CN109468389A - A kind of molecular biology method and its special primer pair for identifying bollworm and oriental tobacco budworm larva - Google Patents
A kind of molecular biology method and its special primer pair for identifying bollworm and oriental tobacco budworm larva Download PDFInfo
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- CN109468389A CN109468389A CN201811599392.2A CN201811599392A CN109468389A CN 109468389 A CN109468389 A CN 109468389A CN 201811599392 A CN201811599392 A CN 201811599392A CN 109468389 A CN109468389 A CN 109468389A
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Abstract
The invention discloses a kind of molecular biology methods and its special primer pair for identifying bollworm and oriental tobacco budworm larva.The special primer pair includes the primer pair first being made of SEQ ID NO.1 and SEQ ID NO.2, the primer pair B being made of SEQ ID NO.3 and SEQ ID NO.4.The present invention is difficult to identify for bollworm and oriental tobacco budworm Larva Morpho. Logy, the status of scientific and effective control measure cannot be provided, a kind of scientific and effective, simple and easy to operate, accurate and inexpensive rapid identification method and its special primer pair are provided, it is significant in terms of plant quarantine, cultural control.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of molecule for identifying bollworm and oriental tobacco budworm larva
Biological method and its special primer pair.
Background technique
Tobacco is one of the mainstay of China's finance, oriental tobacco budworm (Helicoverpa assulta) and bollworm
It (H.armigera) is primary pest common on tobacco leaf production.The two is that sibling species occur for same area, belongs to Lepidoptera noctuid
Section.It is worth noting that, the two larva is all extremely similar in terms of morphology, behaviouristics and ecology, their identification is always
It is a difficult technical bottleneck in bollworm and oriental tobacco budworm research.Accurate species identification is species distribution investigation and kind mass-sending
The premise of raw dynamic studies, and carry out the basis of the researchs such as biology, integrated control in a deep going way.
The identification method of traditional bollworm and oriental tobacco budworm difference worm state depends on their morphological feature, but exists
Biggish limitation: (1) distinguishing characteristics between sibling species is few and unobvious, and conventional method be easy to cause wrong identification；(2) with biography
The method of system can not almost accomplish to identify the different biotypes of pest of the same race；(3) traditional identification method is difficult to provide for two
The identification of kind pest larva or incomplete individual.Therefore, the method for two kinds of sibling species of conventional identification is not only time-consuming and laborious, and normal acceptor
The interference of sight factor, easily obscures error.
With being gradually improved for the rapid development of molecular biology, especially round pcr, more and more molecular biology
Technology is applied to species identification and classification, but these study ununified molecular labeling, thus the scope of application is extremely limited.Closely
Nian Lai has researcher to propose using DNA bar code (Mitochondrial cytochrome c oxidase subunit I) gene order as two kinds
The method of insect identification, this method is then sequenced using the PCR amplification genetic fragment, by comparing sequence in the two segment
The difference of column identifies insect.However this method needs to carry out DNA sequencing, the step have led to the time consuming nature of qualification result and
High cost.It also there is no quicker, accurate, general and easy, inexpensive discrimination method both at home and abroad at present.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide the molecule of a kind of identification bollworm and oriental tobacco budworm larva is raw
Object method and its special primer pair may be implemented to the quick, accurate and inexpensive of cotton bollworm larvae and oriental tobacco budworm larva
Identification.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides the primer combination of a kind of identification cotton bollworm larvae and oriental tobacco budworm larva, described to draw
Object combination includes: primer pair first and/or primer pair B；
In the primer pair first, the nucleotide sequence of forward primer is as shown in SEQ ID NO.1, the nucleotide of reverse primer
Sequence is as shown in SEQ ID NO.2；
In the primer pair B, the nucleotide sequence of forward primer is as shown in SEQ ID NO.3, the nucleotide of reverse primer
Sequence is as shown in SEQ ID NO.4.
The second aspect of the present invention provides above-mentioned primer combination in the examination of preparation identification cotton bollworm larvae and oriental tobacco budworm larva
Application in agent box.
The third aspect of the present invention provides a kind of kit for identifying cotton bollworm larvae and oriental tobacco budworm larva, the reagent
Contain at least pair of primers in above-mentioned primer pair first and primer pair B in box.
Preferably, in mentioned reagent box, in the primer pair first, the molar ratio of forward primer and reverse primer is 1:1；
In the primer pair B, the molar ratio of forward primer and reverse primer is 1:1.
