CN105505942B - A kind of specific gene and its method for identifying molecules of peacefulness pincers midge - Google Patents
A kind of specific gene and its method for identifying molecules of peacefulness pincers midge Download PDFInfo
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- CN105505942B CN105505942B CN201610011253.8A CN201610011253A CN105505942B CN 105505942 B CN105505942 B CN 105505942B CN 201610011253 A CN201610011253 A CN 201610011253A CN 105505942 B CN105505942 B CN 105505942B
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention discloses the specific gene and its method for identifying molecules of a kind of peaceful pincers midge, it is characterised in that the 28S rDNA gene order that the pincers midge is obtained using molecular biology method carries out Identification of Species to the pincers midge by the comparison of gene order similitude.Peacefulness pincers midge method for identifying molecules provided by the invention, is advantageously implemented peaceful pincers midge and accurately and rapidly identifies.
Description
Technical field
The present invention relates to species identification fields, and in particular to the special gene sequence of peaceful pincers midge and its Molecular Identification side
Method.
Background technique
Midge class is the microminiature insect in Diptera, is commonly called as small sting.Currently, being relied primarily on to the identification of midge class experienced
Taxology expert identify that morphological feature is realized, once encountering sibling species and morphological feature is grasped not the full Specimen identification
It is very intractable；Moreover, need to complete by production slide sample to the Morphological Identification of blood sucking midge, it is complex for operation step, the period is long,
Difficulty is big, therefore, needs to seek a kind of new method, to make up the defect of conventional sorting methods.
Molecular Identification technology is to illustrate the difference between species so that the analysis of hereditary material DNA sequence is foundation, from molecular water
Quickly and accurately identify species on flat.It is the product that molecular biology, computer science are combined with traditional taxonomy, is made
For a kind of brand-new taxology technology, the defect of traditional form identification is greatly compensated for, more and more biologists have been caused
Attention.In recent years, with the foundation of the determined dna sequence method on the basis PCR and being widely used, rDNA
(rDNA) it is gradually taken seriously with the genes such as mitochondrial DNA (mtDNA), and is answered extensively in the Molecular Identification of blood sucking midge
With.The technology is proved each other with traditional classification method, can not only solve the problems such as sample is incomplete in identification, but also can be real
The Rapid identification of existing blood sucking midge.Peacefulness pincers midge special gene sequence provided by the invention is advantageously implemented the quick mirror of peaceful pincers midge
It is fixed, shorten qualification time.
Summary of the invention
The purpose of the present invention is to provide the special gene sequences and its method for identifying molecules of a kind of peaceful pincers midge.By dividing
Sub- biological method obtain peaceful pincers midge (Forcipomyia anningensis) specific gene -28S rDNA gene order,
By the comparison of gene order similitude, peaceful pincers midge can be accurately and rapidly identified.
To achieve the above object, the present invention is achieved through the following technical solutions:
Using small insects minim DNA method for preparing template, by improved PCR reaction condition, by the primer synthesized
The 28S rDNA gene of peaceful pincers midge is expanded, PCR product sequence is sent to be measured by professional biotech firm.Sequencing result passes through manual school
To, sequence assembly, and Blast similarity searching is carried out in NCBI, it is ensured that gained sequence is target sequence.It is of the present invention
Peaceful pincers midge specific gene, be 28S rDNA gene, the 28S rDNA gene order of the pincers midge such as SEQ ID No.1 institute
Show (without primer):
The PCR primer of peacefulness pincers midge 28S rDNA gene of the present invention, it is characterised in that PCR primer sequence are as follows:
5 ' TCCGTAACTTCGGGATAAGGATT 3 ' of forward primer
5 ' TGTACCGCCCCAGTCAAACT 3 ' of reverse primer.
The method for identifying molecules of peacefulness pincers midge of the present invention, comprising:
1) the separation and Extraction DNA from insect tissue to be measured；
2) using the DNA as template, the primer of use are as follows: 5 ' TCCGTAACTTCGGGATAAGGATT 3 ' of forward primer, instead
To 5 ' TGTACCGCCCCAGTCAAACT 3 ' of primer, the 28S rDNA gene of the insect is gone out by polymerase chain reaction amplification；
3) the 28S rDNA gene agarose electrophoresis point for then taking the polymerase chain reaction amplification of appropriate step 2 to go out
From, observed under ultraviolet lamp after ethidium bromide staining, according to the size of amplified production determine result.If can correspondingly specificity expand
Increase the band that size out is about 819 bp, biotech firm is sent to be sequenced；
4) according to sequencing result, if the similitude of target gene sequence and SEQ ID No.1 98% or more, can be sentenced
The test serum of breaking is peaceful pincers midge.
