CN105063198B - A kind of mottle clam species specificity detection primer and application - Google Patents
A kind of mottle clam species specificity detection primer and application Download PDFInfo
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- CN105063198B CN105063198B CN201510473256.9A CN201510473256A CN105063198B CN 105063198 B CN105063198 B CN 105063198B CN 201510473256 A CN201510473256 A CN 201510473256A CN 105063198 B CN105063198 B CN 105063198B
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Abstract
The invention belongs to biological technical field, more particularly to a kind of mottle clam species specificity detection primer and application.A kind of mottle clam species specificity detection primer, its sequence are：5’‑ggttgaacagtctaccctcc‑3’（Upstream）；5’‑gctaacatactactacgcaaaa‑3’（Downstream）.A kind of application of mottle clam species specificity detection primer, using I genes of Cox of mottle clam species specificity detection primer PCR amplification mottle clam, the specific electrophoretic band of a 187bp size can be produced after PCR amplification through agarose gel electrophoresis, so that mottle clam component is carried out specificity identification.Specific primer design of the present invention is reasonable, is detected for mottle clam species, and specificity is good, and detection sensitivity is high, i.e., is analyzed by PCR, you can mottle clam component is carried out precise Identification, has specificity well.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of mottle clam species specificity detection primer and application.
Background technology
Mottle clam is shellfish type seafood, is subordinate to Veneridae, scientific name Ruditapes philippinarum, and south is commonly called as spending clam, and Liaoning claims a species of small clam living in fresh water
Son, Shandong claim clam.Mottle clam is widely distributed in China north and south sea area, it grows rapidly, and the culture-cycle is short, adaptable（Extensively
Warm, wide salt, wide distribution）, out-of-water survival time length, is a kind of excellent shellfish for being suitable for artificial high-density breeding, is China master
Want one of marine products economic shellfish.It is the conventional method identified to mottle clam by morphological feature judge, but these shapes
The plasticity of state feature is strong, affected by environment big, has artificial subjective tendency, and there are abundant nearly edge species, closely
The morphological differences of edge inter-species is trickle, so traditional morphological feature recognition methods has identification difficulty with identifying asking for mistake
Topic.
It is molecule skill the most popular and with fastest developing speed in species identification method using DNA technique identification animal species
Art.Tachytelic evolution and in matrilinear inheritance mitochondrial DNA be Population Genetics and evolutionary genetics preferable research object.It is variegated
The sequence similarity of clam and other nearly source species is between 85-94%, and I gene orders of Cox have larger difference, and at present, shellfish moves
The Molecular Detection of thing focuses primarily upon this sequence.The detection method delivered includes regular-PCR method and real-time fluorescence PCR side
Method etc., these methods or very high there are cross reaction, or to equipment requirement at present still can be with without a kind of easy PCR method
The gene of mottle clam and other shell-fish genes are distinguished completely.
The content of the invention
The purpose of the present invention is overcome above-mentioned insufficient problem, there is provided a kind of mottle clam species specificity detection primer and application
Method.The present invention can detect the minim DNA from animal sample, distinguish the gene of mottle clam and other shell-fishes completely
Gene, detection sensitivity is high, and method is quick, easy to operate.
The used to achieve the above object technical solution of the present invention is：A kind of mottle clam species specificity detection primer,
It is characterized in that：Its sequence is：
Sense primer：5’-ggttgaacagtctaccctcc-3’；
Anti-sense primer：5’-gctaacatactactacgcaaaa-3’.
A kind of application of the mottle clam species specificity detection primer, it is characterised in that：Utilize the mottle clam species
Specific detection primer carries out mottle clam species PCR specific detections.
Further, the mottle clam species PCR method for detecting specificity is：Drawn using the detection of mottle clam species specificity
I genes of Cox of thing PCR amplification mottle clam, can produce the special of 187bp size after PCR amplification through agarose gel electrophoresis
Property electrophoretic band so that by mottle clam component carry out specificity identification.
Further, 25 μ L reaction systems such as table 1 below of I genes of Cox of the PCR amplification mottle clam：
The reaction system of I genes of Cox of 1. PCR amplification mottle clam of table
Further, the response parameter such as table 2 below of I genes of Cox of the PCR amplification mottle clam：
The response parameter of I genes of Cox of 2. PCR amplification mottle clam of table
Specific primer design of the present invention is reasonable, is detected for mottle clam species, and specificity is good, and detection sensitivity is high.Adopt
Mottle clam animal component is detected the results show that using mottle clam species specificity detection primer PCR amplification mottle clam with this method
I genes of Cox, the specific electrophoretic band of a 187bp size can be produced after PCR amplification through agarose gel electrophoresis, you can
Mottle clam component is subjected to precise Identification, there is specificity well.