Further, also include in the kit: Taq polymerase, PCR buffer and deionized water.
The fourth aspect of the present invention provides the combination of above-mentioned primer and/or kit in following a1)-a12) in it is any described
Application；
A1) the product of preparation identification cotton bollworm larvae and oriental tobacco budworm larva；
A2 cotton bollworm larvae and oriental tobacco budworm larva) are identified；
A3) preparation identifies that larva to be measured is the product of cotton bollworm larvae or oriental tobacco budworm larva；
A4) identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva；
A5) the product of preparation identification oriental tobacco budworm larva；
A6 oriental tobacco budworm larva) is identified；
A7) preparation identify larva to be measured whether be oriental tobacco budworm larva product；
A8) identify whether larva to be measured is oriental tobacco budworm larva；
A9) the product of preparation identification cotton bollworm larvae；
A10 cotton bollworm larvae) is identified；
A11) preparation identify larva to be measured whether be cotton bollworm larvae product；
A12) identify whether larva to be measured is cotton bollworm larvae.
The fifth aspect of the present invention provides and a kind of identifies or assist to identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm
The method of larva, comprising the following steps:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using above-mentioned primer pair first；
If PCR amplification obtains the target fragment that size is 390bp, larva to be measured is or candidate is oriental tobacco budworm larva；
If the target fragment that PCR amplification is 390bp less than size, larva to be measured is or candidate is cotton bollworm larvae.
Preferably, the condition of the PCR amplification are as follows: 95 DEG C of initial denaturation 3min；94 DEG C of denaturation 1min, 48 DEG C of annealing 1min,
72 DEG C of extension 1min, 30 recycle, and extend 10min after last 72 DEG C.
Further, above-mentioned to identify or assist to identify larva to be measured for the method for cotton bollworm larvae or oriental tobacco budworm larva also
Include the steps that above-mentioned primer pair B is used to be verified；
The method of the verifying is as follows: less than size being the gene of the larva to be measured of the target fragment of 390bp with PCR amplification
Group DNA is template, carries out PCR amplification using above-mentioned primer pair B；If PCR amplification obtains the target fragment that size is 316bp,
Verifying larva to be measured is cotton bollworm larvae.
The sixth aspect of the present invention, provide it is a kind of identify or assist to identify larva to be measured whether be oriental tobacco budworm larva side
Method, comprising the following steps:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using above-mentioned primer pair first；
If PCR amplification obtains the target fragment that size is 390bp, larva to be measured is or candidate is oriental tobacco budworm larva；
If the target fragment that PCR amplification is 390bp less than size, larva to be measured is not or candidate is not oriental tobacco budworm children
Worm.
The seventh aspect of the present invention, provide it is a kind of identify or assist to identify larva to be measured whether be cotton bollworm larvae side
Method, comprising the following steps:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using above-mentioned primer pair B；
If PCR amplification obtains the target fragment that size is 316bp, larva to be measured is or candidate is cotton bollworm larvae；
If the target fragment that PCR amplification is 316bp less than size, larva to be measured is not or candidate is not bollworm children
Worm.
In the above method, the genomic DNA can be used in the prior art conventional genome DNA extracting reagent kit or
Phenol-chloroform method extracts.To avoid pollution, the cephalothorax of larva to be measured is preferably taken to carry out the extraction of genomic DNA.
End of each primer in PCR reaction system in the above method, in the primer pair first or the primer pair B
Concentration is 0.2pM (0.2pmol/ul).
In the above method, the size is the nucleotide sequence of the target fragment of 390bp as shown in SEQ ID NO.5；Institute
The nucleotide sequence for the target fragment that size is 316bp is stated as shown in SEQ ID NO.6.
The eighth aspect of the present invention provides a kind of for identifying the molecular labeling of cotton bollworm larvae and oriental tobacco budworm larva, institute
The nucleotide sequence of molecular labeling is stated as shown in SEQ ID NO.5 and/or SEQ ID NO.6.
The ninth aspect of the present invention provides above-mentioned molecular labeling in following b1)-b12) in any application；
B1) the product of preparation identification cotton bollworm larvae and oriental tobacco budworm larva；
B2 cotton bollworm larvae and oriental tobacco budworm larva) are identified；
B3) preparation identifies that larva to be measured is the product of cotton bollworm larvae or oriental tobacco budworm larva；
B4) identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva；
B5) the product of preparation identification oriental tobacco budworm larva；
B6 oriental tobacco budworm larva) is identified；
B7) preparation identify larva to be measured whether be oriental tobacco budworm larva product；
B8) identify whether larva to be measured is oriental tobacco budworm larva；
B9) the product of preparation identification cotton bollworm larvae；
B10 cotton bollworm larvae) is identified；
B11) preparation identify larva to be measured whether be cotton bollworm larvae product；
B12) identify whether larva to be measured is cotton bollworm larvae.
