CN105177153B - A kind of Rattusflauipectus PCR identification primers and identification method - Google Patents
A kind of Rattusflauipectus PCR identification primers and identification method Download PDFInfo
- Publication number
- CN105177153B CN105177153B CN201510642939.2A CN201510642939A CN105177153B CN 105177153 B CN105177153 B CN 105177153B CN 201510642939 A CN201510642939 A CN 201510642939A CN 105177153 B CN105177153 B CN 105177153B
- Authority
- CN
- China
- Prior art keywords
- rattusflauipectus
- pcr
- identification
- primer
- result
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
Abstract
The present invention provides a kind of Rattusflauipectus PCR identification primers and identification method, so as in the case where list is difficult to judge mouse kind from form, simple and accurate Rattusflauipectus identification is carried out.The Rattusflauipectus PCR primer, the nucleotides sequence of its sense primer are classified as：" 5 ' CCTCGCACCAACTCCTGAA 3 ' ", the nucleotides sequence of primer is classified as downstream：“5’‑TGGCTACCTAAACCTAAAGCAACT‑3’”.Using the method for Rattusflauipectus PCR identification primer identification Rattusflauipectus, including 1) the DNA sample collection and the extraction of STb gene of thing to be identified；2) using the STb gene extracted in step 1) as template, combined using above-mentioned primer and carry out PCR amplification；3) result after reaction, is judged as the positive of Rattusflauipectus in the 503bp specific amplification bands occurred with the agarose gel electrophoresis result of PCR product.The method result is stable, easy to operate, efficient and cost is low.
Description
Technical field
The present invention relates to biological detection identification technology, relates in particular to identify the PCR of Rattusflauipectus primer and use should
The method that primer identifies Rattusflauipectus.
Background technology
Muroid is the reservoir of the multiple pathogens such as the plague, Hemorrhagic fever, leptospirosis, the life to the mankind
Production, life and health form serious threat.Some pathogen have host certain specificity, mouse kind right pop disease not of the same race
Learning aid has different meanings.City incity mouse is then based on the coexistent rodents such as Rattusflauipectus, Rattus norvegicus and house mouse【1】.Wherein, yellow chest
Mouse is the dominant rat in current most cities, and it is small that it is active in each diet restaurant, hotel dining room, food processing factory and resident
Area, is the sanitary city, Establishment of Civilized City main chief culprit in getting rid of the 4 pests.In addition, the Rattusflauipectus resistance to the action of a drug is strong, easily go out to anticoagulation
Mouse agent is developed immunity to drugs【2】, therefore the improvement to Rattusflauipectus becomes more and more difficult.Therefore, it is possible to Rapid identification Rattusflauipectus,
Can some put taking measures for mistake, effectively preventing is carried out, to reach the deratization effect to get twice the result with half the effort.
At present, the identification of mouse kind is still only limitted to traditional morphological method.This method is not only time-consuming and laborious, Er Qie
Some aspects have significant limitation, such as：Rattusflauipectus, Rattus norvegicus and its young rat period form are very close, only by morphology
Individual Chang Wufa provides exact conclusion.
Every field has been widely used in using the gene amplification method of PCR.PCR methods be using the DNA of denier as template,
The method that target DNA region is expanded to hundreds thousand of times in short time, medicine and micro- life are used in because its precision is high extensively
Thing field.VKORC1 (vitamin epoxide reductase complex subunit 1) is one and the relevant gene of vitamin K【3】,
Although mankind VKORC1 Study on gene polymorphism report is relatively more【4】, and the research to muroid VKORC1 genes rarely has report both at home and abroad
Road【5、6】.And muroid VKOCR1 Gene sequence comparisons are guarded, there is important reference value in mouse classification.
Bibliography：
[1] Zhang Shijun, Wang Zhiquan, Chen Yu, wait analysis [J] of Nantong Cities Mousetrap capture and mouse cage method mousing situation Chinese
Hygienic biocide medical instruments, 2013,19 (2)：152-154.