The Identification of Species of peaceful pincers midge is realized by Morphological Identification, and is checked through authoritative expert, to guarantee to finish
The reliability of fruit.
The primer is synthesized according to document, 5 ' TCCGTAACTTCGGGATAAGGATT 3 ' of forward primer, reverse primer 5 '
TGTACCGCCCCAGTCAAACT 3’。
The present invention can detect amplification with agarose gel electrophoresis detection method.
Advantages of the present invention are as follows:
1) present invention is combined using traditional Morphological Identification with molecular classification method, is identified the pincers midge,
It can ensure that the accuracy and reliability of species identification.
2) compared with traditional Morphological Identification method, the peaceful pincers midge method for identifying molecules that the present invention establishes can effectively contract
The qualification time of short peacefulness pincers midge.
Detailed description of the invention
Fig. 1 is peaceful pincers midge 28S rDNA gene PCR amplification electrophoretic identification.Number is described as follows: swimming lane 1-2 is
The 28S rDNA gene of peaceful pincers midge female adult, the band that detection size is about 819 bp.M is the DNA marker of DL 2000.
Fig. 2 is unknown pincers midge female adult 28S rDNA gene PCR amplification electrophoretic identification.Number is described as follows: swimming lane 1
For the 28S rDNA gene of unknown pincers midge female adult, the band that size is about 819 bp is detected.M is the DNA marker of DL 2000.
Specific embodiment
The peaceful pincers midge of embodiment 1 (Forcipomyia anningensis) 28S rDNA gene order acquisition
1, the acquisition of peaceful pincers midge sample
Peaceful pincers midge is the sample that field acquisition obtains, and is directly stored in 95% alcohol after chilled execution.Sample is made
It is identified after slide through authoritative expert, it is ensured that the accuracy of qualification result.Before preparation of specimen, picking peacefulness pincers midge chest end and abdomen
The former sections in portion are placed in 95% alcohol, are extracted for DNA.
2, prepared by DNA profiling
Peacefulness pincers midge DNA(Wen Lizhang is extracted using small insects trace DNA templet preparation method and edits entomology research
Method and the Beijing technology introduction [M]: Science Press, 2010. pp278-286), the DNA sample of extraction is stored in -20 DEG C
It is spare.
3, primer synthesizes
The present embodiment the primer is as follows:
5 ' TCCGTAACTTCGGGATAAGGATT 3 ' of forward primer
5 ' TGTACCGCCCCAGTCAAACT 3 ' of reverse primer.
4, PCR amplification
The PCR reaction system of the present embodiment is as shown in table 1.
The peaceful pincers midge 28S rDNA gene PCR reaction system (50uL system) of table 1
| Ingredient | Volume (uL) |
| Template | 2 |
| Primer 1 | 1 |
| Primer 2 | 1 |
| 10 × buffer | 5 |
| dNTPs | 1 |
| Taq archaeal dna polymerase | 2.5 |
| ddH2O | 37.5 |
The response procedures of the present embodiment are as shown in table 2.
The peaceful pincers midge 28S rDNA gene PCR response procedures of table 2
| Step | Reaction temperature (DEG C) | Time | Recurring number |
| Initial denaturation | 94 | 3 min | — |
| Denaturation | 94 | 45 s | 35 |
| Annealing | 48 | 45 s | 35 |
| Extend | 72 | 60 s | 35 |
| Extend | 72 | 7 min | — |
PCR product is saved backup in 4 DEG C.
5, pcr amplification product detects
5 μ L pcr amplification products are taken, with 1% agarose gel electrophoresis (130V voltage, 35 min of electrophoresis).Ethidium bromide staining,
Gel imaging system detection, electrophorogram is referring to attached drawing 1.
6, gene sequencing
Choose the preferable pcr amplification product of 3-5 effect send and be sequenced after purification by biotech firm (be sequenced the primer and
PCR the primer is identical), it is 28S rDNA gene order-SEQ of peaceful pincers midge after the calibrated splicing of gained gene order
ID No.1。
The identification of the unknown pincers midge of embodiment 2
1, the acquisition and preservation of sample
Using net method is waved or lamp lures method to be acquired, directly it is stored in 95% alcohol after the chilled execution of the sample of harvest.
2, prepared by DNA profiling
Under anatomical lens or stereomicroscope, chooses a female pincers midge at random from collected midge class sample, use
Small insects trace DNA templet preparation method extracts pincers midge DNA(Wen Lizhang chief editor's entomology research method and technology introduction
Beijing [M]: Science Press, 2010. pp278-286), the DNA sample of extraction is saved backup in -20 DEG C, every part of sample one
A number.
