CN105296483A - Primer, kit and method for identifying walnut bark beetles - Google Patents
Primer, kit and method for identifying walnut bark beetles Download PDFInfo
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- CN105296483A CN105296483A CN201510890145.8A CN201510890145A CN105296483A CN 105296483 A CN105296483 A CN 105296483A CN 201510890145 A CN201510890145 A CN 201510890145A CN 105296483 A CN105296483 A CN 105296483A
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Abstract
The invention relates to the field of identifying walnut bark beetles, and in particular relates to a primer, a kit and a method for identifying the walnut bark beetles. The primer for identifying the walnut bark beetles consists of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair; and the base sequences of four pairs of primers are shown by SEQ ID No.1-8. The primers are obtained by virtue of multiple times of verification of standard substances according to systematic biology in combination with known sequences of close species of the walnut bark beetles, are strong in specificity, high in sensitivity and good in repeatability, and can be used for identifying the walnut bark beetles very stably and quickly. The kit for identifying the walnut bark beetles, provided by the invention, is convenient and quick, and the method for identifying the walnut bark beetles takes the first primer pair, the second primer pair, the third primer pair and the fourth primer pair as primers to perform PCR (polymerase chain reaction) amplification on a genome extract of a to-be-detected sample, and then detect PCR amplification products, is convenient and quick and is high in reliability.
Description
Technical field
The present invention relates to the tiny moth-eaten qualification field of walnut, in particular to a kind of primer, test kit and the method thereof of identifying the tiny moth of walnut.
Background technology
At present, to the qualification of the tiny moth of walnut mainly according to following morphological specificity:
(1) bodily form is small, health total length 1.5-1.9mm, and be about the wide 2.8-3.1 of body doubly, female polypide look yellowish-brown, the nearly black of male worm.Feeler hammer shape portion the 1st, have between Section 2 embedding every.Face amount is slightly to epistome edge lift, and lateral margin dashes forward into semicircle shape, with the distance of eyes-affinity be the 2-3 of ommatidium diameter doubly; Female worm face amount is flat or slightly recessed, smooth surface, and punctum is fine and closely woven well-balanced, and fine hair is intensive, and antermarginal fine hair is longer; Male worm face amount is wide and recessed, and punctum is coarse, and fine hair is short and thin, more unobtrusively.Bristle is about and reaches half eye distance most.
(2) pronotum: be about as wide 1.10-1.16 times, half lateral margin anyhow from base portion, and leading edge bends into wide semicircle.Leading edge is had an appointment 18 sawtooth.In the middle part of backboard top is positioned at or middle part is to the front, and forebody is lepidoma district, and knurl has more incises, row's nearly concentric circles arrangement clearly in 4-6, backboard leading edge raw lepidoma number more than 12, is connected, often occurs overlapping near dorsomeson with the adjacent lepidoma of row base portion of being everlasting.Backboard latter half is area punctate, and surface is mild and smooth, and punctum is slightly large and close.Backboard front portion table is longer by bristle, and with lepidoma proper alignment, rear portion bristle is comparatively dredged thin.
(3) elytrum: be about as wide 1.8-1.9 times, be about pronotum 1.7 double-length, 3/4 lateral margin anyhow from base portion, the blunt circle of elytrum end.Elytrum inclined-plane is comparatively steep, and punctum is comparatively large, and be arranged in rows and form punctum ditch, wherein the 1st, the 2nd ditch punctum is more shallow, and except the 1st punctum ditch is except inclined-plane place is sagging, all the other punctum ditches do not sink.Between ditch, portion is level and smooth, and width is about 1 times or 1.5 times of punctum ditch; Between the 1st ditch, portion is very wide, and obviously upwards swells; Portion and 1 wide, level and smooth slightly in keratin between the 2nd ditch; 1st, the punctum between the 3rd ditch, portion respectively having 1 row extremely to dredge.Be positioned at the short and thin bristle of punctum ditch and extend to elytrum base portion, between ditch, bristle major part in portion's is arranged on the radix punctum ditch on elytrum inclined-plane comparatively thinly.Between male worm the 1st, the 2nd ditch, respectively there is 1 row small-particle in portion.