[2] Wang Zhiquan, Zhang Shijun, Chen Yu, wait resistance to the action of a drug research [J] China health of the Nantong Cities Rattusflauipectus to Warfarin
Insecticidal pharmaceutical Machinery, 2014,20 (3):223-225.
[3] Wang Zhiquan, Li Haibo, Zhang Shijun, wait Nantong Cities coexistent rodents VKORC1 genes and its drug-fast correlative study
[J] China hygienic biocide medical instruments, 2014,20 (1):23-25.
[4] He Baoxia, Shi Lei, Zhao Shu are ground into .CYP2C9 and VKORC1 gene pleiomorphisms and warfarin personalized medicines
Study carefully progress [J] Guangdong medicine .2008,29 (4) 684-686.
[5]Tanaka K.D.,Kawai Y.K.,Ikenaka Y.,et al.“A novel mutation in
VKORC1and its effect on enzymatic activity in Japanese warfarin-resistant
rats[J].J.Vet.Med.Sci.,2013,5(2):135-139.
[6]Rost S.,Pelz H.J.,Menzel S.,et al.“Novel mutations in the
VKORC1gene of wild rats and mice–a response to 50years of selection pressure
by warfarin”.BMC Genetics,2009,10:4.
The content of the invention
The present invention provides a kind of Rattusflauipectus PCR identification primers and identification method, is difficult to by reaching to be directed in the form of
On the muroid that is judged carry out the purpose of simple and accurate Rattusflauipectus identification.This method is stable, efficient and cost is low.
To achieve these goals, the technical scheme is that：
The present invention provides a kind of Rattusflauipectus PCR to identify primer, it is characterised in that its nucleotide sequence is respectively：
Sense primer is：5’-CCTCGCACCAACTCCTGAA-3’；
Anti-sense primer is：5’-TGGCTACCTAAACCTAAAGCAACT-3’.
It is including following present invention also offers a kind of method using above-mentioned Rattusflauipectus PCR identification primer identification Rattusflauipectus
Step：
1) STb gene of thing to be identified is extracted；
2) using the STb gene extracted in step 1) as template, PCR is carried out using above-mentioned Rattusflauipectus PCR identification primer combinations
Amplification；
3) use agarose gel electrophoresis detecting step 2) in produce PCR product, to identify Rattusflauipectus.Above-mentioned PCR productions
There is specific amplification band in 503bp in the agarose gel electrophoresis result of thing, then judges result for the positive of Rattusflauipectus.
Technical solution provided by the invention has reached following beneficial effect：1) using than more conservative muroid VDORC1 bases
Because of sequence design, PCR primer, its pcr amplification product are specific to Rattusflauipectus mouse kind, can distinguish Rattusflauipectus and other mouse kinds
Come；2) muroid for being difficult to be judged from form can be directed to using the PCR identification methods of the primer and carries out Rattusflauipectus mirror
It is fixed；3) sample size needed for the experimental method is few, and collection of specimens is easy to implement, and method is easy to operate, as a result stable and accurate, and
And cost is low.
Brief description of the drawings
Fig. 1 is different mouse kind PCR agarose gel electrophoresis result specific outcomes.
Fig. 2 is the fragments molecules amount table of comparisons of molecular marked compound DL2000.
In figure, 1,2. Rattusflauipectus；3rd, 4. Rattus norvegicus；5th, 6. big white mouse；7. Rattus edwardsi；8. Apodemus agrarius；9.DL2000
Embodiment
In order to clarify the technical solutions and technical objectives of the present invention, below in conjunction with the accompanying drawings and embodiment is to the present invention
It is described further.
First, according to VKORC1 gene orders, by trnL sequence designs, Rattusflauipectus PCR diagnostic primers, its nucleosides are devised
Acid sequence is：
Sense primer is：5’-CCTCGCACCAACTCCTGAA-3’；
Anti-sense primer is：5’-TGGCTACCTAAACCTAAAGCAACT-3’.
