CN105255906A - Trichodorus hangzhouensis n.sp. RFLPs detection and identification method and corresponding gene - Google Patents
Trichodorus hangzhouensis n.sp. RFLPs detection and identification method and corresponding gene Download PDFInfo
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Abstract
The invention discloses a trichodorus hangzhouensis n.sp. gene which has a rDNA-ITS1 sequence (1802bp) and a rDNA-ITS2 sequence (874bp). The invention also provides a trichodorus hangzhouensis n.sp. RFLPs detection and identification method. The method comprises the steps that the DNA template of the detected nematode is extracted for rDNA-ITS1 and rDNA-ITS2 region PCR reaction, a PCR product is purified, and it is judged that the detected nematode is trichodorus hangzhouensis n.sp. if the purified PCR product can obtain the rDNA-ITS1 sequence and the rDNA-ITS2 sequence at the same time. Enzymolysis can also be conducted on the purified PCR product so that RFLPs analysis can be conducted, in other words, whether the detected nematode is trichodorus hangzhouensis n.sp. is judged according to the size of a fragment obtained after digestion.
Description
Technical field
The invention belongs to agricultural Defect inspection technical field, be specifically related to a kind of burr and belong to RFLPs (restriction fragment length polymorphism) the Testing and appraisal method of nematode novel species and the rDNA-ITS1 sequence in corresponding Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) and rDNA-ITS2 sequence.
Background technology
Burr belongs to (TrichodorusCobb, 1913) nematode is under the jurisdiction of Nematoda [Nematoda (Rudolphi, 1808) Lankester, 1877], without side tail gland guiding principle (AdenophoreavonLinstow, 1905), three lance order (TriplonchidaCobb, 1920), hymeniderm suborder (DiphtherophorinaCoomans & Loof, 1970), burr Superfamily [TrichodorideaThorne, 1935 (Siddiqi, 1974)], burr section [TrichodoridaeThorne, 1935 (Siddiqi, 1961)], burr subfamily (TrichodorinaeThorne, 1935), it is the important plant needle nematode of a class, plant root can be parasitized, affect root growth, root growth is even caused to stagnate, cause the short and thick root disease of plant.Many populations in genus or the vector of some plant viruses, as the vector that T.primitivus is Tobacco rattle virus (TRV) and pea brown virus morning (PEBV), many cash crop such as legume crop, alfalfa, cucumber, tomato, beet can be made to catch an illness, cause serious financial loss.In recent years, day by day frequent along with international trade contacts, pass in and out seedling and plant propagation material thereof increase, and the various Plant diseases chance that especially nematodiasiss is propagated increases greatly, makes Plant nematode become the object of countries in the world extensive concern.
Belong to regard to nematode with regard to burr, describe first burr from famous nematologist deMan in 1880 and belong to nematode Dorylaimusprimitivus, after Cobb established burr Turbatrix in 1913, describe a lot of burr all over the world and belong to nematode novel species, this genus describes 55 effectively kinds altogether up to now.The Testing and appraisal of this genus nematode current mainly relies on typoiogical classification method, namely based on morphological feature and the morphometric of female worm and male worm, but owing to belonging to, to there is much morphology between interior nematode kind very similar or be difficult to the feature observed, as female worm vaginal orifice is ossify the quantity that has that it's too late in the shape of block and size, lateral body hole, in male worm abdomen cervical papilla quantity and with the form that has that it's too late of the position relationship of excretory pore, copulatory spicules decorative pattern, the form etc. of sperm, make the difference between planting not be clearly sometimes, add the difficulty of qualification.Based on this, people start to attempt adopting the methods such as round pcr to study this genus nematode, as Boutsika etc. utilizes pcr amplification Paratrichodorusmacrostylus, P.pachydermus, the 18SRNA district of Trichodorusprimitivus and T.similis, rDNA-ITS1 district and rDNA-ITS2 district gene, and gained sequence is analyzed, obtain the species-specific primer of these 4 kinds; The relation that Holeva etc. utilize real-time fluorescence PCR method to have studied burr to belong to vector nematode and Tobacco rattle virus.
Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) is that the burr being collected in Hangzhou, Zhejiang province city loquat rhizosphere belongs to nematode novel species, is a record kind, there is no its systematic study report at present both at home and abroad.By its called after Trichodorusn.sp. in existing article.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of burr and belongs to the RFLPs Testing and appraisal method of nematode novel species and corresponding gene.
In order to solve the problems of the technologies described above, the invention provides a kind of Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) gene, there is the rDNA-ITS1 sequence (1802bp) described in SEQIDNO:1.
Improvement as Hangzhou of the present invention burr nematode (Trichodorushangzhouensisn.sp.) gene: also there is the rDNA-ITS2 sequence (874bp) described in SEQIDNO:2.
The present invention also provides a kind of Hangzhou burr nematode RFLPs Testing and appraisal method simultaneously, in turn includes the following steps:
1. the DNA profiling of nematode to be measured (particularly burr nematode), is extracted;
2., carry out rDNA-ITS1 and rDNA-ITS2 district PCR to react:
The primer that the amplification of rDNA – ITS1 region sequence adopts is:
V1：5′-TTGATTACGTCCCTGCCCTTT-3′，
ITSB：5′-GCTGCGTTCTTCATCGAT-3′；
The primer that the amplification of rDNA-ITS2 region sequence adopts is:
ITSA：5′-ATCGATGAAGAACGCAGC-3′
V2：5′-TTTCACTCGCCGTTACTAAGG-3′；
3., carry out PCR primer purifying, obtain PCR purified product;
4., judge:
What PCR purified product can obtain rDNA-ITS1 sequence and rDNA-ITS2 sequence simultaneously is Hangzhou burr nematode (Trichodorushangzhouensisn.sp.); Otherwise, then judge it is not Hangzhou burr nematode (Trichodorushangzhouensisn.sp.).
As the improvement of Hangzhou of the present invention burr nematode RFLPs Testing and appraisal method, this RFLPs Testing and appraisal method is further comprising the steps of:
One, the PCR purified product of step 3. gained is carried out enzymolysis (object carries out RFLPs analysis):
The restriction enzyme that rDNA – ITS1 district PCR primer endonuclease reaction adopts is respectively: Ava I, Bg II, Hind III, Kpn I, Nci I, Pst I;
The restriction enzyme that rDNA-ITS2 district PCR primer endonuclease reaction adopts is respectively: Dde I, Hinf I, Mva I, Nci I, Pst I, Rsa I;
Two, the enzymolysis solution of rDNA-ITS1, rDNA-ITS2 gained is respectively carried out respectively the reading of endonuclease reaction result;
The enzymolysis solution of rDNA-ITS1, rDNA-ITS2 gained is respectively carried out following steps respectively:
10 μ l enzymolysis solutions and 2 μ l6 × loadingbuffer are mixed, carries out electrophoresis; Then carry out dyeing, observe under ultraviolet lamp again, taking pictures (is specially: in 1.3% sepharose (w/v), 1 × TAE electrophoretic buffer, electrophoresis under 75V voltage stabilizing, after electrophoresis terminates, be placed in 0.1% EB solution (w/v) dyeing 10min, after under ultraviolet lamp observe, take pictures; )
When result rDNA-ITS1, rDNA-ITS2 enzyme met described in following table 1 cuts rear clip size, judge that this nematode to be measured is as Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Otherwise, when result rDNA-ITS1, rDNA-ITS2 enzyme do not met described in following table 1 cuts rear clip size, judge that this nematode to be measured is not Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Table 1, rDNA-ITS1, rDNA-ITS2 enzyme cut rear clip size
As Hangzhou of the present invention burr nematode RFLPs Testing and appraisal further improvements in methods:
Described step 2. in:
The PCR reaction system of 25 μ L is: DNA profiling 2 μ L, 10 × PCRbuffer are (containing Mg
2+) 2.5 μ L, 2.5mmol/LdNTP2 μ L, 40 μm of ol/ μ L each primer 1 μ L, 5U/ μ LTaq enzyme 0.3 μ L, ddH
2o complements to 25 μ L (that is, 16.2 μ L);
Pcr amplification program is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 52 DEG C (ITS1 districts) or 55 DEG C of (ITS2 district) 50s that anneal, 72 DEG C of extension 2min, 35 circulations, 72 DEG C of total elongation 10min.
