CN110093437A - The fluorescence PCR detection reagent and method of golden larch - Google Patents
The fluorescence PCR detection reagent and method of golden larch Download PDFInfo
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- CN110093437A CN110093437A CN201910246628.2A CN201910246628A CN110093437A CN 110093437 A CN110093437 A CN 110093437A CN 201910246628 A CN201910246628 A CN 201910246628A CN 110093437 A CN110093437 A CN 110093437A
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- 241000218682 Pseudolarix amabilis Species 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 42
- 239000000284 extract Substances 0.000 claims abstract description 5
- 239000006228 supernatant Substances 0.000 claims description 30
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 230000001376 precipitating effect Effects 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 claims description 6
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
- 238000011529 RT qPCR Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000012047 saturated solution Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 238000010790 dilution Methods 0.000 abstract description 5
- 239000012895 dilution Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 125000006853 reporter group Chemical group 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000012807 PCR reagent Substances 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 241000218689 Podocarpus Species 0.000 description 1
- 241000218681 Pseudolarix Species 0.000 description 1
- -1 chloroform isoamyl alcohols Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000012360 testing method Methods 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention discloses the fluorescence PCR detection reagent of golden larch and methods, the fluorescence PCR detection reagent primer sequence is respectively TTGTCTCTGC ATTTGGATTC GT and CCTGCTCCCT GGCGATTA, probe sequence is TGCCCCTTCG TTGCGTGATG C, probe contains FAM fluorescent reporter group, detection method sample DNA extracts, fluorescent PCR detects and identifies, the fluorescence PCR detection reagent is to golden larch specificity and sensitivity, the detection method can quickly detect golden larch, especially morphology is difficult to the fallen leaves stage identified, and 10‑1~10‑5Times dilution sample standard deviation can obtain typical amplification curve.
Description
Technical field
The present invention relates to the identifications of golden larch, and in particular to the fluorescence PCR detection reagent and method of golden larch.
Background technique
Pseudolarix Pinaceae Pseudolarix is the distinctive single platymiscium in China, is put into Chinese Precious, Rare, Endangered protection and plants
Name record, belongs to national II grade of focuseds protection species, and golden larch is not only important garden plant, while and a kind of application before
The extensive medicinal plant of scape.Ningbo Port exports about 6000 basin of golden larch potted landscape every year, checks based on Morphological Identification at present,
And exporting season is the stage of falling leaves in winter, therefore is badly in need of formulating " the golden larch identification side that morphology and molecular biology combine
Method ", work is checked for species resource of passing in and out, and Law Enforcement Technology support is provided.The molecular biology research of golden larch is at present to analyze
Based on its genetic diversity, molecular biological variety identification method there is no.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of fluorescence PCR detection reagent of golden larch and methods, this is glimmering
Light PCR reagent can quick, special, sensitive identification golden larch, especially morphology be difficult to the fallen leaves stage identified.
The technical scheme of the invention to solve the technical problem is: the fluorescence PCR detection reagent of golden larch, this is glimmering
The nucleotide sequence of light PCR detection reagent primer and probe is as follows:
Upstream primer: 5 '-TTGTCTCTGCATTTGGATTCGT-3 '
Downstream primer: 5 '-CCTGCTCCCTGGCGATTA-3
Probe: 5 '-FAM-TGCCCCTTCGTTGCGTGATGC-Eclipse-3 '.
Primer and probe is according to listed golden larch in GenBank (http://www.ncbi.nlm.nih. gov)
Gene order-endogenous gene referring to 18SrRNA sequence in SN/T 1204-2016, homology ratio is carried out using Blast program
Compared with obtaining the specificity of golden larch using 3.0 software design of Primer Express in the high conservative region of homology sequence and draw
FAM fluorescent reporter group is contained at object and probe, the end of probe 5 ', and not fluorescent quenching group Eclipse is contained at 3 ' ends.
