CN104878109B - Detection primer of culex wolbachia, detection method and detection kit thereof - Google Patents
Detection primer of culex wolbachia, detection method and detection kit thereof Download PDFInfo
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- CN104878109B CN104878109B CN201510318826.7A CN201510318826A CN104878109B CN 104878109 B CN104878109 B CN 104878109B CN 201510318826 A CN201510318826 A CN 201510318826A CN 104878109 B CN104878109 B CN 104878109B
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- 241000604961 Wolbachia Species 0.000 title claims abstract description 61
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 241000256054 Culex <genus> Species 0.000 title claims abstract description 27
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 239000012807 PCR reagent Substances 0.000 claims description 2
- 230000007850 degeneration Effects 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 241000256111 Aedes <genus> Species 0.000 abstract 1
- 241000255925 Diptera Species 0.000 description 20
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 241000256118 Aedes aegypti Species 0.000 description 9
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 241000256173 Aedes albopictus Species 0.000 description 4
- 241000256057 Culex quinquefasciatus Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000001364 causal effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000005570 vertical transmission Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a detection primer of culex wolbachia, a detection method and a detection kit thereof. Of culex wolbachiaThe detection primers are used for rapidly, efficiently and specifically detecting culex wolbachia according to the detection method, the specificity is strong, the specificity is high (only the culex wolbachia is detected, but the aedes wolbachia is not amplified), the sensitivity is high, and the minimum detection limit is 855 × 10‑5ng/. mu.l. Therefore, the method has the advantages of high speed and efficiency, simple and convenient operation, high specificity, high sensitivity, simple and convenient identification and the like.
Description
Technical field：
The invention belongs to molecular biology fields, and in particular to a kind of detection primer of culex Wolbachia and its inspection
Survey method and detection kit.
Background technology：
Wolbachia (Wolbachia) is the symbiotic microorganism being distributed widely in arthropod body, it may be
Monoid the abundantest in insect symbiotic microorganism.It is distributed in coleoptera, Diptera, Semiptera, Homoptera, Hymenoptera, squama
In the castes such as wing mesh.It using vertical transmission as its host's intergenerational transmission basic model.It is steadily present in
In the reproduction cell of host, host's filial generation is passed to by egg cell, and can be as not affine, female such as cytoplasm in several ways
Change the reproduction activity with modulate hosts such as gametocides.Promote its wide-scale distribution in host population by these regulating and controlling effects.
During host's reproductive behavio(u)r caused by Wolbachia (Wolbachia) changes, cytoplasm is not affine
(Cytoplasmic Incompatibility, CI) is the most common type type.It shows as when by Wolbachia
(Wolbachia) when the male mosquito that infects and be uninfected by female mosquito or infects the male and female mosquito of variety classes Wolbachia and mates, sperm with
Ovum combines rear embryo that can not develop.The incompatible characteristic of cytoplasm of Wolbachia (Wolbachia) can cause
The mosquito of Wolbachia infection can invade the mosquito swarm progress population compacting for being uninfected by or infecting Wolbachia not of the same race and population is replaced
Generation.Its application has huge value to the prevention of mosquito matchmaker's disease.Recent studies have found that Wolbachia (Wolbachia) is no
Only have outside above-mentioned characteristic, while some important causal organism (such as Dengues that it can also be in mosquito body with mosquito carrying
Virus, plasmodium etc.) interaction, inhibit their increments in mosquito body and diffusion to block these causal organisms to pass
It is sowed at the mankind.This inhibiting effect of Wolbachia (Wolbachia) is related in the distribution of mosquito in-vivo tissue to it, institute
With understanding Wolbachia (Wolbachia) contributes to quickly to filter out transmission blockage effect in mosquito distribution
The mosquito of best Wolbachia (Wolbachia) infection, it helps monitoring Wolbachia (Wolbachia) exists
Vertical transmission in mosquito swarm, while being also beneficial to expeditiously monitor the Wolbachia of extensive big territorial scope
(Wolbachia) infection conditions in mosquito is organized.
