CN102337336A - Primers for PCR (polymerase chain reaction) detection of domestic francolin (rock partridge)-derived components - Google Patents
Primers for PCR (polymerase chain reaction) detection of domestic francolin (rock partridge)-derived components Download PDFInfo
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- CN102337336A CN102337336A CN201110290476XA CN201110290476A CN102337336A CN 102337336 A CN102337336 A CN 102337336A CN 201110290476X A CN201110290476X A CN 201110290476XA CN 201110290476 A CN201110290476 A CN 201110290476A CN 102337336 A CN102337336 A CN 102337336A
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Abstract
The invention relates to the technical field of identification of animal species and animal derived components, specifically relates to a detection method for detecting domestic francolin (rock partridge)-derived components, and in particular relates to a pair of primers for detecting a cell mitochondria nucleotide sequence of the domestic francolin (rock partridge). The primers aim at identifying whether the domestic francolin (rock partridge)-derived components are contained in the products such as a forage, a food and the like. By designing a specific amplification primer, a PCR (polymerase chain reaction) detection technology is used for detecting a mitochondria DNA (deoxyribonucleic acid) which is relatively independent on the heredity, does not have a repeated sequence and does not have a tissue specificity in an organism individual, and a PCR detection method of the domestic francolin (rock partridge)-derived components is built, so that the purpose for quickly and exactly detecting the domestic francolin (rock partridge)-derived components is achieved.
Description
Technical field
The present invention relates to animal species and animal derived materials authenticate technology field; Be specifically related to a kind of detection method that is used to detect domestic francolin (chukar) derived component, relate in particular to a pair of primer that is used to detect domestic francolin (chukar) cell mitochondrial nucleotide sequence.
Background technology
Countries in the world government is regarded as food safety, feed safety to relate to the problem of national public safety mostly; And strengthen regulatory efforts one after another; Particularly aspect the inspection and quarantine work of entry and exit and livestock and poultry meat food, feed and the commodity thereof in market; Strengthened the animal derived product safety Study on Monitoring Technology of monitoring livestock and poultry meat food and feed thereof, the research of this detection technique more and more comes into one's own with application.
The pcr amplification purpose fragment of utilizing primer of the present invention to carry out is a mtdna sequence.Mitochondrial DNA is the unique extranuclear inheritance material of higher animal; Be that the more satisfactory species that are used for are differentiated the target-gene sequence that detects; Its major cause is: a large amount of plastosomes is all contained in (1) in all histocytes; Can obtain a large amount of mtDNA, mtDNA has the variability of height between different species; The Mitochondrial Genome Overview that has many copies in each zooblast, when the DNA sample of taking equal volume carried out the PCR detection, the template number of Mitochondrial Genome Overview was much higher than the template number of cell nucleus gene group, and this has just improved the sensitivity that PCR detects greatly.(2) mtDNA mainly constitutes with encoding sequence, and the different plasmagene in planting seldom.(3) though there is the high conservative zone in different types of animal mitochondria genome sequence height homology, also there is certain variation zone.
Summary of the invention
The object of the present invention is to provide the primer of the domestic francolin of a pair of detection (chukar) derived component.To solve accurately, fast, identify domestic francolin (chukar) derived component with round pcr reliably.
The object of the invention can be realized through adopting following technical scheme:
1. design primer: the primer that is used to detect the chukar derived component is to design according to the conservative 12S rRNA sequence of Mitochondrial DNA camber, and concrete primer sequence is following:
Upstream primer F:5 '-GCCCCGGCTAAAGACGAGGTAA-3 ';
Downstream primer R:5 '-GAATTTTTGATATTTAGGGTGAG-3 '.
2. extraction sample DNA: adopt chloroform extraction method or commercial DNA extraction test kit to extract sample DNA.
3. adopt step 1 designed primer to carry out pcr amplification: the PCR reaction system is 25 μ L; Comprise 12.5 μ L, 2 * PCRBuffer; 5 μ L dNTPs (2mmol/L), 1 μ L KOD FX archaeal dna polymerase (1U/ μ L), each 1 μ L (final concentration is 0.4 μ M) of upstream and downstream primers F/R; Dna profiling 2 μ L, ultrapure water is mended to 25 μ L.Can adopt business-like PCR reaction mother liquor.The PCR response procedures is: preparatory 94 ℃ of 2min of sex change; 94 ℃ of 30sec then, 57 ℃ of 30sec, 72 ℃ of 40sec circulations 35 times; 72 ℃ of 3min.After accomplishing, reaction preserves down at 4 ℃.
4. electrophoresis detection: the PCR product detects through 1.5% agarose gel electrophoresis.If contain domestic francolin (chukar) derived component in animal product and the feed, amplification purpose band then appears in electrophorogram 184bp position.
The invention has the beneficial effects as follows, Live Animals, consumption animal product, non-consumption animal product, animal derived feed and fodder additives etc. are carried out cultivar identification.Utilize primer of the present invention to adopt PCR method can effectively detect domestic francolin (chukar) derived component in animal product and the feed, and differentiate detection, its purpose band length that increases is 184bp, detects minimumly to be limited to 0.01%.