Beneficial effects of the present invention:
(1) the method for the present invention is accurate and reliable, and the bollworm and oriental tobacco budworm that can be precisely difficult to differentiate between to form are identified.
(2) the method for the present invention does not need to be sequenced, therefore appraisal cost is greatly saved；
(3) the method for the present invention is easy to operate, and qualification result and other molecular biology methods (about 2 can be obtained half a day
It time) it compares, substantially reduce qualification time；
(4) the method for the present invention is unobvious for form diagnostic characteristics or incomplete individual is equally applicable, have it is accurate it is quick,
The characteristics of facilitating operation.
Detailed description of the invention
Fig. 1: detection effect of the special primer (YQCBF1/R1) to oriental tobacco budworm.Wherein, YQ1-YQ10 respectively indicates number and is
The oriental tobacco budworm of 1-10.
Fig. 2: detection effect of the special primer (MLCBF1/R1) to bollworm.Wherein, ML1-ML10 respectively indicates number and is
The bollworm of 1-10.
Fig. 3: the detection effect of primer pair MLCOIF1/R1, MLCOIF2/R2, MLCOIF3/R3 to bollworm.Wherein,
ML17, ML18, ML19, ML20, ML21, ML22 and ML24 respectively indicate the cotton boll that number is 17,18,19,20,21,22 and 24
Worm.
Fig. 4: amplification of the special primer (YQCBF1/R1) to larva to be measured.Wherein, swimming lane 4,6,12,17,19,20,
21,22,23,24 be using the total DNA of oriental tobacco budworm as template, and oriental tobacco budworm special primer (YQCBF1/R1) is expanded
Product；Swimming lane 1,2,3,5,7,8,9,10,11,13,14,15,16,18 is using the total DNA of bollworm as template, and oriental tobacco budworm is special
The product that different primer (YQCBF1/R1) is expanded.
Fig. 5: expanding effect of the special primer (MLCBF1/R1) to larva to be measured.Wherein, swimming lane 4,6,12,17,19,20,
21,22,23,24 be using the total DNA of oriental tobacco budworm as template, and bollworm special primer (MLCBF1/R1) is expanded
Product；Swimming lane 1,2,3,5,7,8,9,10,11,13,14,15,16,18 is using the total DNA of bollworm as template, and bollworm is special
The product that different primer (MLCBF1/R1) is expanded.
Fig. 6: the sensitivity technique result of oriental tobacco budworm primer pair (YQCBF1/YQCBR1).Wherein, swimming lane YQ1 is dense
Degree is the DNA profiling of 8.5ng/ μ L；YQ1-1 is the DNA profiling that concentration is 0.6ng/ μ L；YQ2 is that concentration is 30.5ng/ μ L
DNA profiling；YQ2-1 is the DNA profiling that concentration is 2.6ng/ μ L.
Fig. 7: the sensitivity technique result of bollworm primer pair (MLCBF1/MLCBR1).Wherein, swimming lane ML1 is dense
Degree is the DNA profiling of 9.8ng/ μ L；ML1-1 is the DNA profiling that concentration is 0.8ng/ μ L；ML2 is the DNA that concentration is 3.9ng/ μ L
Template；ML2-1 is the DNA profiling that concentration is 0.1ng/ μ L.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part is introduced, bollworm and oriental tobacco budworm are the important pests on China crops.They
Lepidoptera, Noctuidae, Helicoverpa are belonged to, affiliation is extremely close.The two has similar form and feeding habit, they it
Between there are many identical host plant, be easy to obscure in production, interfere the accuracy of agricultural pests prediction.Especially
The similitude of cotton bollworm larvae and oriental tobacco budworm larva reaches the degree that human eye can not almost distinguish, therefore, how to identify cotton
Earworm larva and oriental tobacco budworm larva are always technical difficult point.
Based on this, the object of the present invention is to provide a kind of scientific and effective, simple and easy to operate, accurate and inexpensive quick mirror
The method for determining bollworm and oriental tobacco budworm larva.