3, the acquisition of objective gene sequence
The synthesis of 3.1 primers
Using the unknown pincers midge DNA of acquisition as template, synthetic primer, the primer sequence is as follows:
5 ' TCCGTAACTTCGGGATAAGGATT 3 ' of forward primer
5 ' TGTACCGCCCCAGTCAAACT 3 ' of reverse primer.
3.2 PCR amplification
The PCR reaction system of the present embodiment is as shown in table 3.
The unknown pincers midge 28S rDNA gene PCR reaction system (50uL system) of table 3
| Ingredient | Volume (uL) |
| Template | 2 |
| Primer 1 | 1 |
| Primer 2 | 1 |
| 10 × buffer | 5 |
| dNTPs | 1 |
| Taq archaeal dna polymerase | 2.5 |
| ddH2O | 37.5 |
The response procedures of the present embodiment are as shown in table 4.
The unknown pincers midge 28S rDNA gene PCR response procedures of table 4
| Step | Reaction temperature (DEG C) | Time | Recurring number |
| Initial denaturation | 94 | 3 min | — |
| Denaturation | 94 | 45 s | 35 |
| Annealing | 48 | 45 s | 35 |
| Extend | 72 | 60 s | 35 |
| Extend | 72 | 7 min | — |
PCR product is saved backup in 4 DEG C.
The detection of 3.3 pcr amplification products and gene sequencing
5 μ L pcr amplification products are taken, with 1% agarose gel electrophoresis (130V voltage, 35 min of electrophoresis).Ethidium bromide dye
Color.Gel imaging system detection, electrophorogram is referring to attached drawing 2.Find out that the unknown pincers midge of embodiment 2 also can specificity by attached drawing 2
Amplify the product that size is about 819 bp, send biotech firm to be sequenced the product that size is about 819 bp, as the result is shown with
The similitude of gene order SEQ ID NO. 1 is 99.9%, thus can determine that the unknown pincers midge for peaceful pincers midge.
Claims (2)
1. a kind of peacefulness pincers midge specific gene, which is characterized in that the gene is 28S rDNA gene, is gene as described below
Sequence SEQ ID No.1:
。
2. a kind of method for identifying molecules of peacefulness pincers midge, comprising:
1) the separation and Extraction DNA from insect tissue to be measured；
2) using the DNA as template, to the primer of 28S rDNA gene use are as follows: 5 ' TCCGTAACTTCGGGATAAGGA of forward primer
TT 3 ', 5 ' TGTACCGCCCCAGTCAAACT 3 ' of reverse primer go out peaceful pincers midge 28S by polymerase chain reaction amplification
RDNA gene；
3) the 28S rDNA gene for then taking the polymerase chain reaction amplification of appropriate step 2 to go out is separated with agarose electrophoresis, is passed through
It observes, is determined according to the size of amplified production as a result, if energy specific amplification goes out size under ultraviolet lamp after ethidium bromide staining
For the band of 819 bp, biotech firm is sent to be sequenced；
4) according to sequencing result, if accordingly 28S rDNA gene order and the similitude of SEQ ID No.1 are 98% or more, i.e.,
It can determine whether the test serum for peaceful pincers midge.
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| CN108486121B (en) * | 2018-01-25 | 2020-02-11 | 遵义医学院 | Specific DNA sequence of Cuicoides spinuloides and molecular identification method thereof |
| CN107988232A (en) * | 2018-01-25 | 2018-05-04 | 遵义医学院 | The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge |
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Non-Patent Citations (6)
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| GenBank.Atrichopogon sp.MAB-2008 28S ribosomal RNA gene,partial sequence |
| Genbank:FJ040575.1.《NCBI》.2008, |
| Phylogenetics and temporal diversification of the earliest true flies (Insecta: Diptera) based on multiple nuclear genes;Matthew A,et al;《Systematic Entomology》;20081001;第33卷(第4期);摘要、补充数据、图2 |
| 云南省安宁蠓类一新种及一新记录（双翅目：蠓科）;余静等;《中国媒介生物学及控制杂志》;20150812;2摘要、说明书第499页左栏 |
| 吸血蠓分子分类研究进展;王飞鹏等;《中国人兽共患病学报》;20121215;第28卷(第12期);全文 |
| 福建省重要口岸吸血蠓类本底调查初报;张述铿等;《中国国境卫生检疫杂志》;20090425;第32卷(第2期);全文 |
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