2nd, rat-tail DNA extraction method (pillar centrifugal pipe process, JaRa genome extracts kit)
1st, by end flap of the rat-tail portion grind into powder of 0.5~1cm in liquid nitrogen, and it is transferred to the centrifuge tube of 1.5ml
In.
2 and then Proteinase K (protease) of 400 μ l lysates of addition and 10 μ l
3rd, concussion mix 1 minute, be subsequently placed in 55 DEG C of water-baths 1~3 it is small when, can suitably take out mixing during this period, have
Help fully crack.
4th, sample is taken out, mixing is gently shaken when room temperature is down to.
5th, in the sample by having pre-processed, 600ul extracting solutions is added, are firmly shaken up, then 12,000rpm centrifugations 5
Minute.Solution will be layered, and upper strata is aqueous layer, and lower floor is organic solvent layer, and partly precipitated layer is might have among two layers of solution,
DNA is in upper strata aqueous phase.
6th, upper strata aqueous phase solution is carefully sucked into centrifugal column with the pipette tips of sterilizing, avoids being drawn onto the heavy of intermediate layer as far as possible
Form sediment.
7th, 8,000rpm is centrifuged 1 minute, is removed centrifugal column, is outwelled waste liquid in collecting pipe.
8th, centrifugal column is put back in collecting pipe, add 500 μ l Wash Solution (cleaning solution), 8,000rpm, room temperature
Centrifugation 1 minute.
9th, repeat step 8 is once.
10th, centrifugal column is removed, discards the waste liquid in collecting pipe.Column is put back in collecting pipe, 12,000rpm, room temperature centrifugation 1
Minute, to remove residual Wash Solution (cleaning solution).
11st, centrifugal column is put into new clean 1.5ml centrifuge tubes, 50~100 μ l Elution is added in column center
Buffer (elution buffer), room temperature or 55 DEG C are placed 2 minutes.Then 12,000rpm, room temperature centrifuges 1 minute.In centrifuge tube
Liquid is genomic DNA.According to purposes, sample can be in 4 DEG C or -20 DEG C preservations.
3rd, PCR amplification system and PCR amplification program
1st, PCR amplification system
Using STb gene as template, PCR amplification is carried out, is contained in the reaction system of 25 μ L：2xTaq PCR MaterMix
12.5 μ L, 10 μM of Forward Primer 1 μ L, 10 μM of 1 μ L of Reverse Primer, DNA sample 1 μ L, Water
(nuclease-free)9.5μL。
2nd, pcr amplification reaction condition
The reaction condition of PCR amplification is：95 DEG C of pre-degeneration, 5min, then 15sec, 55 DEG C of renaturation 15sec are denatured through 95 DEG C,
72 DEG C of extension 30sec, 35 circulations, last 72 DEG C of extensions 5min.After reaction, amplified production is obtained by agarose gel electrophoresis
To qualification result.
4th, electrophoretic procedures
1st, prepared by 1.0% Ago-Gel：
1g agaroses are weighed in conical flask, add 1 × tbe buffer liquids of 100ml, micro-wave oven is heated to dissolving completely, cold
But to 60 DEG C or so, add appropriate ethidium bromide storage liquid, gently shake up, slowly pouring into frame has in the electrophoresis offset plate of comb, should not
There is bubble, stand cooling more than 30min, being put into electrophoresis tank can loading, electrophoretic buffer and the buffer solution for preparing gel
Concentration is identical.
2nd, point sample, electrophoresis
10 × Loading buffer of 1/10 volume are added in electrophoresis Sample, are loaded after mixing.5-7V/ is used during electrophoresis
The voltage of cm, electrophoresis 30min.Gel is taken out after electrophoresis, observes in the UV lamp and takes a picture (see Fig. 1).
