of the main diseases original, this nematode is a kind of transport property endoparasitism nematode, the underground part of main harm crop.China is as far back as nineteen thirty-seven just report generation sweep stem nematode, once (the Liu Xianbao caused by D.dipsaci was once being thought, Ge Jianjun, Tan Zhiqiong, Cao Aixin. Ditylenchus destructor endangers the reported first of potato at home. plant protection, 2006, 32 (6): 157-158), until 1979, Zhang Xiangrong think cause of disease be Ditylenchus destructor (Zhang Xiangrong. pachyrhizus haulm characters. Jinan: Shandong Science Press, 1979), later fourth is good fortune and Lin Maosong again, Yin Guangde and Zhang Yun U.S. have carried out the qualification of system to cause of disease, confirm Shandong, Ditylenchus dipsaci cause of disease on the sweet potato of Jiangsu is Ditylenchus destructor (Ding Zaifu, Lin Maosong. the qualification of the Ditylenchus dipsaci on sweet potato potato and peppermint. plant protection, 1982, 9 (3): 169172, Yin Guangde, Zhang Yunmei. correcting of sweep stem nematode pathogenic nematode. Shandong agricultural university journal (natural science edition), 1983,4,117-127).This nematode of the third-class report of Chen Pin cause Radix Angelicae Sinensis fiber crops stomatosis (Chen Pinsan, Zheng Jingwu. Radix Angelicae Sinensis fiber crops stomatosis causes a disease the identification research of Ditylenchus dipsaci. plant protection, 1988,14 (6): 12-14).Ditylenchus destructor host range widely, comprise potato, sweet potato, pea, more than 100 kind of host (the Peng H such as peanut, Gao B-l, Kong L-a, Yu Q, Huang W-k, et al. (2013) Exploring the Host Parasitism of the Migratory Plant-ParasiticNematode Ditylenchus destuctor by Expressed Sequence Tags Analysis.PLoS ONE8 (7): e69579.), bring huge financial loss every year, and harm is being risen year by year, (People's Republic of China (PRC) enters the territory Plant Quarantine harmful organism register to be divided into quarantine harmful organisms in China and other countries of the world and area, 2007, EPPO, 2008Diagnostics & Diagnostic:Ditylenchus destructor and Ditylenchus dipsaci).In addition, Ditylenchus destructor also infects block root and the stem tuber of flowers; D.destructor is at South Africa infecting peanut, cause huge loss (Lin Maosong, Wen Ling, Fang Zhongda. potato rot nematode and sweep stem nematode. Jiangsu's agriculture journal, 1999,15 (3): 186-190), in addition, there was reported Ditylenchus destructor both at home and abroad and can endanger potato and peanut (Liu Xianbao, Ge Jianjun, Tan Zhiqiong, Cao Aixin. Ditylenchus destructor endangers the reported first of potato at home. plant protection, 2006,32 (6): 157-158; Cheng Yun, Zhang Shaosheng. pathogenic to peanut of rot stem nematodes, University Of Agriculture and Forestry In Fujian's journal (natural science edition), 2007,36(5) 454-457).