As noted previously, as the affiliation of bollworm and oriental tobacco budworm is extremely close, the sequence similarity of contained gene is also very
It is high, it is difficult to find the molecular labeling that can be used for that taxonomic history is carried out to bollworm and oriental tobacco budworm.According to the target of existing report
Primers, then amplified fragments or too short or difference in length are unobvious, all cannot achieve to cotton bollworm larvae and oriental tobacco budworm
The quick identification of larva.
The present invention is found surprisingly that, oriental tobacco budworm, bollworm mitochondrial cytochrome B (CytB) gene order on deposit respectively
It, can be in the otherness nucleotide fragments that size is 390bp (shown in SEQ ID NO.5) and 316bp (shown in SEQ ID NO.6)
As the molecular labeling for identifying oriental tobacco budworm and bollworm.
Based on the molecular labeling of above-mentioned discovery, the present invention devises oriental tobacco budworm specific primer YQCBF1/YQCBR1 and cotton
Earworm specific primer MLCBF1/MLCBR1, to be identified for the molecular biosciences of oriental tobacco budworm larva and cotton bollworm larvae.
The primer sequence of design is as follows:
Oriental tobacco budworm primer sequence (primer pair first) is as follows:
YQCBF1:5 '-TTTTGAGGAGCTACTGTTAT-3 ' (SEQ ID NO.1)；
YQCBR1:5 '-TTGAATATGAACTGGTGTAA-3 ' (SEQ ID NO.2)；
Bollworm primer sequence (primer pair B) is as follows:
MLCBF1:5 '-TAAAATTATCTGGATCTCCT-3 ' (SEQ ID NO.3)；
MLCBR1:5 '-GCTATCCCATCATTAGGTGT-3 ' (SEQ ID NO.4).
Specific primer sequence designed by the invention expands sample to be detected, and amplified production is coagulated
Gel electrophoresis detection directly can identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva according to fragment length, without carrying out
Sequencing can directly obtain qualification result.
In practical applications, it can individually be identified larva to be measured for cotton bollworm larvae still according to the amplification of primer pair first
Oriental tobacco budworm larva: if PCR amplification obtains the target fragment that size is 390bp, larva to be measured is or candidate is oriental tobacco budworm larva；
If the target fragment that PCR amplification is 390bp less than size, larva to be measured is or candidate is cotton bollworm larvae.
It can also individually identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm children according to the amplification of primer pair B
Worm: if PCR amplification obtains the target fragment that size is 316bp, larva to be measured is or candidate is cotton bollworm larvae；If PCR expands
Increase the target fragment for being 316bp less than size, then larva to be measured is or candidate is oriental tobacco budworm larva.
The amplification of the amplification or primer pair B that can be combined with primer pair first identifies that larva to be measured is cotton jointly
Earworm larva or oriental tobacco budworm larva: if primer pair first PCR amplification obtains the target fragment that size is 390bp, and primer pair B
The target fragment that PCR amplification is 316bp less than size, then larva to be measured is or candidate is oriental tobacco budworm larva；If primer pair first PCR
Expand less than size be 390bp target fragment, and primer pair B PCR amplification obtain size be 316bp target fragment, then to
Survey larva is or candidate is cotton bollworm larvae.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.The experimental method that detailed conditions are not specified is said according to conventional methods or according to operation proposed by supplier
What bright book carried out.
Embodiment 1, the primer pair and its identification method for identifying cotton bollworm larvae and oriental tobacco budworm larva
One, the primer pair of cotton bollworm larvae and oriental tobacco budworm larva is identified
1, it is based on mitochondrial cytochrome B (CytB) gene
The present invention is based on the mitochondrial cytochrome gene orders of bollworm, oriental tobacco budworm to devise bollworm specific primer
MLCBF1/MLCBR1 and oriental tobacco budworm specific primer YQCBF1/YQCBR1, with point for cotton bollworm larvae and oriental tobacco budworm larva
Sub- bioassay.The primer sequence of design is as follows:
Oriental tobacco budworm primer sequence is as follows:
YQCBF1:5 '-TTTTGAGGAGCTACTGTTAT-3 ' (SEQ ID NO.1)；
YQCBR1:5 '-TTGAATATGAACTGGTGTAA-3 ' (SEQ ID NO.2)；
Bollworm primer sequence is as follows:
MLCBF1:5 '-TAAAATTATCTGGATCTCCT-3 ' (SEQ ID NO.3)；
MLCBR1:5 '-GCTATCCCATCATTAGGTGT-3 ' (SEQ ID NO.4).