In nature, the type of mosquito has very much, common are two kinds of yellow-fever mosquito and culex.Yellow-fever mosquito be divided into aedes albopictus and
Aedes aegypti, wherein Aedes aegypti do not carry Wolbachia.Since Wolbach Salmonella can only survive in host, no
Can free living in vitro, thus how effectively specifically detection Wolbach Salmonella is indispensable tool to the bacterial studies.
More commonly used detection means has polymerase chain reaction (PCR) method (PCR methods) and immunostaining.Immunostaining takes time and effort, and
Specificity is bad.With popularizing for PCR method, the detection for Wolbachia makes great progress.Different mosquito kinds are taken
With different Wolbachias, the different Wolbachia types that mosquito carries can intuitively be found out by PCR results,
It provides great convenience in this way for our routine testing.It is directed to the method for different Wolbachia PCR detections at present also
It is not perfect.
Invention content：
That the object of the present invention is to provide a species specificities is strong, specificity is high and the culex Wolbachia of high sensitivity
Detection primer.
The detection primer of the culex Wolbachia of the present invention, which is characterized in that including：
wPipF：5 '-GTGCAGCTAATAGCAAGGAACA-3 ' (its nucleotide sequence is as shown in SEQ ID NO.1)；
wPipR：5 '-CCTATCTTTTCTGGCATCTTCG-3 ' (its nucleotide sequence is as shown in SEQ ID NO.2).
Second object of the present invention is to provide a kind of culex Wolbachia of the diagnosing and treating purpose of non-disease
Detection method, which is characterized in that extraction sample DNA, using the sample DNA as template, with above-mentioned culex Wolbachia
Detection primer wPipF and wPipR carries out PCR amplification as PCR primer, after the completion of amplified reaction, judges whether amplification to amplification
Product comes in judgement sample whether contain culex Wolbachia.
The carry out PCR amplification, reaction condition are preferably：94 DEG C of pre-degenerations 5 minutes；95 DEG C of denaturation are moved back for 5 seconds, 60 DEG C
Fire extends 30 seconds for 30 seconds, 72 DEG C, 30 cycles；72 DEG C finally extend 5 minutes.
Third object of the present invention is to provide a kind of detection kits of culex Wolbachia, including detection primer
And PCR reagent, which is characterized in that the detection primer is：
wPipF：5’-GTGCAGCTAATAGCAAGGAACA-3’；
wPipR：5’-CCTATCTTTTCTGGCATCTTCG-3’.
Using the present invention detection primer detection method according to the invention can be rapidly and efficiently single-minded detect culex
Wolbachia has specificity strong, and specificity is high (only to detect culex Wolbachia, and yellow-fever mosquito Wo Erbakeshi
Body does not expand), high sensitivity, lowest detection is limited to 855 × 10-5ng/μl。
Therefore, the present invention have many advantages, such as rapidly and efficiently, easy to operate, high specific, high sensitivity, identification it is easy.
Description of the drawings：
Fig. 1 is specificity experiments as a result, wherein M is Marker, and 1-3 is the Wolbachia of Guangzhou Culex quinquefasciatus, 4-6
For the Wolbachia of Guangzhou aedes albopictus.
Fig. 2 is sensitivity experiment as a result, wherein M is Marker, and 1 is the DNA sample of original concentration, 2,3,4,5,6 difference
It is 10,100,1000,10000,100000 times of diluted DNA samples.
Specific implementation mode：
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1：Specificity
1, taking 6 to be respectively provided with 20 μ l DNA extracting solutions, (formula of DNA extracting solutions is：30mM NaOH、0.25mM
EDTA, 15mMTris-HCl, solvent are water, similarly hereinafter) 0.2ml EP pipe；
2, three Guangzhou Culex quinquefasciatus (containing culex Wolbachia) and three aedes albopictus are (containing yellow-fever mosquito Wall bar
Kirschner body) after carbon dioxide is smoked and is swooned, abdomen is dissected, is respectively put into above-mentioned 6 EP pipes；
3, after brief centrifugation, 95 DEG C of warm bath 10 minutes；
4, after brief centrifugation, DNA extracts are obtained, -20 DEG C of preservations obtain the Wolbachia of six mosquitoes respectively
Genomic DNA (be specially：The genomic DNA of three parts of culex Wolbachias, the gene of three parts of yellow-fever mosquito Wolbachias
Group DNA), respectively PCR reactions are carried out by template of the genomic DNA of the Wolbachia of six mosquitoes.