Using the method for the fluorescence PCR detection reagent detection golden larch of golden larch, its step are as follows:
Sample DNA extracts: it takes 100mg powder sample in 1.5mL centrifuge tube, 600 μ L CTAB extracting solutions is added, sufficiently oscillation mixes,
65 DEG C of water-bath 30min, are mixed by inversion frequently；Then 10000r/min is centrifuged 5min, takes supernatant, supernatant with the supernatant in equal volume
The extracting of chloroform isoamyl alcohol mixed liquor, extracting finish and take supernatant, are added 2 times of Volume CT AB precipitated liquids of the supernatant, precipitation at room temperature 1h,
12000r/m is centrifuged 10min, abandons supernatant；350 μ L NaCl saturated solutions dissolution precipitating is added, then mixed with 350 μ L chloroform isoamyl alcohols
Liquid extracting is closed, supernatant is taken, adds the isometric isopropanol of the supernatant in supernatant, 4 DEG C of precipitating at least 10min, then 12000r/
M is centrifuged 10min, abandons supernatant；After drying, it is heavy that 100 μ L TE buffer solutions are added in the ethanol washing of mass concentration 70% 1~2 time
It forms sediment, obtain sample DNA templates, 4 DEG C save backup, chloroform in above-mentioned chloroform isoamyl alcohol mixed liquor: isoamyl alcohol volume ratio is
24:1；
Fluorescent PCR detection: taking 4 μ L of sample DNA templates, and following detection reagents: Probe qPCR Master Mix-2 × slow are added
10 μ L of fliud flushing, concentration are 10 μm of 0.4 μ L of ol/L upstream primer, and concentration is 10 μm of 0.4 μ L of ol/L upstream primer, and concentration is 10 μ
0.8 μ L, ROX0.4 μ L of mol/L probe, is mended with pure water to 20 μ L；50 DEG C of 2 min that depollute detect response parameter are as follows: initial denaturation
95℃ 10min；95 DEG C of 15s, 60 DEG C of 30s, 40 circulations；
Identify: it is then golden larch that fluorescent PCR detection, which has fluorescence response, can also further analyze amplification curve, there is typical expansion
Increasing curve is just golden larch.
Above-mentioned CTAB extracting solution, CTAB precipitated liquid, Probe qPCR Master Mix-2 × buffer and ROX are bought
In Beijing Quan Shijin biotech firm.
Compared with the prior art, the advantages of the present invention are as follows the fluorescence PCR detection reagent of golden larch and method, the fluorescence
PCR detection reagent primer sequence is respectively TTGTCTCTGC ATTTGGATTC GT and CCTGCTCCCT GGCGATTA, probe sequence
It is classified as TGCCCCTTCG TTGCGTGATG C, probe contains FAM fluorescent reporter group, the extraction of detection method sample DNA, fluorescence
PCR detection and identification, the fluorescence PCR detection reagent can quickly detect golden larch specificity and sensitivity, the detection method
Golden larch, especially morphology are difficult to the fallen leaves stage identified, and 10-1~10-5Times dilution sample standard deviation can obtain typical amplification
Curve.
Specific embodiment
Present invention is further described in detail with reference to embodiments.
Embodiment 1
Sample DNA extracts: it takes 100mg powder sample in 1.5mL centrifuge tube, 600 μ L CTAB extracting solutions is added, sufficiently oscillation mixes,
65 DEG C of water-bath 30min, are mixed by inversion frequently；Then 10000r/min is centrifuged 5min, takes supernatant, supernatant with the supernatant in equal volume
Chloroform isoamyl alcohol mixed liquor (chloroform: isoamyl alcohol volume ratio is 24:1) extracting, extracting, which finishes, takes supernatant, and the supernatant 2 is added
Times Volume CT AB precipitated liquid, precipitation at room temperature 1h, 12000r/m are centrifuged 10min, abandon supernatant；It is molten that 350 μ L NaCl saturated solutions are added
Solution precipitating, then extracted with 350 μ L chloroform isoamyl alcohol mixed liquors, supernatant is taken, the isometric isopropanol of the supernatant is added in supernatant,
4 DEG C of precipitating at least 10min, then 12000r/m is centrifuged 10min, abandons supernatant；The ethanol washing of mass concentration 70% 1~2 time, dries in the air
After dry, 100 μ L TE buffer solutions precipitating is added, obtains sample DNA templates, 4 DEG C save backup.