As Hangzhou of the present invention burr nematode RFLPs Testing and appraisal further improvements in methods:
The endonuclease reaction system of 10 μ L is: PCR purified product 6 μ L, restriction enzyme 1 μ L, 10 × damping fluid (damping fluid carried for often kind of restriction enzyme) 1 μ L, ddH
2o2 μ L;
Endonuclease reaction program is: centrifuge is after 3 ~ 5 seconds, in 37 ± 0.5 DEG C of incubator inside holding at least 5h.
As Hangzhou of the present invention burr nematode RFLPs Testing and appraisal further improvements in methods:
Step DNA profiling is 1. extracted as:
2 nematodes to be measured are put into containing 18 μ l10 × PCR-buffer (Mg
2+free) in 0.5 μ leppendorf pipe, add 2 μ l600 μ g/ml Proteinase Ks and be placed on-70 DEG C, after 20min, proceed to 65 DEG C of 60min in PCR instrument, the centrifugal 2min of 95 DEG C of 10min, rear 12000r/min, obtain DNA profiling.
Hangzhou of the present invention burr nematode RFLPs Testing and appraisal method, in the leaching process of DNA profiling, the nematode (nematode to be measured) of direct picking 2 entry is in eppendorf pipe, but not draw nematode fragment with pipettor again shred object nematode on slide glass after, considerably reduce the loss amount of operation steps and object nematode DNA; RDNA-ITS1 and rDNA-ITS2 district is endonuclease reaction analysis after pcr amplification, T.hangzhouensis and other nematodes is easy to make a distinction, and traditional form authentication method needs female, each about 10 of male imago, method of the present invention is time saving and energy saving compared with traditional method province worm; Provide the Molecular Detection authentication method that burr belongs to a nematode novel species.
The invention provides the method utilizing PCR-RFLPs Testing and appraisal burr Turbatrix one novel species-Hangzhou burr nematode, operator are without the need to possessing more nematology expertise, workable, it is time saving and energy saving that more traditional Morphological Identification method economizes worm, is a kind of easy and simple to handle, high efficiency pathogenic nematode Testing and appraisal novel method.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is rDNA-ITS1 sequence (1802bp);
Fig. 2 is rDNA-ITS2 sequence (874bp);
Fig. 3 is the rDNA-ITS1-RFLPs collection of illustrative plates in Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Fig. 4 is the rDNA-ITS2-RFLPs collection of illustrative plates in Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Fig. 5 is the rDNA-ITS1-RFLPs collection of illustrative plates of Trichodorus nanjinensis (T.nanjingensis);
Fig. 6 is the rDNA-ITS2-RFLPs collection of illustrative plates of Trichodorus nanjinensis (T.nanjingensis);
Fig. 7 is the rDNA-ITS1-RFLPs collection of illustrative plates of Pakistani burr nematode (T.pakistanensis);
Fig. 8 is the rDNA-ITS2-RFLPs collection of illustrative plates of Pakistani burr nematode (T.pakistanensis);
Fig. 9 is the rDNA-ITS1-RFLPs collection of illustrative plates of cdear burr nematode (T.cedarus);
Figure 10 is the rDNA-ITS2-RFLPs collection of illustrative plates of cdear burr nematode (T.cedarus).
In figure, U representative is without the ITS1/ITS2PCR product fragment of digestion with restriction enzyme, and namely PCR primer is without digestion with restriction enzyme.
Embodiment
Embodiment 1, a kind of Hangzhou burr nematode RFLPs Testing and appraisal method, carry out following steps successively:
1. DNA profiling is extracted: the identical object nematode of picking 2 (nematode to be measured)-Hangzhou burr nematode is put into containing 18 μ l10 × PCR-buffer (Mg under anatomical lens
2+free), in 0.5 μ leppendorf pipe, rapidly pipe is placed in-70 DEG C of refrigerators after adding 2 μ l600 μ g/ml Proteinase Ks, proceed to 65 DEG C of 60min in PCR instrument after 20min, the centrifugal 2min of 95 DEG C of 10min, rear 12000r/min, namely obtains DNA profiling.