5, PCR amplification system：
6, PCR amplification is carried out after mixing centrifugation；
7, PCR reaction conditions：
8. carrying out agarose electrophoresis after amplification, target stripe size is 262bp.
As shown in Figure 1, swimming lane 1-3 is Guangzhou Culex quinquefasciatus (culex Wolbachia) in Fig. 1,4-6 is concrete outcome
Guangzhou aedes albopictus (yellow-fever mosquito Wolbachia), from figure 1 it appears that only culex Wolbachia can expand
Go out, and yellow-fever mosquito Wolbachia cannot amplify and, and thus illustrate, amplimer high specificity of the invention.
Embodiment 2：Sensitivity
The genomic DNA of a culex Wolbachia of Example 1, then carry out 10,100,1000,10000,
100000 times of gradient dilutions, then using the dilution after dilution as template DNA, expanded according to the PCR method of embodiment 1, have
The results are shown in Figure 2 for body, and the dilution after 100000 times of dilution can amplify PCR product, then carry out concentration calculating, thus
Detection limit is 855 × 10 to the end-5(i.e. the genomic DNA of culex Wolbachia is dense in 100000 times of dilutions by ng/ μ l
Degree).
Claims (4)
1. a kind of detection primer of culex Wolbachia, which is characterized in that including：
wPipF：5’-GTGCAGCTAATAGCAAGGAACA-3’；
wPipR：5’-CCTATCTTTTCTGGCATCTTCG-3’.
2. a kind of detection method of the culex Wolbachia of the diagnosing and treating purpose of non-disease, which is characterized in that extraction
Sample DNA, using the sample DNA as template, with the detection primer wPipF of culex Wolbachia described in claim 1 and
WPipR carries out PCR amplification as PCR primer, after the completion of amplified reaction, judges whether that amplification carrys out judgement sample to amplified production
In whether contain culex Wolbachia.
3. detection method according to claim 2, which is characterized in that the carry out PCR amplification, reaction condition are：
94 DEG C of pre-degenerations 5 minutes；95 DEG C of denaturation are annealed 30 seconds, 72 DEG C for 5 seconds, 60 DEG C to be extended 30 seconds, 30 cycles.
4. a kind of detection kit of culex Wolbachia, including detection primer and PCR reagent, which is characterized in that described
Detection primer be：
wPipF：5’-GTGCAGCTAATAGCAAGGAACA-3’；
wPipR：5’-CCTATCTTTTCTGGCATCTTCG-3’.
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106386736B (en) * | 2016-08-30 | 2020-04-07 | 广州威佰昆生物科技有限公司 | Method for releasing aedes albopictus partitions carrying culex wolbachia |
| CN106367495A (en) * | 2016-08-30 | 2017-02-01 | 广州威佰昆生物科技有限公司 | Method for monitoring release ratio of culex aedes albopictus carrying culex wolbachia |
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2015
- 2015-06-11 CN CN201510318826.7A patent/CN104878109B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| Molecular discrimination of Wolbachia in the Culex pipiens complex: evidence for variable bacteriophage hyperparasitism;Y. O. Sanogo;《Insect Molecular Biology》;20040831;第13卷(第4期);365-369 * |
| WO bacteriophage transcription in Wolbachia-infected Culex pipiens;Yibayiri O. Sanogo;《Insect Biochemistry and Molecular Biology》;20061231;第36卷;80-85 * |
| 广东省蚊虫感染沃尔巴克氏体初步调查研究;林立丰;《中国媒介生物学及控制杂志》;20141231(第02期);全文 * |
| 白纹伊蚊实验种群感染沃尔巴体Wolbachia的研究;张东京;《寄生虫与医学昆虫学报》;20121231;12-18 * |
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