CN109735554A - It is a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata - Google Patents
It is a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata Download PDFInfo
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- CN109735554A CN109735554A CN201910105087.1A CN201910105087A CN109735554A CN 109735554 A CN109735554 A CN 109735554A CN 201910105087 A CN201910105087 A CN 201910105087A CN 109735554 A CN109735554 A CN 109735554A
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- 241000351615 Triatoma rubrofasciata Species 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 24
- 101150087323 COI gene Proteins 0.000 title claims abstract description 14
- 238000012408 PCR amplification Methods 0.000 claims abstract description 23
- 239000002773 nucleotide Substances 0.000 claims abstract description 15
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 15
- 230000003321 amplification Effects 0.000 claims abstract description 10
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Abstract
The invention discloses a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata, and the nucleotide sequence of the COI genetic fragment is as shown in SEQ ID NO:1.The method for identifying molecules includes the following steps: that S1. extracts sample gene to be tested group DNA；S2. using genomic DNA described in step S1 as template, PCR amplification is carried out with primer pair shown in NO:2~3 SEQ ID；S3. the pcr amplification product electrophoresis detection of appropriate step S2 is taken, if specific amplification goes out the band that size is about 439bp, which is sequenced；If S4. the similitude of the pcr amplification product of step S3 and SEQ ID NO.1 are 95% or more, that is, it can determine whether that sample to be tested is triatoma rubrofasciata.The professional classification knowledge that method for identifying molecules of the invention does not need traditional classification related fields simple and quick effectively can carry out Molecular Identification to triatoma rubrofasciata in molecular biology level；And low in cost, easy to operate, identification accuracy rate height.
Description
Technical field
The invention belongs to field of molecular biotechnology, more particularly, to a kind of for identifying the COI gene of triatoma rubrofasciata
And method for identifying molecules.
Background technique
Triatome bug (Triatomiane), the common name of Insecta Semiptera Heteroptera Reduviidae Triatominae insect are to propagate
The main media of American trypanosomiasis.American trypanosomiasis (American trypanosomiasis) is also known as american trypanosomiasis (Chagas
Disease), be by schizotrypanum cruzi (Trypanosoma cruzi) caused by one kind is chronic, systemic tropical parasitic parasitosis, slowly
The property phase is mainly shown as myocarditis.Since American trypanosomiasis the infected symptom similar with AIDS occurs at illness initial stage, very
Difficulty is noticeable and has incubation period many years, therefore also referred to as novel AIDS.
This cause of disease is mainly in America, especially common with Hispanic remote districts.However in the past ten years, this disease
Epidemiology has occurred that great change, and disease is gradually spread from Latin American to the whole world.Today, American trypanosomiasis is
Become a kind of emerging infectious disease in Europe, North America and the Pacific region (Japan, Australia, New Zealand etc.).?
China, there are mainly four types of the triatome bugs that can propagate schizotrypanum cruzi: triatoma rubrofasciata, Chinese triatome bug, particle triatome bug and horizontal wrinkle triatome bug, they
Coastal area is widely distributed in south China, and there are biggish potential risks for the invasion and propagation to American trypanosomiasis.Therefore, right
The precise Identification of triatoma rubrofasciata is the important foundation for controlling disease and the core premise of prevention and control of diseases.
But since triatome bug is many kinds of, Morphological Identification is more complicated, the limitation vulnerable to the stage of development causes classification to be reflected
Fixed difficulty, and it is cumbersome, strongly professional, and the only veteran taxology expert of small part could effectively identify its kind
Class, therefore there is an urgent need to a kind of new methods for the identification of triatoma rubrofasciata, to make up the deficiency of traditional form classification method,
The efficiency and accuracy of classification are improved, and facilitates the investigation of potential medium, carries out scientific and effective anti-system, is China America
The prevention and control of trypanosomiasis provide theoretical foundation.
With the fast development of molecular biology and bioinformatics, using DNA sequence analysis as the Molecular Identification of foundation
As the common technology means of species identification, the defect of traditional taxonomy identification is greatly compensated for.DNA bar code technology (DNA
Barcoding) refer to in genome a segment standard, short DNA fragmentation identify a Molecular Identification new technology of species,
Get rid of the obstacle that traditional form identification method relies on protracted experience, it is easy to operate, high-efficient, using wide, can quickly, it is accurate
Carry out species identification.Standard target gene as DNA bar code must satisfy two conditions: first is that must have certain
Conservative designs and carries out large-scale PCR amplification convenient for universal primer；Second is that have enough variability, to distinguish difference
Species, especially sibling species.