Embodiment
Identify primer and its identification method to Rattusflauipectus, Rattus norvegicus, big white mouse, white abdomen using Rattusflauipectus provided by the invention
The DNA samples of huge mouse and Apodemus agrarius have carried out Rattusflauipectus identification according to above-mentioned specific experiment method, as a result as follows：
In the electrophoresis result analysis for carrying out the judgement of mouse kind according to purpose fragment size in Fig. 1 and Fig. 2, carried from Rattusflauipectus
The DNA (see mark in Fig. 11 and 2) taken occurs special after PCR amplification at 1.0% agarose gel electrophoresis offset plate 503bp
Property band, and from other muroids, such as：Rattus norvegicus (see mark in Fig. 13 and 4), big white mouse (see mark in Fig. 15 and 6), white abdomen are huge
Mouse (see mark 7 in Fig. 1) Apodemus agrarius (see mark 8 in Fig. 1), the DNA of extraction is after PCR amplification, in 1.0% Ago-Gel
There is specific band at electrophoresis offset plate 593bp.This explanation Rattusflauipectus and Rattus norvegicus, big white mouse, Rattus edwardsi, Apodemus agrarius
The Second Exon genetic fragment size of VKORC1 genes has differences, and the difference is specific to Rattusflauipectus.
Based on the means and method of the diversified detection PCR product increasingly updated, the identification method that the present embodiment is lifted
One of only.Identification primer provided by the invention and thus obtained Rattusflauipectus and other mouse kinds can be distinguished
Genetic fragment, is also suitable for being applied in other associated Rattusflauipectus identifications.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, the present invention
Claimed scope is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Sequence table：
<110>Nantong Municipal Disease Control and Prevention Center
<120>A kind of Rattusflauipectus PCR identification primers and identification method
<160>1
<170>PatentIn version 3.5
<210>1
<211>503
<212>DNA
<213>Rattus flavipectus
<400>1
cctcgcacca actcctgaaa agttattgat ggcctgtgat tccttcgcac ccccatcctg 60
aaaagttatt gatggcctgt gatgacaggc cagtaagagt agaggacaag gtcgctgacc 120
tttggattct tggtgggtgg cgcttcttgc taatcactct ttctagaaaa aaaagctccg 180
gaggcaccag gctgactgca cttacctggt tctttcactt ccaccaggtg gggccggggc 240
tttgggctgg tggagcatgt gttaggagct gacagcatcc tcaaccaatc caacagcata 300
tttggttgca tgttctacac cttacagctg ttgttaggtg agtggctttg ccccctcatg 360
acctgtcttc tgaaatccag accagcccca cctcatgtct tggtgaccca gacagttgac 420
agccagctgc taatactgag caatttcctc acaaggagaa cacttagcag gagtatccaa 480
gttgctttag gtttaggtag cca 503
Claims (2)
1. a kind of Rattusflauipectus identification method, it is characterised in that comprise the following steps：
1) STb gene of thing to be identified is extracted；
2) using the STb gene extracted in step 1) as template, identified using the Rattusflauipectus PCR with nucleotide sequence as follows
Primer carries out PCR amplification：
Sense primer is：5’-CCTCGCACCAACTCCTGAA-3’；
Anti-sense primer is：5’-TGGCTACCTAAACCTAAAGCAACT-3’；
3) reaction condition of PCR amplification is：95 DEG C of pre-degeneration, 5min, then through 95 DEG C of denaturation 15sec, 55 DEG C of renaturation 15sec, 72
DEG C extension 30sec, 35 circulation, it is last 72 DEG C extension 5min；
4) there is specific amplification band in 503bp in the PCR product produced in electrophoresis detection step 3), its electrophoresis result.