2, it is based on mitochondrial cytochrome oxidase subunit 1 (COI) gene
The base that the present invention passes through the chondriogen Cytochrome oxidase subunit 1 (COI) of comparison bollworm and cabbage caterpillar
Because of sequence, COI sequence design according to bollworm multipair primer, primer sequence are as follows:
MLCOIF1:5 '-CGATAAATTAAAATATAATTATTACCAT-3 ' (SEQ ID NO.7)；
MLCOIR1:5 '-TCAATTACCAAATCCACCAA-3 ' (SEQ ID NO.8)；
MLCOIF2:5 '-ACGATTTTGACCATTACTATTATTATC-3 ' (SEQ ID NO.9)；
MLCOIR2:5 '-GAAGTTCCCACTATTCCTGC-3 ' (SEQ ID NO.10)；
MLCOIF3:5 '-ATTTCTTTTTTATCGATCCAGATGA-3 ' (SEQ ID NO.11)；
MLCOIR3:5 '-ATTAAAGATCCAGGATTACC-3 ' (SEQ ID NO.12).
Two, the method for bollworm and oriental tobacco budworm larva is identified
1, the identification result of the primer pair based on the design of mitochondrial cytochrome B (CytB) gene
(1) total DNA of oriental tobacco budworm is extracted using EasyPure Genomic DNA Kit kit.With the total of oriental tobacco budworm
DNA is template, carries out PCR amplification using YQCBF1/YQCBR1 primer pair, obtains pcr amplification product.
PCR reaction system is 50 μ L, wherein DNA profiling about 25ng, PCR buffer (TransGen Biotech,
China) 5 μ L, primer YQCBF1, primer YQCBR1, Taq polymerase (TransGen Biotech, China) add deionized water
It is adjusted to 50 μ L of final volume, system is covered with paraffin oil.The final concentration of two primer YQCBF1/YQCBR1 in the reaction system is
0.2pM (0.2pmol/ μ L), the final concentration of 0.08U/ μ L of Taq polymerase in the reaction system.Reaction is in BioRad thermal cycle
It is completed on instrument.
PCR reaction system is as follows: 95 DEG C of initial denaturation 3min；94 DEG C of denaturation 1min, 48 DEG C of annealing 1min, 72 DEG C extend
1min.Extend 10min after 30 circulations plus after 72 DEG C.
1% detected through gel electrophoresis pcr amplification product.As a result as shown in Figure 1.As can be seen from the figure: PCR amplification obtains greatly
The small purpose band (SEQ ID NO.5) for 390bp.
(2) total DNA of bollworm is extracted using EasyPure Genomic DNA Kit kit.With the total of bollworm
DNA is template, carries out PCR amplification using MLCBF1/MLCBR1 primer pair, obtains pcr amplification product.
PCR reaction system is 50 μ L, wherein DNA profiling about 25ng, PCR buffer 5 μ L, primer MLCBF1, primer
MLCBR1, Taq polymerase add deionized water to be adjusted to 50 μ L of final volume, cover system with paraffin oil.Two primer MLCBF1/
The final concentration of MLCBR1 in the reaction system is 0.2pM (0.2pmol/ μ L), the final concentration of Taq polymerase in the reaction system
For 0.08U/ μ L.Reaction is completed on BioRad thermal cycler.
PCR reaction system is as follows: 95 DEG C of initial denaturation 3min；94 DEG C of denaturation 1min, 45 DEG C of annealing 1min, 72 DEG C extend
1min.Extend 10min after 30 circulations plus after 72 DEG C.
1% detected through gel electrophoresis pcr amplification product.As a result as shown in Figure 2.As can be seen from the figure: PCR amplification obtains greatly
The small purpose band (SEQ ID NO.6) for 316bp.
2, the identification result of the primer pair based on the design of (COI) gene of mitochondrial cytochrome oxidase subunit 1
The total DNA of bollworm is extracted using EasyPure Genomic DNA Kit kit.It is with the total DNA of bollworm
Template is respectively adopted primer pair MLCOIF1/R1, MLCOIF2/R2, MLCOIF3/R3 and carries out PCR amplification, obtains PCR amplification production
Object.