2. carry out rDNA-ITS1 and rDNA-ITS2 district PCR to react:
The primer that the amplification of rDNA – ITS1 region sequence adopts is:
V1 (5 '-TTGATTACGTCCCTGCCCTTT-3 ') and ITSB (5 '-GCTGCGTTCTTCATCGAT-3 '),
The primer that the amplification of rDNA-ITS2 region sequence adopts is:
ITSA (5 '-ATCGATGAAGAACGCAGC-3 ') and V2 (5 '-TTTCACTCGCCGTTACTAAGG-3 ').
PCR reaction system (25 μ L) is: DNA profiling 2 μ L, 10 × PCRbuffer are (containing Mg
2+) 2.5 μ L, 2.5mmol/LdNTP2 μ L, 40 μm of ol/ μ L each primer 1 μ L, 5U/ μ LTaq enzyme 0.3 μ L, ddH
2o16.2 μ L.
Pcr amplification program is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 52 DEG C (ITS1 districts) or 55 DEG C of (ITS2 district) 50s that anneal, 72 DEG C of extension 2min, 35 circulations, 72 DEG C of total elongation 10min.
3. PCR primer purifying is carried out:
After PCR has reacted, respectively following purifying is carried out to 2 kinds of PCR primer of step 2. gained:
25 μ lPCR products and 5 μ l6 × loadingbuffer are mixed, in 1.0% sepharose (w/v), 1 × TAE electrophoretic buffer, electrophoresis is carried out under 90V voltage stabilizing, after electrophoresis terminates (namely, until terminate electrophoresis during the 4th lattice reciprocal), be placed in EB solution (w/v) the dyeing 10min of 0.1%, test kit carries out reclaiming, purifying to select the UNIQ-10 pillar DNA glue of Shanghai Sangon company to reclaim.
Obtain the sequence of (that is, SEQIDNO:1 and SEQIDNO:2) described in Fig. 1 and Fig. 2 respectively.
4. RFLPs analysis is carried out:
The restriction enzyme that rDNA – ITS1 district PCR primer endonuclease reaction adopts is respectively: Ava I, Bg II, Hind III, Kpn I, Nci I, Pst I;
The restriction enzyme that rDNA-ITS2 district PCR primer endonuclease reaction adopts is respectively: Dde I, Hinf I, Mva I, Nci I, Pst I, Rsa I.
Endonuclease reaction system (10 μ L) is: PCR purified product 6 μ L, restriction enzyme 1 μ L, 10 × damping fluid (damping fluid carried for often kind of restriction enzyme) 1 μ L, ddH
2o2 μ L.Endonuclease reaction program is: flick tube wall with finger, of short duration centrifugal 3 ~ 5 seconds of whizzer, after pipe is placed in 37 DEG C of incubator inside holding at least 5h.
RDNA-ITS1, rDNA-ITS2 enzyme is cut rear clip size and is described in table 1 below:
Table 1, rDNA-ITS1, rDNA-ITS2 enzyme cut rear clip size
5. the reading of endonuclease reaction result:
After endonuclease reaction completes, 10 μ l enzymolysis solutions and 2 μ l6 × loadingbuffer are mixed, in 1.3% sepharose (w/v), 1 × TAE electrophoretic buffer, electrophoresis under 75V voltage stabilizing, after electrophoresis terminates, be placed in 0.1% EB solution (w/v) dyeing 10min, after under ultraviolet lamp observe, take pictures.
Acquired results respectively as described in fig. 3 and fig. 4.
When result rDNA-ITS1, rDNA-ITS2 enzyme met described in table 1 cuts rear clip size (Fig. 3+Fig. 4), judge that this nematode to be measured is as Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Otherwise, when result rDNA-ITS1, rDNA-ITS2 enzyme do not met described in table 1 cuts rear clip size (Fig. 3+Fig. 4), judge that this nematode to be measured is not Hangzhou burr nematode (Trichodorushangzhouensisn.sp.).