The sweep stem nematode caused by Ditylenchus destructor is one of the important disease in China sweet potato producing region, the general morbidity field underproduction 20% ~ 50%, grave illness field has no harvest substantially without receiving (Chen Pinsan. sweet potato chaff rotten haulm characters .1995,512-517).Generation is all had in provinces and cities such as Beijing of China, Tianjin, Shandong, Shanxi, Hebei, Henan, Jiangsu, Zhejiang, Fujian, Liaoning, Gansu, occur the most serious with Shandong, Hebei two province, the sweet potato producing region onset area of some areas reaches 25%, sickness rate is about 30%, severe patient sickness rate can up to 80%, the morbidity general underproduction 20-30% in plot, severe patient reaches more than 50%, even has no harvest; This disease not only causes harm in field and directly causes the underproduction, but also cause storage later stage rotten cellar for storing things (Chen Fawei, Han Xianghong, Li Xiangsheng. the integrated control technique of sweep stem nematode. plant protection technology and popularization, 1996:16 (6): 12).Shandong investigation in 1977, this disease of the whole province is throughout 9 areas (city), 57 counties.The regional field potato seed the had underproduction of rotting reaches more than 80%, that stores rotten cellar for storing things is lost in more than 50% (Li Jianzhong, Peng Deliang, Liu Shuyan, Huang Wenkun. Ditylenchus destructor is in the suitable natural disposition venture analysis of China. Liao Jinling, Peng Deliang, Zheng Jingwu etc. Chinese nematology research (volume Two). Beijing: Scientia Agricultura Sinica technology press, 20082:137-142).Therefore, Ditylenchus destructor has become crushing nematodiasiss on China North China and East China sweet potato, seriously constrains the development of China's Sweet Potato Industry (in model essay, Sun Shuyun, Sun Yanmei. vigilant sweep stem nematode is popular. Jilin agricultural sciences, 1999,24 (4): 32; Leaf pine branch, Li Yuejie, bang unit. the occurrence and control technology of sweep stem nematode. plant protection technology and popularization, 1997,17 (3), 20-21).After this nematode invades sweet potato, esophageal gland secretes the cell wall degradation enzymes such as some polygalacturonases, amylase and proteolytic enzyme, injected in sweet potato body by lancet, make sweet potato cell destruction, organize and rot, along with other fungus and bacterium aggravation harm, normal formation " the chaff heart " or be full of cracks, make sweet potato lose edibleness (Lin Maosong, Wen Ling, Fang Zhongda. potato rot nematode and sweep stem nematode. Jiangsu's agriculture journal, 1999,15 (3): 186-190.).

The quick diagnosis that the fast development of the Protocols in Molecular Biology based on PCR is Plant nematode and detection provide strong instrument.Auele Specific Primer PCR molecular detection technology achieves very large development in the Molecular Detection of Plant nematode, can be detected the population of the one or more Plant nematodes in sample, shorten detection time greatly, improve detection efficiency by pcr amplification.In the rapid molecular of Plant nematode detects, the rDNA (rDNA) based on Plant nematode is target and the method for detecting specificity that builds achieves huge progress.Bulman & Marshall(Bulman SR, Marshall JW.Differentiation of Australasian potato cyst nematode (PCN) populations using the polymerase chain reaction (PCR) New Zealand Journal of Crop and Horticultural Science.1997; 25:123 – 129) on the sequential structure basis of rDNA-ITS analyzing globodera rostochiensis and G.pallida, construct the Auele Specific Primer PITSr3 of globodera rostochiensis and the Auele Specific Primer PITSp4 of G.pallida, universal primer ITS5 and PITSr3 can increase the specific fragment of 343bp globodera rostochiensis, can amplify the DNA fragmentation of 265bp specific recognition G.pallida with PITSp4.In a PCR reaction, use these 3 primers of PITSr3, PITSp4 and ITS5, even if the content of hairworm DNA is only one of percentage of white line worm in compound population, also can detect globodera rostochiensis from compound population.(the Subbotin such as Subbotin, S.A., Peng, D.and Moens, M.2001A rapid method for the identification of the soybean cyst nematode Heterodera glycines using duplex PCR.Nematology3,365 – 370) construct based on the soy bean cyst roundworm specific primer GlyF1 on rDNA-ITS region, apply two groups of primer D3A and D3B and soy bean cyst roundworm species-specific primer GlyF1 and primer rDNA2 and successfully molecular diagnosis research has been carried out to soy bean cyst roundworm.Soy bean cyst roundworm species-specific primer GlyF1 susceptibility is very high, can amplify soy bean cyst roundworm species specificity fragment from the minim DNA of wall scroll larva.(the Peng Deliang such as Peng Deliang, Wang Qiong, Geng Lifeng, Li Jia, Peng Huan, Zhang Dongsheng. a kind of detection method of early molecule of radopholus similes thorne, patent No. ZL201010113930.X) the Auele Specific Primer RsF1/RsR1 of radopholus similes thorne is devised according to the sequence difference of radopholus similes thorne ITS, in conjunction with the universal primer (D3A/D3B) of the D3 expansion area in 28S district as interior mark, adopt an one-step dual PCR method can detect radopholus similes thorne specifically, the method can be used in soil sample and the direct-detection of plant of falling ill simultaneously.