Have the insect of obvious form diagnostic characteristics, experienced insect qualification worker can identify result within the shortest time.The most scabrous problem of identification of morphology is sibling species or the difference allied species with nuance, especially for the assessor that qualification experience is enriched not, more difficult.Due to form fairly similar, the form diagnostic characteristics that thus often can not find.So need molecules authentication method to go further differentiation.
Current molecules authentication method can distinguish the species of different order, section accurately, but still faces huge challenge for the differentiation of the multiple near edge species belonged to together.The primer that the nearly edge species similar to sequence height design only amplification species is difficult; PCR is very sensitive to false negative, false positive results for tradition species specificity.
In view of this, special proposition the present invention.
Summary of the invention
The first object of the present invention is to provide a kind of primer identifying the tiny moth of walnut, and this primer specificity is strong, and susceptibility is high, reproducible, and whether can be very stable identify is the tiny moth of walnut.
The second object of the present invention is to provide a kind of test kit identifying the tiny moth of walnut, for the qualification of the tiny moth of walnut provides convenient.
The third object of the present invention is to provide a kind of method identifying the tiny moth of walnut, and the qualification of the method combining form and molecular biology identification technique, qualification accuracy rate is high, convenient and swift, for the tiny moth of walnut provides the taxonomic identification foundation of science.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
Identify a primer for the tiny moth of walnut, be made up of with the 4th primer pair the first primer pair, the second primer pair, three-primer;
Described first primer pair is made up of upstream primer P1-F and downstream primer P1-R, and the base sequence of described upstream primer P1-F is as shown in SEQIDNo.1; The base sequence of described downstream primer P1-R is as shown in SEQIDNo.2;
Described second primer pair is made up of upstream primer P2-F and downstream primer P2-R, and the base sequence of described upstream primer P2-F is as shown in SEQIDNo.3; The base sequence of described downstream primer P2-R is as shown in SEQIDNo.4;
Described three-primer forms by upstream primer P3-F and downstream primer P3-R, and the base sequence of described upstream primer P3-F is as shown in SEQIDNo.5; The base sequence of described downstream primer P3-R is as shown in SEQIDNo.6;
Described 4th primer pair is made up of upstream primer P4-F and downstream primer P4-R, and the base sequence of described upstream primer P4-F is as shown in SEQIDNo.7; The base sequence of described downstream primer P4-R is as shown in SEQIDNo.8.
The primer of the tiny moth of qualification walnut provided by the invention, according to biosystem biology, in conjunction with the known array of the nearly edge species of the tiny moth of walnut, obtained by the multiple authentication of standard substance, this primer specificity is strong, susceptibility is high, reproducible, can identify whether be the tiny moth of walnut very stable, fast.
Present invention also offers a kind of test kit identifying the tiny moth of walnut, containing the first primer pair described in claim 1, the second primer pair, three-primer to and the 4th primer pair at least one primer pair.
As: in certain embodiments, the test kit of qualification walnut tiny moth, can contain the first primer pair, the second primer pair, three-primer to and the 4th primer pair in any one; In certain embodiments, the test kit of qualification walnut tiny moth, can contain the first primer pair, the second primer pair, three-primer to and the 4th primer pair in any two; In certain embodiments, the test kit of qualification walnut tiny moth, can contain the first primer pair, the second primer pair, three-primer to and the 4th primer pair in wantonly three; In certain embodiments, the test kit of qualification walnut tiny moth, containing the first primer pair, the second primer pair, three-primer to and the 4th primer pair.
For the ease of application, preferably, described test kit is also containing Taq enzyme, dNTPs and PCRbuffer.
Present invention also offers a kind of method identifying the tiny moth of walnut, molecular biology identification is carried out to detected sample; Described molecular biology identification carries out pcr amplification with the first primer pair in claim 1, the second primer pair, three-primer to the genome extract of the 4th primer pair to detected sample, then detects pcr amplification product.
Current molecules authentication method can distinguish the species of different order, section accurately, but still faces huge challenge for the differentiation of the multiple near edge species belonged to together.Present invention improves over traditional species specificity PCR, the medium Auele Specific Primer of the many covers of design, determines the identity of species by the result comprehensively analyzing several PCR.The multiple PCR set up by medium Auele Specific Primer are called assembly PCR.The advantage of the more traditional species specificity PCR of assembly PCR method that the present invention sets up is:
(1) primer that similar to sequence height nearly edge species design only amplification species is difficult, and assembly PCR requires the lower several species of primer amplification that namely allow to primer specificity, therefore, design of primers is relatively easy, the theory of this design primer, the selection space not only increasing primer reduces the difficulty of design of primers, and can make full use of the evolution difference of gene different zones, makes analysis more objective and accurate.