Brief description of the drawings
Fig. 1 is 5 kinds of primer PCR result figures, in figure：M.DL2000marker, 1.P1P2 primer amplification result, 2.P3P4 draw
Thing amplification, 3.P5P6 primer amplification results, 4.P7P8 primer amplification results, 5.P9P10 primer amplification results, 6. is negative right
According to 7. blank controls.
Fig. 2 is the PCR specific detection result figures of mottle clam and other marine animals, in figure：M.mark, 1. mottle clams,
2. mottle clam, 3. summers razed scallop, 4. Anthocidaris crassispinas, 5. Pacific oysters, 6. stalwart blood clams, 7. negative controls, 8. blank controls.
Fig. 3 is sensitivity analysis result figure, in figure：M.DL2000marker, 1. 10 ng/ μ L amplifications, 2. 5ng/
μ L amplifications, 3. 1 ng/ μ L amplifications, 4. 0.5ng/ μ L amplifications, 5. 0.1 ng/ μ L amplifications, 6. is negative
Control, 7. blank controls.
Embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but the invention is not limited in specific implementation
Example.
The mottle clam species specificity detection primer utilized in the present embodiment, its sequence are：
5’-ggttgaacagtctaccctcc-3’（Upstream）；
5’-gctaacatactactacgcaaaa-3’（Downstream）.
A kind of application of the mottle clam species specificity detection primer, i.e., detected using the mottle clam species specificity
Primer carries out mottle clam species PCR specific detections, comprises the following steps that：
1. mottle clam species specificity detection primer synthesizes：Draw in the synthesis mottle clam species specificity detection of precious biotech firm
Thing 5 is right, its sequence（It is shown in Table 3）：
The primer sequence of I genes of Cox of 3. PCR amplification mottle clam of table：
2. the extraction of mottle clam DNA：Using DNA extraction kit（It is purchased from precious biotech firm）Mottle clam template DNA is extracted, use is micro-
The concentration for measuring spectrophotometer Detection and Extraction mottle clam template DNA is 100ng.
3.PCR expands I genes of Cox of mottle clam：Prepare 25 μ L PCR reaction systems such as table 4 below：
The reaction system of I genes of Cox of 4. PCR amplification mottle clam of table
Use the response parameter such as table 5 below that PCR is set during the mottle clam species specificity detection primer：
The response parameter of I genes of Cox of 5. PCR amplification mottle clam of table
4. agarose gel electrophoresis detects：With 5 couples of specific primer PCR such as P1P2, P3P4, P5P6, P7P8, P9P10
After I genes of Cox for expanding mottle clam, it is detected with 2% agarose gel electrophoresis, the results show（As shown in Figure 1）, through agar
The specific electrophoretic band of 182bp, 178bp, 180bp, 212bp, 269bp size can be produced after sugared detected through gel electrophoresis respectively.
Specific PCR amplification is carried out using P1P2 as upstream and downstream primer, electrophoretic effects are best, PCR amplification efficiency highest, therefore choose
Specificity amplification primers of the P1P2 as amplification system.
When being detected in real time using the Species composition of mottle clam species specificity detection primer progress mottle clam, using this
Method detect mottle clam animal component the results show that using mottle clam species specificity detection primer PCR amplification detection mottle clam,
Summer razes the samples such as scallop, Anthocidaris crassispina, Pacific oyster, stalwart blood clam, through agarose gel electrophoresis after PCR amplification, as shown in Fig. 2, miscellaneous
Color clam sample produces the specific electrophoretic band of a 187bp size, other samples are without electrophoretic band, you can by mottle clam component
Precise Identification is carried out, there is specificity well.
The template DNA of each sample to be tested is extracted using DNA extraction kit, is detected and is carried respectively with micro-spectrophotometer
The template DNA gradient dilution taken, is prepared into 10 ng/ μ L, 5 ng/ μ L, 1 ng/ μ L, 0.5 ng/ μ L, 0.1 ng/ μ L 5
Concentration gradient sample, carries out PCR amplification according to reaction system described in above-mentioned steps 3 and response parameter respectively, after with 2% agar
Sugared gel electrophoresis is detected, and to determine the detection sensitivity of this standard method, the results are shown in Figure 3, and caused size is
The specific electrophoretic band of 182bp is mottle clam, can be with specific amplified, i.e. the standard side as DNA concentration >=1ng/ μ L
The detection sensitivity of method is 1ng/ μ L.The method accurately can identify the component of mottle clam have sensitive well
Degree.
Above content is to combine the further description that optimal technical scheme is the present invention, it is impossible to assert invention
Specific implementation is only limitted to these explanations.For general technical staff of the technical field of the invention, the present invention is not being departed from
Design on the premise of, can also make it is simple deduce and replace, should all be considered as protection scope of the present invention.