Embodiment 2
Sensitivity technique: the sample DNA templates measurement nucleic acid concentration of embodiment 1 is 1.6 ng/ μ L, and sample DNA templates are formed
10-1~106Times gradient dilution, with fluorescence PCR detecting method: taking 4 μ L of each sample, following detection reagents: Probe qPCR are added
10 μ L of Master Mix-2 × buffer, concentration are 1 μ 0mol/L upstream primer, 0.4 μ L, and concentration is 10 μm of ol/L upstream primers
0.4 μ L, concentration are 10 μm of 0.8 μ L, ROX0.4 μ L of ol/L probe, are mended with pure water to 20 μ L；50 DEG C of 2 min that depollute, detection are anti-
Answer parameter are as follows: 95 DEG C of 10min of initial denaturation；95 DEG C of 15s, 60 DEG C of 30s, 40 circulations；Amplification shows sample DNA templates
With 10-1~10-5Times dilution sample standard deviation can obtain typical amplification curve, and threshold line is arranged according to negative control, obtains its Ct
Value is respectively 19.35,22.71,25.94,29.48,32.79,35.55.10-6 DNA dilution sample is not expanded typically
Curve is determined as feminine gender, and the related coefficient of the standard curve of the sensitivity test is 0.994.
Embodiment 3
Specific detection: the sample DNA templates of embodiment 1, in addition the phase Tongfang of podocarpus, wet-land pine tree and five-leaved pine embodiment 1
Method extracts DNA profiling, then is detected respectively with the fluorescence PCR detecting method of embodiment 2, as the result is shown the sample DNA mould of embodiment 1
Plate has fluorescence response, and has typical amplification curve, and other three kinds have no fluorescence response, illustrates that the present invention has golden larch
Specificity.
Sequence table
<110>Ningbo Institute of Inspection and Quarantine Science Technology
<120>fluorescence PCR detection reagent and method of golden larch
<160> 3
<170> PatentIn version 3.1
<210>1
<211> 22
<212>DNA
<213>artificial sequence
<220>
<223>fluorescent PCR of golden larch detects upstream primer
<400> 1
TTGTCTCTGC ATTTGGATTC GT
210>2
<211> 18
<212>DNA
<213>artificial sequence
<220>
<223>fluorescent PCR of golden larch detects downstream primer
<400> 2
CCTGCTCCCT GGCGATTA
210>3
<211> 21
<212>DNA
<213>artificial sequence
<220>
<223>the fluorescent PCR detection probe of golden larch
<400>3
TGCCCCTTCG TTGCGTGATG C 21
Claims (2)
1. the fluorescence PCR detection reagent of golden larch, it is characterised in that the nucleotides sequence of the fluorescence PCR detection reagent primer and probe
Column are as follows: upstream primer is TTGTCTCTGC ATTTGGATTC GT, and downstream primer is CCTGCTCCCT GGCGATTA,
Probe is FAM-TGCCCCTTCG TTGCGTGATG C-Eclipse.