In the Molecular Detection of Ditylenchus destructor, (the Wendt such as Wendt, K., Vrain, T.C. & Webster, J.M.1993.Separation of three species of Ditylenchus and some host races of D.dipsaci by Restriction Fragment Length Polymorphism.Journal of Nematology25,555-563) develop the technology of ITS-RFLP, utilize multiple digestion with restriction enzyme ITS sequence, Ditylenchus destructor and lepisphere Ditylenchus dipsaci can be distinguished accurately.This laboratory finds in the research in early stage, the ITS of Ditylenchus destructor has notable difference, obvious 2 types can be divided into: A type and Type B, design 2 pairs of Auele Specific Primers accordingly and detect A type and Type B, A type Ditylenchus destructor special primer is DDS1/DDS2, and specific amplified fragment is 252bp; Type B Ditylenchus destructor special primer is DDL1/DDL2, and specific amplified fragment is 485bp; Introduce nematode universal primer D3A/D3B and make interior mark, work out molecular engineering and the method for a dissimilar colony of one-step dual PCR specific detection Ditylenchus destructor, this molecular detection technology has high specificity, method is reliable, susceptibility is high, easy and simple to handle, the tolerance range of detection reaches the level of wall scroll nematode.As if (luxuriant and rich with fragrance, Peng Deliang, Yang Yuwen, He Yueqiu. Ditylenchus destructor specific molecular detection technique research .2008 Plant Pathology, 38 (3): 263-270).(the M.Marek such as Marek, M.Zouhar, O.Douda, J.Mazakova and P.Rysanek 2010.Bioinformatics-assisted characterization of the ITS1-58S-ITS2segments of nuclear rRNA gene clusters, and its exploitation in molecular diagnostics of European crop-parasitic nematodes of the genus Ditylenchus.Plant Pathology59, 931-943) etc. develop the Auele Specific Primer DipU-F/DipU-R that simultaneously can detect above-mentioned two kinds of nematodes according to the difference of D.destructor and D.dipsaci, Des2-F/Des1-R, the fragment detecting 333bp from D.dipsaci that wherein DipU-F/DipU-R can be special, Des2-F/Des1-R can be special the fragment detecting 453bp from Ditylenchus destructor.But the result of study in later stage shows that Des2-F/Des1-R is merely able to detect Ditylenchus destructor (the Subbotin S.A. of Type B, Inserra R.N., Marais M., Mullin P., Powers T., Roberts P.A., Van den Berg E., Yeates G.W. & Baldwin, J.G.2011.Diversity and phylogenetic relationships within the spiral nematodes of Helicotylenchus Steiner, 1945 (Tylenchida:Hoplolaimidae) as inferred from analysis of the D2-D3expansion segments of28S rRNA gene sequences.Nematology, 13:773-785).(the Liu Bin such as Liu Bin, plum is round, Zheng is through the research of military .2007. rot stem nematodes kind in-group specific detection. journal of Zhejiang university (agricultural and life science version), 33 (5): 490-496) group-specific primers is devised according to the ITS sequence of Ditylenchus destructor, can amplify the fragment of 346bp, but the sample detected only includes Ditylenchus destructor.Liu Qian etc. (Liu Qian, Niu Junhai, letter is permanent, Guo Quanxin, Chen Changlong. and detect test kit and the application thereof of sweet potato rot stem nematodes based on ring mediated isothermal amplification, CN102260746A) develop the LAMP detection technique of Ditylenchus destructor.

The research report in the ITS region of existing Ditylenchus destructor rDNA both at home and abroad, but the primer major part developed at present is merely able to detect a certain type of Ditylenchus destructor, does not also have the PCR method for detecting specificity that can detect category-A and Type B Ditylenchus destructor at present simultaneously.