(2) traditional species specificity PCR is very sensitive to false negative, false positive results, and indivedual PCR result occurs that false negative or false positive results can't have the impact of essence on qualification result in assembly PCR, still can according to the doubtful identity of the result judgement sample of assembly PCR;
(3) assembly PCR make use of multiple object fragment simultaneously, from multiple angle reflection sample identity, higher than traditional species specificity PCR accuracy; When normal PCR differentiates species, required Auele Specific Primer quantity generally equals even to be greater than the quantity waiting to distinguish species, and owing to intersecting effect between each PCR in assembly PCR, it can differentiate multiple species with relatively few reaction.
A kind of method identifying the tiny moth of walnut provided by the invention, using the first primer pair, the second primer pair, three-primer to the 4th primer as primer, pcr amplification is carried out to the genome extract of detected sample, then pcr amplification product is detected, convenient and swift, reliability is high.
Further, before described molecular biology identification, first Morphological Identification is carried out to detected sample, after determining to meet the morphological specificity of the tiny moth of walnut, then carry out described molecular biology identification.
First adopt morphological method to carry out preliminary evaluation, its advantage has:
(1) error is little: Senile Mouse is directly perceived, and middle-chain is few, causes the possibility of personal errors minimum, as long as keep the integrity of insect specimen, different people can carry out repeated observation, can contrast if desired with type specimen, to reduce qualification mistake;
(2) find that insect numbers is huge, accumulation documents and materials are many: in the centuries of classification of insect research, acquire a large amount of samples, name a large amount of type species, the caste described up to now has more than 1,000,000 kinds, have accumulated a large amount of documents and materials simultaneously, cultivate the expert much having and enrich insect qualification experience;
(3) research is convenient: need instrument less, only need magnifying glass and microscope, simple to operate, the overwhelming majority is had to the insect of obvious form diagnostic characteristics, identification of morphology the most intuitively, the easiest;
(4) qualification cycle is short: to the common insect with having obvious form diagnostic characteristics, and experienced insect qualification worker can identify result within the shortest time.
Meanwhile, the present invention, in order to guarantee the accuracy to species identification further, distinguishes plesiomorphic nearly edge species, adopts molecular biology mirror method for distinguishing to be assisted.
Morphological Identification adopts the morphological specificity of the tiny moth of existing walnut to carry out.Concrete form identification mark is as follows:
The bodily form is small, health total length 1.5-1.9mm, and be about the wide 2.8-3.1 of body doubly, female polypide look yellowish-brown, the nearly black of male worm.Feeler hammer shape portion the 1st, have between Section 2 embedding every.Face amount is slightly to epistome edge lift, and lateral margin dashes forward into semicircle shape, with the distance of eyes-affinity be the 2-3 of ommatidium diameter doubly; Female worm face amount is flat or slightly recessed, smooth surface, and punctum is fine and closely woven well-balanced, and fine hair is intensive, and antermarginal fine hair is longer; Male worm face amount is wide and recessed, and punctum is coarse, and fine hair is short and thin, more unobtrusively.Bristle is about and reaches half eye distance most.
Pronotum: be about as wide 1.10-1.16 times, half lateral margin anyhow from base portion, and leading edge bends into wide semicircle.Leading edge is had an appointment 18 sawtooth.In the middle part of backboard top is positioned at or middle part is to the front, and forebody is lepidoma district, and knurl has more incises, row's nearly concentric circles arrangement clearly in 4-6, backboard leading edge raw lepidoma number more than 12, is connected, often occurs overlapping near dorsomeson with the adjacent lepidoma of row base portion of being everlasting.Backboard latter half is area punctate, and surface is mild and smooth, and punctum is slightly large and close.Backboard front portion table is longer by bristle, and with lepidoma proper alignment, rear portion bristle is comparatively dredged thin.