2. the method for the fluorescence PCR detection reagent detection golden larch using the golden larch of claim 1, it is characterised in that step is such as
Under:
A, sample DNA extracts: taking 100mg powder sample in 1.5mL centrifuge tube, 600 μ L CTAB extracting solutions are added, sufficiently oscillation is mixed
Even, 65 DEG C of water-bath 30min are mixed by inversion frequently；Then 10000r/min is centrifuged 5min, takes supernatant, the supernatant bodies such as the supernatant
Long-pending chloroform isoamyl alcohol mixed liquor extracting, extracting, which finishes, takes supernatant, and 2 times of Volume CT AB precipitated liquids of the supernatant, precipitation at room temperature is added
1h, 12000r/m are centrifuged 10min, abandon supernatant；350 μ L NaCl saturated solutions dissolution precipitating is added, then with 350 μ L chloroform isoamyls
The extracting of alcohol mixed liquor, takes supernatant, adds the isometric isopropanol of the supernatant in supernatant, 4 DEG C of precipitating at least 10min, then
12000r/m is centrifuged 10min, abandons supernatant；After drying, 100 μ L TE buffering is added in the ethanol washing of mass concentration 70% 1~2 time
Liquid dissolution precipitating, obtains sample DNA templates, 4 DEG C save backup, chloroform in above-mentioned chloroform isoamyl alcohol mixed liquor: isoamyl alcohol
Volume ratio is 24:1；
B, fluorescent PCR detects: 4 μ L of sample DNA templates is taken, is added following detection reagents: Probe qPCR Master Mix-2 ×
10 μ L of buffer, concentration are 10 μm of 0.4 μ L of ol/L upstream primer, and concentration is 10 μm of 0.4 μ L of ol/L upstream primer, and concentration is 10 μ
0.8 μ L, ROX0.4 μ L of mol/L probe, is mended with pure water to 20 μ L；50 DEG C of 2 min that depollute detect response parameter are as follows: initial denaturation
95℃ 10min；95 DEG C of 15s, 60 DEG C of 30s, 40 circulations；
C, identify: it is then golden larch that fluorescent PCR detection, which has fluorescence response,.
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| CN201910246628.2A CN110093437A (en) | 2019-03-29 | 2019-03-29 | The fluorescence PCR detection reagent and method of golden larch |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484642A (en) * | 2019-08-20 | 2019-11-22 | 宁波检验检疫科学技术研究院 | A kind of real-time fluorescence PCR detection method of kit, its application and ginkgo |
| CN113718055A (en) * | 2021-10-22 | 2021-11-30 | 南京海关动植物与食品检测中心 | Method and kit for identifying Magasjialong tree |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967807A (en) * | 2017-04-07 | 2017-07-21 | 中国医学科学院药用植物研究所 | A kind of method for identifying molecules of poisonous medicine materical crude slice Golden Larch Bark |
-
2019
- 2019-03-29 CN CN201910246628.2A patent/CN110093437A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967807A (en) * | 2017-04-07 | 2017-07-21 | 中国医学科学院药用植物研究所 | A kind of method for identifying molecules of poisonous medicine materical crude slice Golden Larch Bark |
Non-Patent Citations (4)
| Title |
|---|
| XIAN-ZHAO KAN: "Structural evolution of nrDNA ITS in Pinaceae and its phylogenetic implications", 《MOLECULAR PHYLOGENETICS AND EVOLUTION》 * |
| 佚名: "GenBank登录号: DQ975355.1", 《NCBI》 * |
| 张吉红: "金钱松实时荧光PCR鉴定方法的建立", 《植物检疫》 * |
| 朱水芳: "《实时荧光聚合酶链反应PCR检测技术》", 31 July 2003, 中国计量出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484642A (en) * | 2019-08-20 | 2019-11-22 | 宁波检验检疫科学技术研究院 | A kind of real-time fluorescence PCR detection method of kit, its application and ginkgo |
| CN110484642B (en) * | 2019-08-20 | 2023-05-05 | 宁波检验检疫科学技术研究院 | Kit, application thereof and real-time fluorescence PCR detection method of ginkgo |
| CN113718055A (en) * | 2021-10-22 | 2021-11-30 | 南京海关动植物与食品检测中心 | Method and kit for identifying Magasjialong tree |
| CN113718055B (en) * | 2021-10-22 | 2023-05-26 | 南京海关动植物与食品检测中心 | Method and kit for identifying Gastrodia elata |
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