Summary of the invention

BROAD SUMMARY of the present invention is to provide a kind of Ditylenchus destructor fast PCR molecular detecting method based on ITS sequence difference and application thereof, for formulating the services such as the rapid molecular detection of nematode, the early diagnosis of crop rot stem nematodes disease and assistant identification.

Ditylenchus destructor fast PCR molecular detecting method, comprises PCR reaction system, and it is characterized in that there is a group-specific primers in described PCR reaction system, described Auele Specific Primer is DdF1 and DdR1,

DdF1:5’-GCTCTGTGCCTGGCTAATTTGTG-3’，

DdR1:5’-ACCAAACACTGGACAGCATTATC-3’。

Also containing universal primer D2A and D3B in described PCR reaction system,

D2A:5’-ACAAGTACCGTGAGGGAAAGTTG-3’，

D3B:5’-TCGGAAGGAACCAGCTACTA-3’。

Described PCR reaction system is the Buffer of 10 × PCR containing Mg2+ of 2.5 μ l; 2 μ l10mM dNTP; 1.5 μ l primer pair DdF1/DdR1; 0.2 μ l Taq archaeal dna polymerase; 2 μ l template DNAs; Sterilizing ddH2O complements to 25 μ l.

PCR reaction conditions is 95 DEG C of denaturation 8min, 95 DEG C, 45s, 55 DEG C, 30s, 72 DEG C, 1min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.

Above-mentioned Ditylenchus destructor fast PCR molecular detecting method in censorship sample whether with the application in Ditylenchus destructor and assistant identification.

The present invention utilizes the Ditylenchus destructor in ITS universal primer amplification acquisition 7 country variants and area, comprising 2 Type Bs and 5 A type populations.Download the ITS sequence comparison of multiple Ditylenchus from Genbank after, at Ditylenchus destructor ITS specific regions design primer, the rapid molecular for Ditylenchus destructor detects.

This invention exploits Ditylenchus destructor Auele Specific Primer DdF1 and DdR1, construct fast PCR Molecular Detection PCR system.Adopt Auele Specific Primer DdF1 and DdR1 can carry out specific amplification to A type and Type B Ditylenchus destructor, expanding fragment length is 495bp, and other 7 class Ditylenchus 14 populations all do not detect the specific fragment of 495bp simultaneously.In order to reduce the false negative result of detection, universal primer D2A and D3B of 28S is added while specific detection, for the detection of DNA quality, universal primer D2A and D3B can amplify the fragment that length is about 780bp from all effective DNA, positive sample is by the fragment of the specific fragment that occurs 495bp and 780bp simultaneously, but not Ditylenchus destructor only has the fragment of 780bp, thus add the accuracy of detected result.Simultaneously, the Ditylenchus destructor genomic dna of a series of weaker concns that the single head Ditylenchus destructor DNA of different weaker concn and concentration are determined for the sensitivity technique of Ditylenchus destructor Auele Specific Primer DdF1 and DdR1 of these research and development, thus determines that the Auele Specific Primer of Ditylenchus destructor and the detection threshold of detection method are 1ng/ul and 1/128 nematode DNA.There is very high sensitivity.Auele Specific Primer DdF1 and DdR1 that the present invention builds and detection method high specificity thereof, highly sensitive, quick, accurate can be determined thus.

The present invention's Plant nematode to be measured comprises A type Ditylenchus destructor, Type B Ditylenchus destructor, 2 external Ditylenchus destructor populations, lepisphere Ditylenchus dipsaci, African Ditylenchus dipsaci, D.gagis, D.weischeri and 3 Ditylenchus dipsaci unknown species.

The present invention adopts universal primer TW81 and AB28 to amplify the ITS sequence of Ditylenchus destructor, by designing a pair Auele Specific Primer DdF1 and DdR1 with the ITS sequence comparison of other Ditylenchus dipsacis,, achieve diagnosis and detection while A type and Type B Potato Rot Nemotode.There is high specificity and sensitivity, reducing the erroneous judgement to detecting sample.The method can be applied to the aspect such as the field sample detection of Ditylenchus destructor and the monitoring and warning of occurrence and distribution simultaneously, have actual using value.