2. Rattusflauipectus PCR identification primers as described in claim 1 and its genetic fragment expanded are identified in Rattusflauipectus
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510642939.2A CN105177153B (en) | 2015-09-30 | 2015-09-30 | A kind of Rattusflauipectus PCR identification primers and identification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510642939.2A CN105177153B (en) | 2015-09-30 | 2015-09-30 | A kind of Rattusflauipectus PCR identification primers and identification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105177153A CN105177153A (en) | 2015-12-23 |
| CN105177153B true CN105177153B (en) | 2018-04-24 |
Family
ID=54899575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510642939.2A Expired - Fee Related CN105177153B (en) | 2015-09-30 | 2015-09-30 | A kind of Rattusflauipectus PCR identification primers and identification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105177153B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040367A1 (en) * | 2003-10-14 | 2005-05-06 | Baxter International Inc. | Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives |
| CN104694632A (en) * | 2015-02-05 | 2015-06-10 | 浙江省检验检疫科学技术研究院 | Real-time fluorescent PCR species composition detection method, primer, probe and kit for mice |
-
2015
- 2015-09-30 CN CN201510642939.2A patent/CN105177153B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040367A1 (en) * | 2003-10-14 | 2005-05-06 | Baxter International Inc. | Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives |
| CN104694632A (en) * | 2015-02-05 | 2015-06-10 | 浙江省检验检疫科学技术研究院 | Real-time fluorescent PCR species composition detection method, primer, probe and kit for mice |
Non-Patent Citations (3)
| Title |
|---|
| Analysis of vkorc1 polymorphisms in Norway rats using the roof rat as outgroup;Diaz JC et al.;《BMC Genet》;20100524;第11卷;摘要，结果和讨论，方法 * |
| DNA条形码技术在贵州茂兰鼠类鉴定中的应用;刘润吉等;《中华卫生杀虫药械》;20140220;第20卷(第1期);摘要，材料与方法，结果与分析 * |
| 南通市家栖鼠VKORC1基因及其抗药性的相关研究;王智泉等;《中华卫生杀虫药械》;20140220;第20卷(第1期);摘要，材料与方法，结果与分析，表1-2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105177153A (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bialek et al. | Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays | |
| US9238839B2 (en) | Primer set, method and kit for detecting pathogen in animals or plants | |
| CN106811521A (en) | For the method and system of nucleic acid amplification | |
| KR20200083461A (en) | Devices, systems and methods for biomarker analysis | |
| CN103602757B (en) | The foundation of foot and mouth disease, vesicular stomatitis and swine pox multi-fluorescence RT-PCR detection method and application thereof | |
| CN103789446A (en) | Kit and method for detecting polymorphism of pyrosequencing clopidogrel personalized drug gene | |
| CN102807976B (en) | Rapid nucleic acid extraction kit and applications thereof | |
| CN102796727B (en) | Method for extracting nucleic acid of gram positive bacteria | |
| WO2015096063A1 (en) | Methods and systems for nucleic acid amplification | |
| CN101831494B (en) | Molecular biology authentication method of bulbus fritillariae cirrhosae in Chinese patent medicine containing fritillariae | |
| CN107022611B (en) | Method and special primer for detecting accurate medication of 4 common clinical cardiovascular and cerebrovascular disease medicines | |
| CN107012230A (en) | Differentiate the method for zaocys dhumnade, scorpio and centipede using specific primer | |
| CN109385488B (en) | Multiplex PCR (polymerase chain reaction) primer, method and kit for simultaneously detecting three kinds of toxigenic fungi polluted by traditional Chinese medicinal materials | |
| CN109182600B (en) | Fluorescent quantitative PCR kit for synchronously detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1 | |
| CN101914526A (en) | Extraction method for tiny RNA in serum or plasma | |
| CN105177153B (en) | A kind of Rattusflauipectus PCR identification primers and identification method | |
| CN103571939B (en) | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen | |
| CN104388587A (en) | One-step detection kit for influenza B virus and influenza B virus detection method | |
| CN103789430B (en) | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit | |
| CN109880822A (en) | A kind of idesia high quality DNA extracting method | |
| CN104830856A (en) | Single nucleotide mutant primer group and detection method thereof | |
| CN104830848A (en) | Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof | |
| CN107815510A (en) | The detection method of the type of COxsackie A groups 10 virus in hand-foot-and-mouth disease mixed infection | |
| CN102304574B (en) | Diagnostic method for rifampicin resistant mycobacterium tuberculosis | |
| Posteraro et al. | Profiling the Gastrointestinal Microbiota |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180424 Termination date: 20200930 |