However yet there are no the Molecular Identification means for triatoma rubrofasciata, if can be by Molecular Identification and traditional form
Classification method combines, and mutually proves, can not only solve that sample present in Morphological Identification is incomplete fubaritic to ask
Topic, and it is advantageously implemented quick, the precise Identification of triatoma rubrofasciata, shorten the species identification time.
Summary of the invention
The technical problem to be solved by the present invention is to for defect existing for existing triatoma rubrofasciata species identification aspect and not
Foot, provides a kind of for identifying the COI genetic fragment of triatoma rubrofasciata
A second object of the present invention is to provide a kind of method for identifying molecules of triatoma rubrofasciata.
Above-mentioned purpose of the invention is to give realization by the following technical programs:
It is a kind of for identifying the COI genetic fragment of triatoma rubrofasciata, the nucleotide sequence of the COI genetic fragment such as SEQ ID NO:
Shown in 1.
The research of the invention finds that COI genetic fragment shown in SEQ ID NO:1 has certain guard in triatoma rubrofasciata
Property, while there is also enough variability in different triatoma rubrofasciata samples sources, it can be used to identify triatoma rubrofasciata object
Kind identification.
Therefore, triatoma rubrofasciata COI genetic fragment described in SEQ ID NO:1 in identification triatoma rubrofasciata/or is preparing red tape cone
Application in stinkbug identification kit is also in the scope of the present invention.
It is a kind of for identifying the specificity amplification primer pair of triatoma rubrofasciata, the primer pair includes upstream primer F and downstream
Primer R, nucleotide sequence is successively as shown in NO:2~3 SEQ ID:
F:5 '-TGTAGAAAGAGGAGCGGGAA-3 ' (SEQ ID NO:2)
R:5 '-TCCGGTTTCCATGGCAATAA-3 ' (SEQ ID NO:3).
The present invention is also claimed the specificity amplification primer and reflects in identification triatoma rubrofasciata/or preparing triatoma rubrofasciata
Determine the application in kit.
A kind of method for identifying molecules of triatoma rubrofasciata, includes the following steps:
S1. sample gene to be tested group DNA is extracted；
S2. using genomic DNA described in step S1 as template, PCR amplification is carried out with primer pair shown in NO:2~3 SEQ ID；
S3. the pcr amplification product electrophoresis detection of appropriate step S2 is taken, if specific amplification goes out the band that size is about 439bp,
The pcr amplification product is sequenced；
If S4. the similitude of the pcr amplification product of step S3 and SEQ ID NO.1 are 95% or more, that is, it can determine whether that sample to be tested is
Triatoma rubrofasciata.
Preferably, the method for identifying molecules of above-mentioned triatoma rubrofasciata further includes the triatome bug that similitude in GenBank is greater than to 80%
COI gene order is compared with the step S3 sequence being sequenced, and constructs the phylogenetic tree based on COI nucleotide sequence,
Distinguish the triatome bug not belonged to.
Preferably, PCR amplification system described in step S2 is as follows: 50 μ L of PCR system, including 1 μ of sample to be tested DNA profiling
L, 1.1 × PCR mix 45 μ L, each 2 μ L of 10 μm of ol/L upstream and downstream primers.
Preferably, PCR amplification program described in step S2 are as follows: 98 DEG C of 2 min of initial denaturation；98 DEG C of 10 s of denaturation, 55 DEG C are moved back
Fire 30 s, 72 DEG C of 15 s of extension are recycled 35 times；72 DEG C are continued to extend 3 min, and final reaction is terminated at 4 DEG C.
The present invention also provides a kind of Molecular Identification kits of triatoma rubrofasciata, include specific amplification SEQ ID NO:1 institute
State the primer pair of nucleotide sequence.
Preferably, the primer pair includes upstream primer F and downstream primer R, and nucleotide sequence is successively such as SEQ ID
Shown in NO:2~3.