Accompanying drawing explanation

Fig. 1: the ITS sequence comparison of different Ditylenchus dipsaci kind and specific primer design,

Fig. 2: Ditylenchus destructor specific detection result,

M is stranded DNA molecule mark (DNA marker III); 1-7 is respectively Ditylenchus destructor different groups (being encoded to Dd01, Dd02, Dd03, Dd04, Dd05, Dd06, Dd07); 8-14 is respectively lepisphere Ditylenchus dipsaci different groups (Dp01, Dp02, Dp03, Dp04, Dp05, Dp06, Dp07); 15 is African Ditylenchus dipsaci (being encoded to Da01); 16 be D.weischeri colony (Dw01) 17 is D.gagis colony (Dg01), and 18-20 is the Ditylenchus dipsaci (Dity01, Dity02 and Dity03) of 3 unknown species; CK is negative control;

Fig. 3: the single head Ditylenchus destructor DNA sensitivity technique result of different weaker concn,

M is stranded DNA molecule mark (DNA marker III); 1-8 is respectively 1,1/2,1/4,1/8,1/16,1/32,1/64,1/128 Ditylenchus destructor; CK is negative control.

Fig. 4: the Ditylenchus destructor genomic dna sensitivity technique result of different concns,

M is stranded DNA molecule mark (DNA marker III); 1-8 is Ditylenchus destructor genomic dna (100 μ g/ μ l, 10 μ g/ μ l, 1 μ g/ μ l, 100ng/ μ l, 10ng/ μ l, 1ng/ μ l, 10pg/ μ l and 1pg/ μ l); CK: negative control.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

The material that experiment of the present invention is selected:

7 Ditylenchus destructor colonies be this laboratory from the Inner Mongol, Henan, Jilin, the ground such as Shandong and America & Canada collects, 13 lepisphere Ditylenchus dipsacis, African Ditylenchus dipsaci, D.weishceri, D.gagis and the Ditylenchus dipsaci DNA of other unknown species are that this laboratory worker collects (see table 1) from foreign country.All there is preservation in above-mentioned this laboratory of Ditylenchus destructor DNA, can provide the public.

The Ditylenchus dipsaci Sample code that table 1 detects and colony source

Main agents: Taq archaeal dna polymerase, DNA gel reclaim test kit, DNA marker and PMD18-T vector purchased from TaKaRa company, and primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.

Embodiment 1: the foundation of Ditylenchus destructor specific PCR molecular detection method

The extraction of 1.1 nematode DNA

Hand chooses Ditylenchus dipsaci population single head nematode, put into fill 7 μ l 10 × PCR buffer, in the centrifuge tube in the redistilled water of 3 μ l Proteinase K Solution (600 μ g/ml) and 10 μ l sterilizings ,-20 DEG C of refrigerator and cooled freeze 1h.Ice-out is turned to, then freezing at least 2h at-20 DEG C with the glass stick of 75% alcohol disinfecting.After 2h, centrifuge tube is taken out from refrigerator, incubation 1.5h at putting 65 DEG C, then at 95 DEG C of 10min, centrifugal 1min under last 10000rpm, supernatant liquor saves backup in-20 DEG C.