Test 1, " Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) " will be known as in advance as sample A, and using Trichodorus nanjinensis (T.nanjingensis), Pakistani burr nematode (T.pakistanensis), cdear burr nematode (T.cedarus) as sample B ~ sample D, each sample arranges 3 repetitions; Detect according to mode described in above-described embodiment 1,3 repetition acquired results of each sample are all consistent.Specific as follows:
1), PCR primer after the purifying of gained, only have " Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) " of sample A can obtain sequence described in Fig. 1 and Fig. 2;
And the sequence of sample B ~ sample D gained is all not identical with Fig. 1 with Fig. 2.
2), endonuclease reaction result take pictures after the result of gained be:
Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) of sample A, as shown in Fig. 3+Fig. 4;
The Trichodorus nanjinensis (T.nanjingensis) of sample B, as shown in Fig. 5+Fig. 6;
The Pakistani burr nematode (T.pakistanensis) of sample C, as shown in Fig. 7+Fig. 8;
The cdear burr nematode (T.cedarus) of sample D, as shown in Fig. 9+Figure 10.
According to the contrast of above-mentioned figure, we can find out that the result of sample B ~ sample D is different from the result of sample A gained completely.
Comparative example 1, make the sequence of primer V1, ITSB that the rDNA – ITS1 region sequence amplification in embodiment 1 adopts into following 3 cover primer pairs respectively:
V1-1 (5 '-TTTATTAGGTCCCTGGCCTTT-3 ') and ITSB-1 (5 '-GCTCCCTTCTTCATCGAT-3 ');
V1-2 (5 '-TTGAATACCTCCCTCCCCTTT-3 ') and ITSB-2 (5 '-GCTCCCTTCTTGATCGAT-3 ');
V1-3 (5 '-TTGATTACGACCCAGGGCTTT-3 ') and ITSB-3 (5 '-CCTGCGTTCTTGTTCGAT-3 ').
All the other are equal to embodiment 1.
By the sample A described in experiment 1, sample B ~ sample D, detect according to method described in comparative example 1, acquired results is respectively: with regard to V1-1 and ITSB-1, V1-2 and ITSB-2 primer pair, how to change regardless of annealing temperature and annealing time, sample A ~ sample DPCR amplification all obtains corresponding micro-object fragment, electrophoretogram shows as object fragment faint, and the shape that trails in moderate; And V1-3 and ITSB-3 primer pair, sample A ~ sample DPCR amplification all can not obtain corresponding object fragment, and electrophoretogram shows as serious traction shape.
Comparative example 2, make the sequence of primer I TSA, V2 that the rDNA-ITS2 region sequence amplification in embodiment 1 adopts into following 3 cover primer pairs respectively:
The primer that the amplification of rDNA-ITS2 region sequence adopts is:
ITSA-1 (5 '-ATCGATGAAAAAGGCAGC-3 ') and V2-1 (5 '-TTTCACTCCCCGTTTCTAAGG-3 ');
ITSA-2 (5 '-ATCGATGAAAAACGGGGC-3 ') and V2-2 (5 '-TTTCACTCCCCCTTACAAAGG-3 ');
ITSA-3 (5 '-ATGGAAAAAGAACGCAGC-3 ') and V2-3 (5 '-ATTCACCCCCCGTTACTAAGG-3 ').
All the other are equal to embodiment 1.
By the sample A described in experiment 1, sample B ~ sample D, detect according to method described in comparative example 2, acquired results is respectively: with regard to ITSA-2 and V2-2, ITSA-3 and V2-3 primer pair, how to change regardless of annealing temperature and annealing time, sample A ~ sample DPCR amplification all can not obtain corresponding object fragment, electrophoretogram shows as serious traction shape; And ITSA-1 and V2-1 primer pair, sample A ~ sample DPCR amplification all obtains corresponding micro-object fragment, electrophoretogram shows as object fragment place faint.