Elytrum: be about as wide 1.8-1.9 times, be about pronotum 1.7 double-length, 3/4 lateral margin anyhow from base portion, the blunt circle of elytrum end.Elytrum inclined-plane is comparatively steep, and punctum is comparatively large, and be arranged in rows and form punctum ditch, wherein the 1st, the 2nd ditch punctum is more shallow, and except the 1st punctum ditch is except inclined-plane place is sagging, all the other punctum ditches do not sink.Between ditch, portion is level and smooth, and width is about 1 times or 1.5 times of punctum ditch; Between the 1st ditch, portion is very wide, and obviously upwards swells; Portion and 1 wide, level and smooth slightly in keratin between the 2nd ditch; 1st, the punctum between the 3rd ditch, portion respectively having 1 row extremely to dredge.Be positioned at the short and thin bristle of punctum ditch and extend to elytrum base portion, between ditch, bristle major part in portion's is arranged on the radix punctum ditch on elytrum inclined-plane comparatively thinly.Between male worm the 1st, the 2nd ditch, respectively there is 1 row small-particle in portion.
First Morphological Identification is carried out to detected sample, with cost-saving; Then to the sample binding molecule biological assay technical evaluation that be not easy distinguish close especially with the tiny moth-eaten form of walnut, to distinguish, reduce the requirement to qualification worker, make qualification work more convenient easy.
In order to make qualification more convenient easy, and reduce the interference of extraneous factor to result, preferably, described first primer pair, the second primer pair, three-primer carry out the pcr amplification with the 4th primer pair simultaneously.Generally that the PCR system of each primer pair is carried out pcr amplification in same PCR instrument.
Preferably, the annealing temperature that described pcr amplification is used is 50 DEG C ± 1 DEG C.Through verification experimental verification, carry out pcr amplification with this annealing temperature, specificity is good.
Further, the volume of described PCR reaction system is 15-50 μ l.Preferably, the volume of described PCR reaction system is 20-25 μ l.
As, the volume of PCR system is 25 μ l, and its composition is as follows:
Wherein, if the PCR system of the first primer pair, then PrimerF is P1-F, PrimerR is P1-R; If the PCR system of the second primer pair, then PrimerF is P2-F, PrimerR is P2-R; If the PCR system that three-primer is right, then PrimerF is P3-F, PrimerR is P3-R; If the PCR system of the 4th primer pair, then PrimerF is P4-F, PrimerR is P4-R.
Wherein, the concentration of each primer can have variation by a small margin, as about 10% float all can obtain good pcr amplification effect.
In addition, correspondingly, the volume of PCR system can also be 15 μ l, 20 μ l, 30 μ l, 50 μ l etc., and correspondingly, each composition in system reduces accordingly or expands.
The genome extract of detected sample is that whole detected sample is extracted genome, extracting method conventionally or the commercially available prod method of recommending carry out.Because the tiny moth of walnut and resemblance thereof are all occur in groups, every group of individualities are all consistent, and therefore, of getting wherein detects, and can judge whether cluster individuality is the tiny moth of walnut.
Because detected sample individuality is smaller, extract the genomic content obtained limited, preferably, in described PCR reaction system, the content of the genome extract of detected sample is more than 50ng, is preferably 100-200ng.Carry out PCR detection using the genome extract of this content as template, obvious band can be obtained, thus judge.
Preferably, the program of described pcr amplification is: 94-95 DEG C of denaturation 3-5min, enters amplification cycles; Prior to 94-95 DEG C of sex change 28-30s in each circulation, after be cooled to 50 DEG C ± 1 DEG C annealing 30-35s, then be warming up to 72 DEG C and extend 45-50s, altogether 35-40 circulation; Finally keep 5-10min in 72 DEG C.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention adopts assembly PCR to require the several species of lower i.e. permission primer amplification to primer specificity, therefore, design of primers is relatively easy, the theory of this design primer, the selection space not only increasing primer reduces the difficulty of design of primers, and the evolution difference of gene different zones can be made full use of, make analysis more objective and accurate.
(2) the present invention is by analyzing species sibship, devises four pairs of primers, and these primer specificity are strong, and susceptibility is high, reproducible, can identify whether be the tiny moth of walnut, convenient and swift, reliability is high very stable, fast.
(3) the present invention first carries out Morphological Identification to species, accuracy is high, qualification cycle is short, convenient and easy, in order to guarantee the accuracy to species identification further, distinguishing plesiomorphic nearly edge species, adopting molecular biology mirror method for distinguishing to be assisted, qualification result reliability is high, is applicable to popular detection.