1.2 Ditylenchus destructor specific primer design and screenings

Adopt universal primer TW81(5 '-TTGATTACGTCCCTGCCCTTT-3 ') and AB28(5 ' TTTCACTCGCCGTTACTAAGG-3 '), with the DNA of the Ditylenchus dipsaci of said extracted for template, amplification Ditylenchus destructor ITS sequence.The reaction system of PCR is that 10 × Buffer is (containing Mg ²⁺) 5 μ l, 10mM dNTP4 μ l, ddH2O32.6 μ l, primer TW81 and AB28(20 μM ol/L) each 1.5 μ l, Taq enzyme (5U/ μ l, Takara) 0.4 μ l, template DNA 5 μ l, amounts to 50 μ l.Pcr amplification condition is: 95 DEG C, 8min, 56 DEG C, 30s, 72 DEG C, 1min; 94 DEG C, 45s, 50 DEG C, 30s, 72 DEG C, 2min, 35 circulations; 72 DEG C, 10min, 10 DEG C of preservations.After pcr amplification, get 5 μ l amplified productions and add 1 μ l sample loading buffer on 1.5% sepharose, electrophoresis 40 minutes under 0.5 × TAE electrophoretic buffer, EB dyes, and observes and take a picture under gel imaging instrument.The ITS sequence of domestic Ditylenchus destructor is all to submit to Genbank to register (EF210367, EF418002, EF210372, EF208211, EF208212).

The rDNA-ITS regional sequence of fuller's teasel Ditylenchus dipsaci, rice stem nematode, D.myceliophagus, leafy stalk nematode, D.askenasyi, African Ditylenchus dipsaci, multiple sibling species nematode such as D.gagis, D.weisheri is downloaded from NCBI gene pool, use the ITS sequencing result of MEGA5.0 to above-mentioned sequence and Ditylenchus destructor different shaped colony of China to compare, adopt primer5.0 to design the specific detection (Fig. 1) of Auele Specific Primer DdF1 and DdR1 for Ditylenchus destructor.

According to the ITS sequence comparison result of multiple Ditylenchus dipsaci, a group-specific primers is designed in the specific regions choosing Ditylenchus destructor, and the length of this group primer is 23 bases.Ditylenchus destructor Auele Specific Primer upstream primer and downstream primer called after DdF1 and DdR1 respectively, its sequence signature is as follows:

DdF1:5’-GCTCTGTGCCTGGCTAATTTGTG-3’，

DdR1:5’-ACCAAACACTGGACAGCATTATC-3’。

The foundation of 1.3 Ditylenchus destructor specific PCR molecular detection system

Adopt the Auele Specific Primer DdF1/DdR1 of the present invention's design, with the genomic dna of Ditylenchus destructor for template, carry out pcr amplification, PCR reaction system: 2.5 μ l10 × PCR Buffer are (containing Mg ²⁺), 2 μ l10mM dNTP(2.5mM), 1.5 μ l primer pairs DdF1/DdR1 (20uM), 0.2 μ l Taq archaeal dna polymerase (5U/ul), 2 μ l template DNAs, sterilizing ddH2O complements to 25 μ l.Adopting without nematode DNA profiling is negative control.PCR amplification instrument increases.PCR reaction conditions is as follows: 95 DEG C, 8min, 95 DEG C, 45s, 55 DEG C, 30s, 72 DEG C, 1min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l amplified productions and add 1 μ l sample-loading buffer electrophoresis on 1.0% sepharose, with 0.5 × TAE as electrophoretic buffer, 120V/40min, with EB dyeing, observes and takes a picture under ultraviolet light.

Embodiment 2: Ditylenchus destructor PCR molecular detecting method specificity and sensitivity technique.

2.1 Ditylenchus destructor PCR molecular detecting method specific detection.

Auele Specific Primer DdF1/DdR1 and the universal primer D2A/D3B of the present invention's design transfer to Shanghai biotechnology company limited to synthesize, PCR reaction system: 2.5 μ l10 × PCR Buffer are (containing Mg ²⁺), 2 μ l10mM dNTP(2.5mM), 1.5 μ l primer pair DdF1/DdR1 (20 μMs), 0.2 μ l Taq archaeal dna polymerase (5U/ μ l), 2 μ l template DNAs, sterilizing ddH2O complements to 25 μ l.Adopting without nematode DNA profiling is negative control.Eppendorf PCR amplification instrument increases.PCR reaction conditions is as follows: 95 DEG C, 8min, 95 DEG C, 45s, 55 DEG C, 30s, 72 DEG C, 1min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l amplified productions and add 1 μ l sample-loading buffer electrophoresis on 1.0% sepharose, with 0.5 × TAE as electrophoretic buffer, 120V/40min, with EB dyeing, observes and takes a picture under ultraviolet light.Simultaneously we adopt universal primer D2A with D3B and above-mentioned identical PCR reaction system and amplification condition with above-mentioned DNA for template increases, and detect the quality of DNA.