Description of drawings:
The domestic francolin of Fig. 1 (chukar) primer specificity PCR detects electrophorogram
M.Marker among the figure; 1. chicken; 2. duck; 3 geese; 4. turkey; 5. dove; 6. ostrich; 7. quail is 8. Ns; 9. goat; 10. pig; 11. sheep; 12. rabbit; 13. fish; 14. deer 15. dogs; 16. donkey; 17. horse; 18. chukar; 19. negative control; M
The domestic francolin of Fig. 2 (chukar) primer sensitivity PCR detects electrophorogram
M:marker DL100 among the figure; 1:10.00% (chukar DNA); 2:1.00%; 3:0.1%;
4：0.01％；5：0.001％；6：0.0001％；7：0.00001％；8：0.000001％
Embodiment:
Most preferred embodiment is made further detailed description to the present invention shown in the following certificate.Should be understood that embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
1. primer design: through kind close in zoological taxonomy such as chicken and quail chondriogen sequence comparing analysis, select the 12S rRNA gene of high conserved region section, primer is also optimized in design, and sequence is following:
The sequence of upstream primer F is: 5 '-CAGGCTTTACCCTACACCCC-3 ';
The sequence of downstream primer R is: 5 '-AGTATCGGCGAGGTATGCCG-3 '.
2. the extraction of sample total DNA
Adopt chloroform extraction method or commercial DNA extraction test kit; Extract the genomic dna of chicken, duck, goose, turkey, pigeon, ostrich, quail, ox, goat, pig, sheep, rabbit, fish, deer, dog, donkey, horse, chukar animal, the genomic dna of extraction is all measured its purity and concentration through ultraviolet spectrophotometer.Measure OD
260/ OD
280Value is about 1.8, explains that DNA purity is higher to meet the pcr amplification requirement.
3. domestic francolin (chukar) derived component PCR reaction
Utilizing the Auele Specific Primer of step 1 design, is that template is carried out pcr amplification with chukar DNA etc.
3.1PCR reaction system is 25 μ L, comprises 12.5 μ L, 2 * PCR Buffer, 5 μ L dNTPs (2mmol/L); 1 μ LKOD FX archaeal dna polymerase (1U/ μ L); Each 1 μ L (final concentration is 0.4 μ M) of upstream and downstream primers F/R, dna profiling 2 μ L, ultrapure water is mended to 25 μ L.Can adopt business-like PCR reaction mother liquor.
3.2PCR response procedures is: preparatory 94 ℃ of 2min of sex change; 94 ℃ of 30sec then, 57 ℃ of 30sec, 72 ℃ of 40sec circulations 35 times; 72 ℃ of 3min.
4. electrophoresis detection: the PCR product detects through 1.5% agarose gel electrophoresis.
5. the specificity of primer checking
Utilize the Auele Specific Primer of the domestic francolin of the identification and detection that designs (chukar) derived component; Total DNA with animals such as chicken, duck, goose, turkey, pigeon, ostrich, quail, ox, goat, pig, sheep, rabbit, fish, deer, dog, donkey, horse, chukars is a template respectively; Carry out the PCR reaction, the specificity of checking primer by above-mentioned 3.Electrophoresis detection result is as shown in Figure 1, and No. 18 sample the purpose amplified band occurs in the 184bp position, and other sample does not have chukar derived component DNA, does not then have the purpose amplified band, shows that above-mentioned primer has good specificity.
6.PCR sensitivity checking
With 10 times of pure chukar meat of gradient dilution of ultrapure water appearance DNA,, carry out PCR reaction, electrophoresis result such as Fig. 2 by above-mentioned 3 until 10-7.Can know that it detects lower bound and can reach 0.01% pure chukar meat appearance DNA concentration at least, the sensitivity of this primer can reach 0.01% pure chukar meat sample.
Claims (1)
1. a pair of primer that is used to detect domestic francolin (chukar) derived component is characterized in that described primer sequence comprises:
Upstream primer F:5 '-GCCCCGGCTAAAGACGAGGTAA-3 '
Downstream primer R:5 '-GAATTTTTGATATTTAGGGTGAG-3 '.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110290476XA CN102337336A (en) | 2011-09-28 | 2011-09-28 | Primers for PCR (polymerase chain reaction) detection of domestic francolin (rock partridge)-derived components |
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| CN201110290476XA CN102337336A (en) | 2011-09-28 | 2011-09-28 | Primers for PCR (polymerase chain reaction) detection of domestic francolin (rock partridge)-derived components |
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| CN102337336A true CN102337336A (en) | 2012-02-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265455A (en) * | 2021-05-19 | 2021-08-17 | 嘉应学院 | Rapid identification method and primer set for sex of American partridges |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006097549A1 (en) * | 2005-03-18 | 2006-09-21 | Universidad De Zaragoza | Dna analysis-based method for the identification of species of partridge and the detection of hybrids of the genus alectoris |
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2011
- 2011-09-28 CN CN201110290476XA patent/CN102337336A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006097549A1 (en) * | 2005-03-18 | 2006-09-21 | Universidad De Zaragoza | Dna analysis-based method for the identification of species of partridge and the detection of hybrids of the genus alectoris |
Non-Patent Citations (2)
| Title |
|---|
| 宗卉等: "鹌鹑源性成分PCR检测方法的建立", 《中国兽医科学》, vol. 38, no. 4, 20 April 2008 (2008-04-20) * |
| 张利平,等: "应用PCR技术检测禽类源性成分", 《中国畜牧杂志》, vol. 44, no. 11, 15 June 2008 (2008-06-15), pages 46 - 49 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265455A (en) * | 2021-05-19 | 2021-08-17 | 嘉应学院 | Rapid identification method and primer set for sex of American partridges |
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Application publication date: 20120201 |