(4) present invention also defines some design parameters of PCR system, to make PCR result more reliable and stable.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is the PCR primer electrophoresis result figure of the tiny moth of walnut and PityophthoruslautusEichhoff in the embodiment of the present invention 1.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercially available acquisition.
Embodiment 1
One, get the white standard model of the tiny moth of walnut and its nearly edge species PityophthoruslautusEichhoff sample, carry out Morphological Identification, Morphological Identification is according to specific as follows:
The bodily form is small, health total length 1.5-1.9mm, and be about the wide 2.8-3.1 of body doubly, female polypide look yellowish-brown, the nearly black of male worm.Feeler hammer shape portion the 1st, have between Section 2 embedding every.Face amount is slightly to epistome edge lift, and lateral margin dashes forward into semicircle shape, with the distance of eyes-affinity be the 2-3 of ommatidium diameter doubly; Female worm face amount is flat or slightly recessed, smooth surface, and punctum is fine and closely woven well-balanced, and fine hair is intensive, and antermarginal fine hair is longer; Male worm face amount is wide and recessed, and punctum is coarse, and fine hair is short and thin, more unobtrusively.Bristle is about and reaches half eye distance most.
Pronotum: be about as wide 1.10-1.16 times, half lateral margin anyhow from base portion, and leading edge bends into wide semicircle.Leading edge is had an appointment 18 sawtooth.In the middle part of backboard top is positioned at or middle part is to the front, and forebody is lepidoma district, and knurl has more incises, row's nearly concentric circles arrangement clearly in 4-6, backboard leading edge raw lepidoma number more than 12, is connected, often occurs overlapping near dorsomeson with the adjacent lepidoma of row base portion of being everlasting.Backboard latter half is area punctate, and surface is mild and smooth, and punctum is slightly large and close.Backboard front portion table is longer by bristle, and with lepidoma proper alignment, rear portion bristle is comparatively dredged thin.
Elytrum: be about as wide 1.8-1.9 times, be about pronotum 1.7 double-length, 3/4 lateral margin anyhow from base portion, the blunt circle of elytrum end.Elytrum inclined-plane is comparatively steep, and punctum is comparatively large, and be arranged in rows and form punctum ditch, wherein the 1st, the 2nd ditch punctum is more shallow, and except the 1st punctum ditch is except inclined-plane place is sagging, all the other punctum ditches do not sink.Between ditch, portion is level and smooth, and width is about 1 times or 1.5 times of punctum ditch; Between the 1st ditch, portion is very wide, and obviously upwards swells; Portion and 1 wide, level and smooth slightly in keratin between the 2nd ditch; 1st, the punctum between the 3rd ditch, portion respectively having 1 row extremely to dredge.Be positioned at the short and thin bristle of punctum ditch and extend to elytrum base portion, between ditch, bristle major part in portion's is arranged on the radix punctum ditch on elytrum inclined-plane comparatively thinly.Between male worm the 1st, the 2nd ditch, respectively there is 1 row small-particle in portion.
Two, the white standard model of the tiny moth of walnut and its nearly edge species PityophthoruslautusEichhoff sample all meet above-mentioned morphological specificity, then carry out molecular biology identification.
1, testing sample genome is extracted
Extracting testing sample genome adopts Hangzhou new scape biological genome DNA small volume of reagent box to extract, and extraction step carries out with reference to product description, and concrete steps are as follows:
1) whole sample to be detected is carried out liquid nitrogen grinding;
2) 250 μ lBufferS1 are added;
3) isopyknic BufferS2 is added, mixing;
4) add 350 μ lBufferS3 to mix, the centrifugal 10min of 12000 × g;
5) draw supernatant and be transferred to preparation pipe (being placed in 2ml centrifuge tube), the centrifugal 1min of 12000 × g, abandons filtrate;
6) putting back centrifuge tube by preparing pipe, adding 500 μ lBufferW1, the centrifugal 1min of 12000 × g, abandons filtrate;
7) putting back centrifuge tube by preparing pipe, adding 700 μ lBufferW2, the centrifugal 1min of 12000 × g, abandons filtrate; Wash once with 700 μ lBufferW2 more in the same way, abandon filtrate;
8) put back in 2ml centrifuge tube by preparing pipe, the centrifugal 1min of 12000 × g;
9) put in the centrifuge tube of the 1.5ml of Hui Xin by preparing pipe, add 20 μ l deionized waters preparing periosteum central authorities, room temperature leaves standstill 1min.The centrifugal 1min of 12000 × g;
10) abandon purification column, filtrate is the genomic dna of wash-out.