SEQUENCE LISTING
<110>It is refined gorgeous, Liu
It is super, ten thousand
Pellet, Jiang
Phoenix is quick, Xu
<120>A kind of mottle clam species specificity detection primer and application
<130> 20150715
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Designed according to sequence-specific, the sense primer as mottle clam species PCR specific detections
<220>
<221> Artificial Sequence
<222> (1)..(20)
<400> 1
ggttgaacag tctaccctcc 20
<210> 2
<211> 22
<212> DNA
<213> Artificial
<220>
<223>Designed according to sequence-specific, the anti-sense primer as mottle clam species PCR specific detections
<220>
<221> Artificial Sequence
<222> (1)..(22)
<400> 2
gctaacatac tactacgcaa aa 22
Claims (5)
- A kind of 1. mottle clam species specificity detection primer, it is characterised in that：Its sequence is：Sense primer：5’-ggttgaacagtctaccctcc-3’；Anti-sense primer：5’-gctaacatactactacgcaaaa-3’；The primer carries out mottle clam species PCR specific detections, and method is：Using mottle clam species specificity detection primer I genes of Cox of PCR amplification mottle clam, the specificity of a 187bp size can be produced after PCR amplification through agarose gel electrophoresis Electrophoretic band, so that mottle clam component is carried out specificity identification.
- A kind of 2. application of mottle clam species specificity detection primer according to claim 1, it is characterised in that：Using institute State mottle clam species specificity detection primer and carry out mottle clam species PCR specific detections.
- 3. the application of mottle clam species specificity detection primer according to claim 2, it is characterised in that：The mottle clam Species PCR method for detecting specificity is：Using I genes of Cox of mottle clam species specificity detection primer PCR amplification mottle clam, The specific electrophoretic band of a 187bp size can be produced after PCR amplification through agarose gel electrophoresis, so that by mottle clam component Carry out specificity identification.
- 4. the application of mottle clam species specificity detection primer according to claim 3, it is characterised in that：The PCR expands Increase the reaction system such as table 1 below of I genes of Cox of mottle clam：The reaction system of I genes of Cox of 1. PCR amplification mottle clam of table。
- 5. according to the application of any mottle clam species specificity detection primer of claim 3 or 4, it is characterised in that：Institute State the response parameter such as table 2 below of I genes of Cox of PCR amplification mottle clam：The response parameter of I genes of Cox of 2. PCR amplification mottle clam of table。
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Citations (4)
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|---|---|---|---|---|
| CN101979536A (en) * | 2010-09-26 | 2011-02-23 | 中国海洋大学 | Method for screening veneridae mitochondria COI gene amplification primers |
| CN102199597A (en) * | 2011-03-09 | 2011-09-28 | 中国海洋大学 | Screening method of bivalve mitochondrion COI gene amplification primers |
| CN103103281A (en) * | 2013-02-04 | 2013-05-15 | 曹际娟 | Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method |
| CN103937802A (en) * | 2014-03-19 | 2014-07-23 | 刘丰铭 | DNA barcoding standard gene sequence of Rizhao Blepharipoda liberata and species identification method based thereon |
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2015
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Patent Citations (4)
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|---|---|---|---|---|
| CN101979536A (en) * | 2010-09-26 | 2011-02-23 | 中国海洋大学 | Method for screening veneridae mitochondria COI gene amplification primers |
| CN102199597A (en) * | 2011-03-09 | 2011-09-28 | 中国海洋大学 | Screening method of bivalve mitochondrion COI gene amplification primers |
| CN103103281A (en) * | 2013-02-04 | 2013-05-15 | 曹际娟 | Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method |
| CN103937802A (en) * | 2014-03-19 | 2014-07-23 | 刘丰铭 | DNA barcoding standard gene sequence of Rizhao Blepharipoda liberata and species identification method based thereon |
Non-Patent Citations (4)
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| Molecular Phylogeny of Giant Clams Based on Mitochondrial DNA Cytochrome C Oxidase I Gene;AGUS NURYANTO等;《HAYATI Journal of Biosciences》;20071231;第14卷(第4期);162-166 * |
| 中国帘蛤目16种经济贝类DNA条形码及分子系统发育的研究;王琳楠等;《大连海洋大学学报》;20131031;第28卷(第5期);摘要、表1，第435页右栏倒数第 2段 * |
| 基于线粒体细胞色素C氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究;程汉良等;《生态学报》;20130531;第33卷(第9期);2744-2753 * |
| 菲律宾蛤仔EST-SSR引物设计及特性;陈丽梅等;《201 1年中国水产学会学术年会论文摘要集》;20111231;129 * |
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