Preferably, the Molecular Identification kit of the triatoma rubrofasciata further includes reagent required for pcr amplification reaction.
Preferably, the Molecular Identification kit of the triatoma rubrofasciata further includes for extracting triatoma rubrofasciata genomic DNA
Reagent.
Compared with prior art, the invention has the following advantages:
The present invention provides a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata, does not need traditional classification phase
The professional classification knowledge in pass field simple and quick effectively can carry out molecule mirror to triatoma rubrofasciata in molecular biology level
It is fixed；And low in cost, easy to operate, identification accuracy rate height.
Detailed description of the invention
Fig. 1 is triatoma rubrofasciata COI gene PCR amplification electrophoretogram, and swimming lane 1 is blank control, and swimming lane 2~5 is from good fortune
The sample of ZhangZhou acquisition is built, swimming lane 6~8 is the sample acquired from Period In Maoming, detects the band that size is about 439bp, and M is
The DNA marker of DL 2000.
Fig. 2 is the systematic growth tree graph constructed based on COI nucleotide sequence and ortho position conflation algorithm (NJ).
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The acquisition of 1 triatoma rubrofasciata COI gene order of embodiment
(1) acquisition of triatome bug sample
It lures method to obtain 4 triatome bug samples from Zhangzhou, Fujian acquisition using cage, obtains 3 triatome bug samples from Period In Maoming acquisition, pass through
Taxology expert appraisal, determination are triatoma rubrofasciata.It is directly stored in 95% alcohol after the above-mentioned chilled execution of sample.Mark
Before this production, the foot of picking triatome bug is used for the extraction of genomic DNA.
(2) extraction of genomic DNA
Triatoma rubrofasciata genomic DNA is extracted using micro-example DNA extraction kit, the DNA sample of extraction is stored in -20 DEG C
It is spare.
(3) PCR amplification of specific gene COI gene
PCR amplification system uses 50 μ L systems, including 1 μ L, 1.1 × PCR mix of sample to be tested DNA profiling 45 μ L, 10 μ
Each 2 μ L of mol/L upstream and downstream primer.The specific base sequence of upstream and downstream primer is as shown in SEQ ID NO.2 and SEQ ID NO.3.
PCR amplification program are as follows: 98 DEG C of 2 min of initial denaturation, (98 DEG C of 10 s of denaturation, 55 DEG C of 30 s of annealing, 72 DEG C extend 15 and s) follow
Ring 35 times, 72 DEG C are continued to extend 3 min, and final reaction is terminated at 4 DEG C, obtains PCR product.
(4) cloning and sequencing
Obtained PCR product is separated with 1.5% agarose gel electrophoresis, observation is as a result, such as Fig. 1 institute under gel imaging system
Show only occur specific fragment in the sample of triatoma rubrofasciata, primer size is in 439bp or so, and blank control does not expand
Any band out illustrates that PCR result is reliable.Using PCR product Purification Kit product, then connect with pMD18-T carrier
It connects, recovers after conversion, by recovery bacterium solution even spread to LB culture medium, flat-plate inverted is placed in 37 DEG C of incubators and is trained overnight
It supports.For bacterial strain after bacterium colony PCR identification in 37 DEG C of overnight incubations of LB culture medium, the random multiple clones of picking send professional biology
Sequencing company is sequenced.
(5) sequence alignment and phylogenetic tree building
Sequencing result after check and correction by hand is subjected to Blast in NCBI, the results showed that corresponding COI gene order and SEQ ID
The similitude of NO.1 is 96%, therefore can determine whether that species to be measured are triatoma rubrofasciata.Similitude in GenBank is greater than to 80% cone
Stinkbug COI gene order is compared with the sequence that sequencing obtains, the system by MEGA software building based on COI nucleotide sequence
Development tree further analyzes the affiliation of triatoma rubrofasciata and other triatome bugs, such as Fig. 2 using Rhodnius species as outgroup
It is shown.As can be seen that the various triatome bugs of Triatoma flock together from the NJ tree of building, their affiliation is closer, and
The triatome bug for climbing Triatoma is distributed on another evolutionary branching, individually at branch, show with the triatome bug affiliation of Triatoma farther out, because
This, the triatome bug of different categories can be distinguished according to phylogenetic tree substantially.From the triatome bug that Zhangzhou, Fujian and Period In Maoming two places acquire
In in same branch, it is polymerized to one with triatoma rubrofasciata, also indicates that the triatome bug sample of this two places acquisition is triatoma rubrofasciata, with tradition
Morphological Identification result it is consistent.