1% detected through gel electrophoresis pcr amplification product.As a result as shown in Figure 3.As can be seen from the figure: all primer pairs are equal
Target fragment is not amplified.Analyzing reason may be since bollworm and oriental tobacco budworm COI sequence similarity are very high, according to COI
Perhaps amplified fragments are too short for the primer of sequence design, thus to the taxonomic history for being not used to bollworm and oriental tobacco budworm.
It is detected by said effect it is found that the primer pair MLCBF1/ based on the design of mitochondrial cytochrome B (CytB) gene
MLCBR1 and YQCBF1/YQCBR1 can effectively identify bollworm and oriental tobacco budworm.It in practical applications can as follows quickly
Identify cotton bollworm larvae and oriental tobacco budworm larva:
1) total DNA of larva to be measured is extracted；
2) with the total DNA template of step 1), PCR amplification is carried out using oriental tobacco budworm primer pair (YQCBF1/YQCBR1)；
If amplification obtains the target fragment that size is 390bp, larva to be measured is oriental tobacco budworm；
If expanding the target fragment for being 390bp less than size, larva to be measured is bollworm.
3) using bollworm primer pair (MLCBF1/MLCBR1) further verification step 2) result:
With the total DNA template of step 1), PCR amplification is carried out using bollworm primer pair (MLCBF1/MLCBR1)；
If amplification obtains the target fragment that size is 316bp, larva to be measured is cotton bollworm larvae；
If expanding the target fragment for being 316bp less than size, larva to be measured is oriental tobacco budworm larva.
The application of the identification method of embodiment 2, cotton bollworm larvae and oriental tobacco budworm larva
One, sample to be tested
24 larva individuals are selected at random as children to be measured in more than 200 a bollworms and oriental tobacco budworm larva mixing individual
Worm sample.Wherein, 10 larva individuals are oriental tobacco budworm larva individual, and 14 larva individuals are cotton bollworm larvae individual.
Two, identification method
1, the total DNA of larva sample to be measured is extracted, respectively using EasyPure Genomic DNA Kit kit to keep away
Exempt to pollute, the cephalothorax of bollworm or oriental tobacco budworm is only taken to extract DNA.
2, the total DNA obtained using step 1 is expanded as template using oriental tobacco budworm primer pair (YQCBF1/YQCBR1)
CytB genetic fragment.PCR amplification system and condition with the step of embodiment 1 two 1.
As a result as shown in Figure 4.10 oriental tobacco budworm larva individuals, which expand, obtains the target fragment that size is 390bp, and 14
A cotton bollworm larvae individual does not expand to obtain the target fragment that size is 390bp.
3, the total DNA obtained using step 1 is expanded as template using bollworm primer pair (MLCBF1/MLCBR1)
CytB genetic fragment.PCR amplification system and condition with the step of embodiment 1 two 2.
As a result as shown in Figure 5.10 oriental tobacco budworm larva individuals do not expand to obtain the target fragment that size is 316bp, and
14 cotton bollworm larvae individuals, which expand, obtains the target fragment that size is 316bp.
It is verified simultaneously with direct sequencing, qualification result of the invention is consistent with sequence verification result, and accuracy rate is
100%.
The above results show: the method for identification cotton bollworm larvae of the invention and oriental tobacco budworm larva is accurate, reliable and cost
It is low, it can Rapid identification cotton bollworm larvae and oriental tobacco budworm larva.
Embodiment 3, sensitivity technique
One, the sensitivity technique of oriental tobacco budworm primer pair (YQCBF1/YQCBR1)
Respectively with concentration for 8.5ng/ μ L (YQ1), 0.6ng/ μ L (YQ1-1), 30.5ng/ μ L (YQ2), 2.6ng/ μ L
(YQ2-1) oriental tobacco budworm genomic DNA carries out PCR amplification as DNA profiling, using YQCBF1/YQCBR1 primer pair, obtains
Pcr amplification product.Final concentration difference of oriental tobacco budworm genomic DNA template YQ1, YQ1-1, YQ2, the YQ2-1 in PCR reaction system
For 0.68ng/ μ L, 0.048ng/ μ L, 2.44ng/ μ L, 0.208ng/ μ L.
1% detected through gel electrophoresis pcr amplification product.As a result as shown in Figure 6.As can be seen from the figure: as the DNA in system
For the concentration of template down to 0.048ng/ μ L, corresponding to oriental tobacco budworm genomic DNA template original content is 0.6ng/ μ L (YQ1-1) Shi Yeke
It realizes and successfully detects.