Comparative example 3, restriction enzyme A va I, Bg II, Hind III, Kpn I, Nci I, Pst I is adopted to make following 3 cover restriction enzymes respectively into the rDNA-ITS1 district PCR primer RFLPs in embodiment 1:
1. T.hangzhouensisn.sp.rDNA-ITS1 district PCR primer enzyme is cut to 26 small segments by Aci I, Bg II, Hind III, Kpn I, Nci I, Pst I, Aci I, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough;
2. T.hangzhouensisn.sp.rDNA-ITS1 district PCR primer enzyme is cut to 11 small segments by Ava I, Bs II, Hind III, Kpn I, Nci I, Pst I, Bs II, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough;
3. T.hangzhouensisn.sp.rDNA-ITS1 district PCR primer enzyme is cut to 15 small segments by Ava I, Bg II, Hinf I, Kpn I, Nci I, Pst I, Hinf I, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough.
Comparative example 4, restriction enzyme Dde I, Hinf I, Mva I, Nci I, Pst I, Rsa I is adopted to make following 3 cover restriction enzymes respectively into the rDNA-ITS2 district PCR primer RFLPs in embodiment 1:
1. T.hangzhouensisn.sp.rDNA-ITS2 district PCR primer enzyme is cut to 17 small segments by CviT I, Hinf I, Mva I, Nci I, Pst I, Rsa I, CviT I, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough;
2. T.hangzhouensisn.sp.rDNA-ITS2 district PCR primer enzyme is cut to 12 small segments by Dde I, Hinf I, Mva I, Nci I, Rsa I, TspR I, TspR I, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough;
3. T.hangzhouensisn.sp.rDNA-ITS2 district PCR primer enzyme is cut to 10 small segments by Dde I, Hinf I, Hpy188 I, Mva I, Nci I, Rsa I, Hpy188 I, and electrophoretogram is rendered as fuzzy a slice, and practicality and aesthetic property are all not good enough.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (7)
1. Hangzhou burr nematode (Trichodorushangzhouensisn.sp.) gene, is characterized in that: have the rDNA-ITS1 sequence described in SEQIDNO:1.
2. Hangzhou according to claim 1 burr nematode (Trichodorushangzhouensisn.sp.) gene, is characterized in that: also have the rDNA-ITS2 sequence described in SEQIDNO:2.
3. Hangzhou burr nematode RFLPs Testing and appraisal method, is characterized in that in turn including the following steps:
1. the DNA profiling of nematode to be measured, is extracted;
2., carry out rDNA-ITS1 and rDNA-ITS2 district PCR to react:
The primer that the amplification of rDNA – ITS1 region sequence adopts is:
V1：5′-TTGATTACGTCCCTGCCCTTT-3′，
ITSB：5′-GCTGCGTTCTTCATCGAT-3′；
The primer that the amplification of rDNA-ITS2 region sequence adopts is:
ITSA：5′-ATCGATGAAGAACGCAGC-3′
V2：5′-TTTCACTCGCCGTTACTAAGG-3′；
3., carry out PCR primer purifying, obtain PCR purified product;
4., judge:
What PCR purified product can obtain rDNA-ITS1 sequence and rDNA-ITS2 sequence simultaneously is Hangzhou burr nematode (Trichodorushangzhouensisn.sp.); Otherwise, then judge it is not Hangzhou burr nematode (Trichodorushangzhouensisn.sp.).