The genomic dna concentration of the white standard model of the tiny moth of the walnut obtained is 134.73ng/ μ l; The genomic dna concentration of PityophthoruslautusEichhoff sample is 150ng/ μ l.
2, pcr amplification
1) four pairs of pcr amplification primers, base sequence is as follows:
P1-F：5'-AGAATTATTGGAGATGACC-3'
P1-R：5'-GTTAGGATGTATATTAATA-3'
P2-F：5'-TTTATAATACTGTAGTAAC-3'
P2-R：5'-AACTGATGAACCTTCATGAG-3'
P3-F：5'-ATTTTTGGAGCATGATCAG-3'
P3-R：5'-TCTCCTCCTCCAGCTGGGT-3'
P4-F：5'-CCTATTATAATAGGGGGAT-3'
P4-R：5'-TAATTGTTCTGGTTTTAT-3'
First primer pair, the second primer pair, three-primer are all configured to the concentration of 10 μMs to the upstream and downstream primer with the 4th primer pair.
2) PCR reaction system
3) program of pcr amplification
Denaturation 95 DEG C of 4min; 95 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 50s:35 circulation; Last 72 DEG C extend 10min.
4) pcr amplification product detected through gel electrophoresis
(1) a certain amount of agarose is taken, with the sepharose of 5 × TBE electrophoretic buffer preparation 2.0%;
(2) in well, add the mixed solution of pcr amplification product 5 μ l, loadingbuffer and dyestuff;
(3) switch on power, 120V, 20min;
(4), after electrophoresis, glue is placed in the clear JS-780 full automatic gel imaging analysis instrument of training, imaging.
Pcr amplification product detected through gel electrophoresis result as shown in Figure 1.
In Fig. 1, digital 1-4 is corresponding in turn to the pcr amplification product of the first primer pair to the 4th primer pair of the tiny moth-eaten standard substance of walnut; Numeral 5-8 is corresponding in turn to the pcr amplification product of the first primer pair to the 4th primer pair of PityophthoruslautusEichhoff sample.M is the D2000DNAMarker purchased from TIANGEN Biotech (Beijing) Co., Ltd..
As can be seen from Figure 1, standard substance four pairs of primers of the tiny moth of walnut all increase and obtain object band, and PityophthoruslautusEichhoff sample only has the second primer pair and the 4th primer pair amplifies to obtain corresponding object band.
The present invention has also carried out above-mentioned PCR checking to the standard substance of the tiny moth of walnut of multiple different institutes preservation with difference near edge species, and the standard substance result of the tiny moth of multiple walnut is all consistent with the standard substance result of the tiny moth of the walnut in Fig. 1; The band of appearance one entry that different nearly edge species have, the band of some appearance two entries, there is not object band in what have, its object band number consistent with the Characterization of the tiny moth of walnut.
In summary, pcr amplification is carried out to detected sample, if four pairs of primers can amplify object product band respectively, the positive (detecting sample is the tiny moth of walnut) can be judged to be; If four pairs of primers only amplify portion of product band or occur without any amplified band, be then judged to be feminine gender (detecting the tiny moth of the non-walnut of sample).
Embodiment 2
Change some parameters in embodiment 1, carry out pcr amplification to the tiny moth of standard substance walnut, concrete change is as follows:
1, the genome concentration of the tiny moth of standard substance walnut is 50ng/ μ l;
2, the genome concentration of the tiny moth of standard substance walnut is 200ng/ μ l;
3, the program of pcr amplification is: 94 DEG C of 5min; 94 DEG C of 30s, 49 DEG C of 30s, 72 DEG C of 45s, totally 40 circulations; 72 DEG C keep 5min;
4, the program of pcr amplification is: 95 DEG C of 3min; 95 DEG C of 28s, 51 DEG C of 35s, 72 DEG C of 50s, totally 38 circulations; 72 DEG C keep 8min;
Change above-mentioned four kinds respectively to the standard substance in embodiment 1, the result obtained is all consistent with embodiment 1 standard substance result.
Embodiment 3
Adopt the method identical with embodiment 1 to detect 10 samples to be detected, namely first carry out morphologic detection, after determining that it meets the morphological specificity of the tiny moth of walnut, then carry out molecular biology identification.