Two, the sensitivity technique of bollworm primer pair (MLCBF1/MLCBR1)
Respectively with concentration for 9.8ng/ μ L (ML1), 0.8ng/ μ L (ML1-1), 3.9ng/ μ L (ML2), 0.1ng/ μ L (ML2-
1) cotton boll molitor genomic dna carries out PCR amplification as DNA profiling, using MLCBF1/MLCBR1 primer pair, obtains PCR amplification
Product.Final concentration of bollworm genomic DNA template ML1, ML1-1, ML2, the ML2-1 in PCR reaction system be respectively
0.784ng/μL、0.064ng/μL、0.312ng/μL、0.008ng/μL。
1% detected through gel electrophoresis pcr amplification product.As a result as shown in Figure 7.As can be seen from the figure: as the DNA in system
For the concentration of template down to 0.008ng/ μ L, corresponding to bollworm genomic DNA template original content is 0.1ng/ μ L (ML2-1) Shi Yeke
It realizes and successfully detects.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>a kind of molecular biology method and its special primer pair for identifying bollworm and oriental tobacco budworm larva
<130> 2018
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
ttttgaggag ctactgttat 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ttgaatatga actggtgtaa 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
taaaattatc tggatctcct 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
gctatcccat cattaggtgt 20
<210> 5
<211> 390
<212> DNA
<213>artificial sequence
<400> 5
ttttgaggag ctactgttat tacaaattta ttatcagcta ttccatcatt aggggtaaaa 60
attgtaactt gaatttgagg gggatttgcg gtagataatg caacattaac tcgattttat 120
actttccatt ttcttttacc atttattatt ttaataataa ctataatcca cttattattt 180
ctccatcaaa caggatcaaa taatccttta ggattaaata gaaatttaga taaaatccct 240
tttcatccat tttttacatt taaagatcta attggattta ttattttatt attcttatta 300
actattttaa ccttaactaa tccatattta ctaggagatc ctgataactt tattccagca 360
aatcctttag ttacaccagt tcatattcaa 390
<210> 6
<211> 316
<212> DNA
<213>artificial sequence
<400> 6
taaaattatc tggatctcct aataaatatg ggttagttaa agttaaaata gttaataaaa 60
ataataaaat aataaatcca attaagtctt taaatacaaa aaatggatgg aaagggattt 120
tatctaaatt tctatttaaa cctaaggggt tatttgatcc tgtttggtga agaaataata 180
aatgaattat agttattatt aaaataataa atggtaaaag aaaatgaaaa gtataaaatc 240
gagttaatgt tgcgttatct actgcaaacc cccctcaaat tcaagttaca atttttacac 300
ctaatgatgg gatagc 316
<210> 7
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tcaattacca aatccaccaa 20
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<400> 9
acgattttga ccattactat tattatc 27
<210> 10
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<212> DNA
<213>artificial sequence
<400> 10
gaagttccca ctattcctgc 20
<210> 11
<211> 25
<212> DNA
<213>artificial sequence
<400> 11
atttcttttt tatcgatcca gatga 25
<210> 12
<211> 20
<212> DNA
<213>artificial sequence
<400> 12
attaaagatc caggattacc 20
Claims (10)
1. the primer combination of a kind of identification cotton bollworm larvae and oriental tobacco budworm larva, which is characterized in that the primer combination includes: to draw
Object is to first and/or primer pair B；
In the primer pair first, the nucleotide sequence of forward primer is as shown in SEQ ID NO.1, the nucleotide sequence of reverse primer
As shown in SEQ ID NO.2；
In the primer pair B, the nucleotide sequence of forward primer is as shown in SEQ ID NO.3, the nucleotide sequence of reverse primer
As shown in SEQ ID NO.4.
2. primer combination answering in preparation identification cotton bollworm larvae and the kit of oriental tobacco budworm larva described in claim 1
With.
3. a kind of kit for identifying cotton bollworm larvae and oriental tobacco budworm larva, which is characterized in that contain in the kit and have the right
It is required that at least pair of primers in 1 in primer pair first and primer pair B.
4. kit according to claim 3, which is characterized in that also include in the kit: Taq polymerase, PCR
Buffer and deionized water.