4. Hangzhou according to claim 3 burr nematode RFLPs Testing and appraisal method, is characterized in that this RFLPs Testing and appraisal method is further comprising the steps of:
One, the PCR purified product of step 3. gained is carried out enzymolysis:
The restriction enzyme that rDNA – ITS1 district PCR primer endonuclease reaction adopts is respectively: Ava I, Bg II, Hind III, Kpn I, Nci I, Pst I;
The restriction enzyme that rDNA-ITS2 district PCR primer endonuclease reaction adopts is respectively: Dde I, Hinf I, Mva I, Nci I, Pst I, Rsa I;
Two, the enzymolysis solution of rDNA-ITS1, rDNA-ITS2 gained is respectively carried out respectively the reading of endonuclease reaction result;
The enzymolysis solution of rDNA-ITS1, rDNA-ITS2 gained is respectively carried out following steps respectively:
10 μ l enzymolysis solutions and 2 μ l6 × loadingbuffer are mixed, carries out electrophoresis; Then carry out dyeing, observe under ultraviolet lamp again, take pictures;
When result rDNA-ITS1, rDNA-ITS2 enzyme met described in following table 1 cuts rear clip size, judge that this nematode to be measured is as Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Otherwise, when result rDNA-ITS1, rDNA-ITS2 enzyme do not met described in following table 1 cuts rear clip size, judge that this nematode to be measured is not Hangzhou burr nematode (Trichodorushangzhouensisn.sp.);
Table 1, rDNA-ITS1, rDNA-ITS2 enzyme cut rear clip size
5. the Hangzhou burr nematode RFLPs Testing and appraisal method according to claim 3 or 4, is characterized in that:
Described step 2. in:
The PCR reaction system of 25 μ L is: DNA profiling 2 μ L, 10 × PCRbuffer are (containing Mg
2+) 2.5 μ L, 2.5mmol/LdNTP2 μ L, 40 μm of ol/ μ L each primer 1 μ L, 5U/ μ LTaq enzyme 0.3 μ L, ddH
2o complements to 25 μ L;
Pcr amplification program is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 52 DEG C (ITS1 districts) or 55 DEG C of (ITS2 district) 50s that anneal, 72 DEG C of extension 2min, 35 circulations, 72 DEG C of total elongation 10min.
6. Hangzhou according to claim 4 burr nematode RFLPs Testing and appraisal method, is characterized in that:
The endonuclease reaction system of 10 μ L is: PCR purified product 6 μ L, restriction enzyme 1 μ L, 10 × damping fluid 1 μ L, ddH
2o2 μ L;
Endonuclease reaction program is: centrifuge is after 3 ~ 5 seconds, in 37 ± 0.5 DEG C of incubator inside holding at least 5h.
7. the Hangzhou burr nematode RFLPs Testing and appraisal method according to claim 3 or 4, is characterized in that:
Step DNA profiling is 1. extracted as:
2 nematodes to be measured are put into containing 18 μ l10 × PCR-buffer (Mg
2+free) in 0.5 μ leppendorf pipe, add 2 μ l600 μ g/ml Proteinase Ks and be placed on-70 DEG C, after 20min, proceed to 65 DEG C of 60min in PCR instrument, the centrifugal 2min of 95 DEG C of 10min, rear 12000r/min, obtain DNA profiling.
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| CN106148542A (en) * | 2016-08-26 | 2016-11-23 | 广东出入境检验检疫局检验检疫技术中心 | A kind of primer sets detecting Japan burr nematicide and application thereof |
| CN106222261A (en) * | 2016-07-28 | 2016-12-14 | 宁波检验检疫科学技术研究院 | The PCR detectable of tool poison burr nematicide and test kit |
| CN108220454A (en) * | 2017-12-26 | 2018-06-29 | 广东出入境检验检疫局检验检疫技术中心 | A kind of smaller kit for intending burr nematode and kidney shape plan burr nematode of while detection |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222261A (en) * | 2016-07-28 | 2016-12-14 | 宁波检验检疫科学技术研究院 | The PCR detectable of tool poison burr nematicide and test kit |
| CN106148542A (en) * | 2016-08-26 | 2016-11-23 | 广东出入境检验检疫局检验检疫技术中心 | A kind of primer sets detecting Japan burr nematicide and application thereof |
| CN108220454A (en) * | 2017-12-26 | 2018-06-29 | 广东出入境检验检疫局检验检疫技术中心 | A kind of smaller kit for intending burr nematode and kidney shape plan burr nematode of while detection |
| CN108220454B (en) * | 2017-12-26 | 2021-08-06 | 广东出入境检验检疫局检验检疫技术中心 | Kit for simultaneously detecting minor Bursaphelenchus-like nematodes and nephroid Bursaphelenchus-like nematodes |
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