Finally obtain there are 4 in 10 samples to be detected for the tiny moth of walnut, namely four pairs of primers of PCR all increase and obtain object band; 6 is the tiny moth of non-walnut, and four pairs of primers of PCR all partly obtain object band.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. identify a primer for the tiny moth of walnut, it is characterized in that, be made up of with the 4th primer pair the first primer pair, the second primer pair, three-primer;
Described first primer pair is made up of upstream primer P1-F and downstream primer P1-R, and the base sequence of described upstream primer P1-F is as shown in SEQIDNo.1; The base sequence of described downstream primer P1-R is as shown in SEQIDNo.2;
Described second primer pair is made up of upstream primer P2-F and downstream primer P2-R, and the base sequence of described upstream primer P2-F is as shown in SEQIDNo.3; The base sequence of described downstream primer P2-R is as shown in SEQIDNo.4;
Described three-primer forms by upstream primer P3-F and downstream primer P3-R, and the base sequence of described upstream primer P3-F is as shown in SEQIDNo.5; The base sequence of described downstream primer P3-R is as shown in SEQIDNo.6;
Described 4th primer pair is made up of upstream primer P4-F and downstream primer P4-R, and the base sequence of described upstream primer P4-F is as shown in SEQIDNo.7; The base sequence of described downstream primer P4-R is as shown in SEQIDNo.8.
2. identify a test kit for the tiny moth of walnut, it is characterized in that, containing the first primer pair described in claim 1, the second primer pair, three-primer to and the 4th primer pair at least one primer pair.
3. test kit according to claim 2, is characterized in that, described test kit is also containing Taq enzyme, dNTPs and PCRbuffer.
4. identify a method for the tiny moth of walnut, it is characterized in that, molecular biology identification is carried out to detected sample; Described molecular biology identification carries out pcr amplification with the first primer pair in claim 1, the second primer pair, three-primer to the genome extract of the 4th primer pair to detected sample, then detects pcr amplification product.
5. the method for the tiny moth of qualification walnut according to claim 4, it is characterized in that, before described molecular biology identification, first Morphological Identification is carried out to detected sample, after determining to meet the morphological specificity of the tiny moth of walnut, then carry out described molecular biology identification.
6. the method for the tiny moth of qualification walnut according to claim 4, is characterized in that, described first primer pair, the second primer pair, three-primer carry out the pcr amplification with the 4th primer pair simultaneously.
7. the method for the tiny moth of qualification walnut according to claim 6, is characterized in that, described pcr amplification annealing temperature used is 50 DEG C ± 1 DEG C.
8. the method for the tiny moth of qualification walnut according to claim 7, is characterized in that, the volume of described PCR reaction system is 15-50 μ l, is preferably 20-25 μ l.
9. the method for the tiny moth of qualification walnut according to claim 8, is characterized in that, in described PCR reaction system, the content of the genome extract of detected sample is more than 50ng, is preferably 100-200ng.
10. the method for the tiny moth of qualification walnut according to claim 6, is characterized in that, the program of described pcr amplification is: 94-95 DEG C of denaturation 3-5min, enters amplification cycles; Prior to 94-95 DEG C of sex change 28-30s in each circulation, after be cooled to 50 DEG C ± 1 DEG C annealing 30-35s, then be warming up to 72 DEG C and extend 45-50s, altogether 35-40 circulation; Finally keep 5-10min in 72 DEG C.
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| CN111826450A (en) * | 2020-09-04 | 2020-10-27 | 中国热带农业科学院香料饮料研究所 | Specific SS-COI primer pair, identification kit and rapid identification method of coffee berry bark beetle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111500745A (en) * | 2020-04-29 | 2020-08-07 | 北京林业大学 | Long fragment amplification primer for eliminating interference of mitochondrion pseudogene of dendroctonus valens |
| CN111500745B (en) * | 2020-04-29 | 2022-04-26 | 北京林业大学 | Long fragment amplification primer for eliminating interference of mitochondrion pseudogene of dendroctonus valens |
| CN111826450A (en) * | 2020-09-04 | 2020-10-27 | 中国热带农业科学院香料饮料研究所 | Specific SS-COI primer pair, identification kit and rapid identification method of coffee berry bark beetle |
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