5. primer described in claim 1 combination and/or kit as claimed in claim 3 are in following a1)-a12) in any institute
The application stated；
A1) the product of preparation identification cotton bollworm larvae and oriental tobacco budworm larva；
A2 cotton bollworm larvae and oriental tobacco budworm larva) are identified；
A3) preparation identifies that larva to be measured is the product of cotton bollworm larvae or oriental tobacco budworm larva；
A4) identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva；
A5) the product of preparation identification oriental tobacco budworm larva；
A6 oriental tobacco budworm larva) is identified；
A7) preparation identify larva to be measured whether be oriental tobacco budworm larva product；
A8) identify whether larva to be measured is oriental tobacco budworm larva；
A9) the product of preparation identification cotton bollworm larvae；
A10 cotton bollworm larvae) is identified；
A11) preparation identify larva to be measured whether be cotton bollworm larvae product；
A12) identify whether larva to be measured is cotton bollworm larvae.
6. a kind of method for identifying or assisting to identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva, which is characterized in that
The following steps are included:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using primer pair first described in claim 1；
If PCR amplification obtains the target fragment that size is 390bp, larva to be measured is or candidate is oriental tobacco budworm larva；
If the target fragment that PCR amplification is 390bp less than size, larva to be measured is or candidate is cotton bollworm larvae；
Preferably, the method also includes using primer pair B described in claim 1 to be verified；
The method of the verifying is as follows: less than size being the genome of the larva to be measured of the target fragment of 390bp with PCR amplification
DNA is template, carries out PCR amplification using above-mentioned primer pair B；If PCR amplification obtains the target fragment that size is 316bp, test
Demonstrate,proving larva to be measured is cotton bollworm larvae.
7. it is a kind of identify or assist to identify larva to be measured whether be oriental tobacco budworm larva method, which is characterized in that including following step
It is rapid:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using primer pair first described in claim 1；
If PCR amplification obtains the target fragment that size is 390bp, larva to be measured is or candidate is oriental tobacco budworm larva；
If the target fragment that PCR amplification is 390bp less than size, larva to be measured is not or candidate is not oriental tobacco budworm larva.
8. it is a kind of identify or assist to identify larva to be measured whether be cotton bollworm larvae method, comprising the following steps:
Using the genomic DNA of larva to be measured as template, PCR amplification is carried out using primer pair B described in claim 1；
If PCR amplification obtains the target fragment that size is 316bp, larva to be measured is or candidate is cotton bollworm larvae；
If the target fragment that PCR amplification is 316bp less than size, larva to be measured is not or candidate is not cotton bollworm larvae.
9. a kind of for identifying the molecular labeling of cotton bollworm larvae and oriental tobacco budworm larva, which is characterized in that the molecular labeling
Nucleotide sequence is as shown in SEQ ID NO.5 and/or SEQ ID NO.6.
10. molecular labeling as claimed in claim 9 is in following b1)-b12) in any application；
B1) the product of preparation identification cotton bollworm larvae and oriental tobacco budworm larva；
B2 cotton bollworm larvae and oriental tobacco budworm larva) are identified；
B3) preparation identifies that larva to be measured is the product of cotton bollworm larvae or oriental tobacco budworm larva；
B4) identify that larva to be measured is cotton bollworm larvae or oriental tobacco budworm larva；
B5) the product of preparation identification oriental tobacco budworm larva；
B6 oriental tobacco budworm larva) is identified；
B7) preparation identify larva to be measured whether be oriental tobacco budworm larva product；
B8) identify whether larva to be measured is oriental tobacco budworm larva；
B9) the product of preparation identification cotton bollworm larvae；
B10 cotton bollworm larvae) is identified；
B11) preparation identify larva to be measured whether be cotton bollworm larvae product；
B12) identify whether larva to be measured is cotton bollworm larvae.
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| Application Number | Priority Date | Filing Date | Title |
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|---|---|---|---|
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134599A (en) * | 2010-12-28 | 2011-07-27 | 红云红河烟草(集团)有限责任公司 | Molecular biology method for quickly identifying Heliothis armigera and Helicoverpa assulta |
-
2018
- 2018-12-26 CN CN201811599392.2A patent/CN109468389B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134599A (en) * | 2010-12-28 | 2011-07-27 | 红云红河烟草(集团)有限责任公司 | Molecular biology method for quickly identifying Heliothis armigera and Helicoverpa assulta |
Non-Patent Citations (2)
| Title |
|---|
| G.T. BEHERE等: "Molecular markers to discriminate among gour pest species of helicoverpa", 《BULLETIN OF ENTOMOLOGICAL RESEARCH》 * |
| 李艳梅等: "AFLP分子标记技术在昆虫学研究中的应用及展望", 《植物保护》 * |
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