D2A:5’-ACAAGTACCGTGAGGGAAAGTTG-3’，

D3B:5’-TCGGAAGGAACCAGCTACTA-3’。

7 Ditylenchus destructor populations amplify a stable band through Auele Specific Primer DdF1 and DdR1, length is 495bp, occur without amplified band in other Ditylenchus dipsaci populations and negative control, DNA quality examination finds, the DNA profiling that the present invention adopts is all intact, universal primer D2A and D3B all can amplify stable band, as shown in Figure 2, illustrates that the Auele Specific Primer of design meets the requirements.

2.2 Ditylenchus destructor PCR molecular detecting method sensitivity technique.

Extract the DNA of wall scroll Ditylenchus destructor, adopt 2 times of dilution methods, DNA is diluted obtain 1/2,1/4,1/8,1/16,1/32,1/64,1/128, the DNA of bar Ditylenchus destructor, respectively get 1 μ l Ditylenchus destructor DNA to be template Auele Specific Primer DdF1/DdR1 and to carry out pcr amplification, with check Auele Specific Primer sensitivity.

Adopt the genomic dna of phenol/chloroform extraction method Ditylenchus destructor, Nanodrop2000 is adopted to measure its genomic dna concentration, adopt water be diluted to 100 μ g/ μ l, 10 μ g/ μ l, 1 μ g/ μ l, 100ng/ μ l, 10ng/ μ l, 1ng/ μ l, 10pg/ μ l and 1pg/ μ l8 concentration, respectively get 1 μ l Ditylenchus destructor DNA be template Auele Specific Primer DdF1/DdR1 carry out pcr amplification with check Auele Specific Primer sensitivity.Ditylenchus destructor ITS Auele Specific Primer DdF1/DdR1 is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.Auele Specific Primer sensitivity technique amplification reaction system and amplification program, as described in 2.1, PCR amplification instrument increase.Electrophoresis result as shown in Figure 3 and Figure 4.

The result of Fig. 3 shows, can both amplify band (swimming lane 1-6) clearly from 1-1/64 bar nematode, and the band of 1/64,1/128 nematode also can may be seen indistinctly, negative control does not have amplified band.The result of Fig. 4 illustrates, the Auele Specific Primer of this Ditylenchus destructor and the detection threshold of detection method are 1ng/ μ l, and this illustrates that the sensitivity of this Auele Specific Primer and detection method is very high, therefore illustrates that the method is very reliable.

Claims

1. Ditylenchus destructor fast PCR molecular detecting method, it is characterized in that using the PCR reaction system containing a group-specific primers to detect, described Auele Specific Primer is DdF1 and DdR1,

DdF1:5’-GCTCTGTGCCTGGCTAATTTGTG-3’，

DdR1:5’-ACCAAACACTGGACAGCATTATC-3’。

2. molecular detecting method according to claim 1, also containing universal primer D2A and D3B in described PCR reaction system,

D2A:5’-ACAAGTACCGTGAGGGAAAGTTG-3’，

D3B:5’-TCGGAAGGAACCAGCTACTA-3’。

3. molecular detecting method according to claim 1, described PCR reaction system is that the 10 × PCR of 2.5 μ l is containing Mg ²⁺buffer; 2 μ l 10mM dNTP; 1.5 μ l primer pair DdF1/DdR1; 0.2 μ l Taq archaeal dna polymerase; 2 μ l template DNAs; Sterilizing ddH ₂o complements to 25 μ l.

4. molecular detecting method according to claim 3, wherein PCR reaction conditions is 95 DEG C of denaturation 8min, 95 DEG C, 45s, 55 DEG C, 30s, 72 DEG C, 1min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.

5. whether claim 1-4 arbitrary Ditylenchus destructor fast PCR molecular detecting method is detecting in censorship sample with the application in Ditylenchus destructor.