Embodiment 2
1, the Molecular Identification kit of a kind of triatoma rubrofasciata, including specific PCR amplimer, the primer pair include that upstream is drawn
Object F and downstream primer R, nucleotide sequence is successively as shown in NO:2~3 SEQ ID:
F:5 '-TGTAGAAAGAGGAGCGGGAA-3 ' (SEQ ID NO:2)
R:5 '-TCCGGTTTCCATGGCAATAA-3 ' (SEQ ID NO:3)；
It further include 1.1 × PCR mix.
2, kit application method includes the following steps:
S1. sample gene to be tested group DNA is extracted；
S2. using genomic DNA described in step S1 as template, PCR amplification is carried out with primer pair shown in NO:2~3 SEQ ID；
S3. the pcr amplification product electrophoresis detection of appropriate step S2 is taken, if specific amplification goes out the band that size is about 439bp,
The pcr amplification product is sequenced；
If S4. the similitude of the pcr amplification product of step S3 and SEQ ID NO.1 are 95% or more, that is, it can determine whether that sample to be tested is
Triatoma rubrofasciata；The sequence that triatome bug COI gene order by similitude in GenBank greater than 80% and step S3 are sequenced simultaneously
It is compared, constructs the phylogenetic tree based on COI nucleotide sequence, distinguish the triatome bug not belonged to.
PCR amplification system described in step S2 is as follows: 50 μ L of PCR system, including sample to be tested DNA profiling 1 μ L, 1.1 ×
PCR mix 45 μ L, each 2 μ L of 10 μm of ol/L upstream and downstream primers.
PCR amplification program described in step S2 are as follows: 98 DEG C of 2 min of initial denaturation；98 DEG C of denaturation 10 s, 55 DEG C of 30 s of annealing,
72 DEG C of 15 s of extension are recycled 35 times；72 DEG C are continued to extend 3 min, and final reaction is terminated at 4 DEG C.
Although the present invention has been described in detail, it will be understood by those skilled in the art that in the technology model of present disclosure
In enclosing, the simple change or equivalence replacement of technical solution can be apparently carried out.However, it should be understood that the present invention remembers
The various aspects of load, each section of different specific embodiments and the various features enumerated can be combined or completely or partially exchange.
In above-mentioned each specific embodiment, those with reference to another embodiment embodiment can suitably with it is other
Embodiment combination, this is will be to understand by those skilled in the art.In addition, it will be understood to those of skill in the art that preceding
The description in face is only the preferable specific embodiment of the present invention, it is no intended to the limitation present invention.
Sequence table
<110>Zhongshan University
<120>a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 530
<212> DNA
<213>triatome bug (Triatomiane)
<400> 1
gaagatttgt agaaagagga gcgggaacag gatgaactgt atatccaccc ttatcaggaa 60
atattgccca tagtggtgca tccgtagatt taactatctt ttcactacat ttagcaggta 120
tttcttcaat cctaggagct gtaaatttta tttctacaat tattaacata cgtccctctg 180
gaatacaacc agaacgaatc cctctatttg tatgatcagt aggaattaca gccctcctcc 240
ttttacttag actccctgtt cttgcaggag ccattaccat actcttaact gatcgaaact 300
tcaatacctc attctttgat ccagcaggag ggggagaccc cattctatat caacatttat 360
tctgattctt tggccatccg gaagtctata ttcttatttt acccggattt ggtcttattt 420
cccatattat tgccatggaa accggaaaaa atgaagcatt tggaacttta ggaataattt 480
aggccatacc tagctattgg attattagga ttccattgta tgagctcatc 530
<210> 2
<211> 20
<212> DNA
<213>triatome bug (Triatomiane)
<400> 2
tgtagaaaga ggagcgggaa 20
<210> 3
<211> 20
<212> DNA
<213>triatome bug (Triatomiane)
<400> 3
tccggtttcc atggcaataa 20
Claims (10)
1. a kind of for identifying the COI genetic fragment of triatoma rubrofasciata, which is characterized in that the nucleotides sequence of the COI genetic fragment
Column are as shown in SEQ ID NO:1.
Triatoma rubrofasciata COI genetic fragment described in 2.SEQ ID NO:1 is in identification triatoma rubrofasciata/or is preparing triatoma rubrofasciata identification examination
Application in agent box.
3. a kind of for identifying the specificity amplification primer pair of triatoma rubrofasciata, which is characterized in that the primer pair includes that upstream is drawn
Object F and downstream primer R, nucleotide sequence is successively as shown in NO:2~3 SEQ ID.
4. specificity amplification primer described in claim 3 triatoma rubrofasciata/or is preparing triatoma rubrofasciata identification kit in identification
In application.
5. a kind of method for identifying molecules of triatoma rubrofasciata, which comprises the steps of:
S1. sample gene to be tested group DNA is extracted；
S2. using genomic DNA described in step S1 as template, PCR amplification is carried out with primer pair shown in NO:2~3 SEQ ID；
S3. the pcr amplification product electrophoresis detection of appropriate step S2 is taken, if specific amplification goes out the band that size is about 439bp,
The pcr amplification product is sequenced；
If S4. the similitude of the pcr amplification product of step S3 and SEQ ID NO.1 are 95% or more, that is, it can determine whether that sample to be tested is
Triatoma rubrofasciata.
6. method for identifying molecules according to claim 5, which is characterized in that further include that similitude in GenBank is big
Triatome bug COI gene order in 80% is compared with the step S3 sequence being sequenced, and constructs based on COI nucleotide sequence
Phylogenetic tree distinguishes the triatome bug not belonged to.
7. method for identifying molecules according to claim 5, which is characterized in that PCR amplification system described in step S2 is as follows:
50 μ L of PCR system, including 1 μ L, 1.1 × PCR mix of sample to be tested DNA profiling, 45 μ L, 10 μm of ol/L upstream and downstream primers
Each 2 μ L.
8. method for identifying molecules according to claim 5, which is characterized in that PCR amplification program described in step S2 are as follows: 98
DEG C 2 min of initial denaturation；98 DEG C of denaturation 10 s, 55 DEG C of annealing 30 s, 72 DEG C of 15 s of extension are recycled 35 times；72 DEG C after reneing
3 min are stretched, final reaction is terminated at 4 DEG C.
9. a kind of Molecular Identification kit of triatoma rubrofasciata, which is characterized in that include core described in specific amplification SEQ ID NO:1
The primer pair of nucleotide sequence.
10. Molecular Identification kit according to claim 8, which is characterized in that the primer pair include upstream primer F and
Downstream primer R, nucleotide sequence is successively as shown in NO:2~3 SEQ ID.
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| JAMES S PATTERSON等: "Phylogenetic multi-locus codon models and molecular clocks reveal the monophyly of haematophagous reduviid bugs and their evolution at the formation of South America", 《MOLECULAR PHYLOGENETICS AND EVOLUTION》 * |
| LYMAN DF等: "Mitochondrial DNA sequence variation among triatomine vectors of Chagas" disease", 《THE AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE》 * |
| MICHAEL W. GAUNT等: "An Insect Molecular Clock Dates the Origin of the Insects and Accords with Palaeontological and Biogeographic Landmarks", 《MOLECULAR BIOLOGY AND EVOLUTION》 * |
| 卜云等: "基于线粒体COⅡ基因的中国蝽科分子系统学研究(半翅目,异翅亚目)", 《动物分类学报》 * |
| 姚亚林等: "基于COI基因序列片段的柔突叶蝉属部分近缘种分子鉴定与系统发育", 《山地农业生物学报》 * |
| 无: "ACCESSION GQ869655.1，Triatoma rubrofasciata isolate 17RB cytochrome oxidase subunit I-like (COI) gene, partial sequence; mitochondrial", 《GENBANK》 * |
| 曹理想: "动物共生放线菌研究展望", 《微生物学通报》 * |
| 李淑娟: "中国猎蝽科昆虫分子分类初探", 《万方学位论文》 * |
| 李聪等: "虫媒病的生物防制", 《热带医学杂志》 * |
| 谢红卫等: "线粒体RNA编辑的研究进展", 《作物研究》 * |
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