CN104017875B - For detecting the primer pair of lolium temulentum, probe and detection method thereof - Google Patents
For detecting the primer pair of lolium temulentum, probe and detection method thereof Download PDFInfo
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Abstract
The present invention relates to agricultural and plant inspection Quarantine Techniques field, a kind of primer pair for detecting lolium temulentum and probe are provided, its sequence is respectively: nucleic acid primer F:5 '-GAATCCCGCGAACCATCGAG-3 ' or its complementary strand, nucleic acid primer R:5 '-TAAAGCGAGGTCGCCTACCA-3 ' or its complementary strand, nucleic acid molecular probe P:5 '-FAM-CACCCCACTAACTTGGTGT-TAMRA-3 ' or its complementary strand.The present invention also provides the real-time fluorescence PCR detection method utilizing this primer pair and probe in detecting lolium temulentum.Utilize primer pair provided by the invention and probe, by real-time PCR detection technology, can complete the detection of lolium temulentum within half working days, not only greatly shorten detection time, the sensitivity of detection also significantly improves.
Description
Technical field
The present invention relates to agricultural and plant inspection Quarantine Techniques field, be specifically related to primer pair and probe that one utilizes molecular Biological Detection technology for detection lolium temulentum (L.temulentum), the invention still further relates to the real-time fluorescence PCR detection method utilizing this primer pair and probe in detecting lolium temulentum.
Background technology
Lolium (Lolium L.), for grass, at home and abroad the plant of the normal lolium occurred has in document at present: lolium temulentum (L.temulentum), English ryegrass (L.perenne), Itanlian rye (L.multiflorum), hard straight rye grass (L.rigidum) etc.Because the many kinds of lolium are widely used in herbage and feed, therefore, the many kinds of this genus comprise China all over the world all have a large amount of cultivation, causes this genus worldwide extensively to distribute.
The seed of each kind of plant of lolium is very similar, but has nothing in common with each other in its hazardness and quarantine status.Lolium temulentum (L.temulentum) in lolium is poisonous plants, and the person poultry poisoning's event caused because eating lolium temulentum by mistake all can occur in the world every year.At inward cargo as in wheat, often can intercept and capture poisonous and injurious weed lolium temulentum, but due to the seed of each kind of plant of lolium and festuca (Festuca L.) etc. very similar, its Seed Identification is easily obscured.
The conventional identification method of lolium temulentum utilizes morphological characteristics of seeds, cell biological characteristics etc. to identify.Mode of appearance qualification becomes one of important means of port quarantine weeds because classifying to not kindred plant according to the surface of different plant easily.But in real work, the external appearance characteristic of the rye grass weed seed be mingled with in agricultural-food is often because transport is worn and distortion, simultaneously due to the impact of environment, weather, cultivation condition, kernel maturing do not cause individual difference on an equal basis, more identification of morphology adds difficulty, and the cycle that cell biology method etc. need is long and be difficult to precise Identification.Up to now, the achievement in research of molecular level is not yet had to can be used for identifying lolium temulentum.
Summary of the invention
One object of the present invention is to overcome the difficulty existed in identification of morphology and identification of cell biology method in above-mentioned prior art, propose to adopt molecular biological method, to detection primer, the probe of rrna the Internal Transcribed Spacer (Internal Transcribed Spacers, ITS) sequence difference design lolium temulentum.
Another object of the present invention is the real-time fluorescence PCR detection method setting up lolium temulentum (L.temulentum).
The nucleic acid primer of detection lolium temulentum provided by the invention is to being that the difference on rDNA internal transcribed spacer sequence designs based on lolium temulentum and its allied species with probe.The Internal Transcribed Spacer ITS of 18S ~ 26S Nuclear ribosomal DNA (nrDNA) is divided into ITS1 and ITS2 two portions, ITS district highly repeats, and whole nrDNA repeating unit comprises 40,000 copies and appears on one or more chromogene site in tandem sequence repeats (tandemrepeats) mode.In angiosperm, ITS district had not only had the variability of nucleotide sequence but also had had conservative property in length, illustrate that the sequence of these transcribed spacers is easy to sort between Relatives, and abundant variation (as: between genus and between planting) can solve botanical system growth problem in lower taxonomic category.Drawn by the comparison of the ITS sequence (from biology database) to different biological group: the difference between species value of the ITS sequence that the most of section of angiosperm belongs to is 1.2% ~ 10.2%, between genus, difference value is 9.6% ~ 28.8%, and this is all more suitable scope concerning phylogeny research.
First the present invention provides a kind of primer pair for detecting lolium temulentum, and its sequence is:
Nucleic acid primer F, i.e. upstream primer: 5 '-GAATCCCGCGAACCATCGAG-3 ' (SEQ ID No1) or its complementary strand,
Nucleic acid primer R, i.e. downstream primer: 5 '-TAAAGCGAGGTCGCCTACCA-3 ' (SEQ ID No2) or its complementary strand.
The present invention also provides a kind of probe for detecting lolium temulentum, and its sequence is:
Nucleic acid molecular probe P:5 '-CACCCCACTAACTTGGTGT-3 ' (SEQ ID No3) or its complementary strand, this nucleic acid molecular probe P is Taqman fluorescence probe, its 5 ' end mark fluorescent group, 3 ' end mark quenching group, described fluorophor can be: Fluoresceincarboxylic acid (FAM), and described quenching group can be: carboxyl tetramethyl-falls rhodamine (TAMRA).
Can know by inference according to nucleotide base pairing rules, the one group of nucleic acid composition formed with the nucleic acid chains of nucleic acid primer F provided by the invention, nucleic acid primer R and nucleic acid molecular probe P nucleotide sequence complete complementary is also applicable in the middle of the present invention.
Further, the invention provides a kind of nucleic acid composition for detecting lolium temulentum, comprising above-mentioned nucleic acid primer F, nucleic acid primer R and nucleic acid molecular probe P:
Nucleic acid primer F, i.e. upstream primer: 5 '-GAATCCCGCGAACCATCGAG-3 ' (SEQ ID No1) or its complementary strand,
Nucleic acid primer R, i.e. downstream primer: 5 '-TAAAGCGAGGTCGCCTACCA-3 ' (SEQ ID No2) or its complementary strand.
Nucleic acid molecular probe P:5 '-CACCCCACTAACTTGGTGT-3 ' (SEQ ID No3) or its complementary strand, this nucleic acid molecular probe P is Taqman fluorescence probe, its 5 ' end mark fluorescent group, 3 ' end mark quenching group.
Above-mentioned fluorophor can be: Fluoresceincarboxylic acid (FAM), and above-mentioned quenching group can be: carboxyl tetramethyl-falls rhodamine (TAMRA).
The present invention also provides the above-mentioned nucleic acid composition comprising nucleic acid primer F, nucleic acid primer R and nucleic acid molecular probe P lolium temulentum being carried out to the purposes in real-time PCR detection.
Further, the invention provides a kind of real-time fluorescence PCR detection method for detecting lolium temulentum, comprising the following steps:
(1) design and synthesis primer and probe: described primer is above-mentioned nucleic acid primer F and nucleic acid primer R, described probe is above-mentioned nucleic acid molecular probe P;
(2) sample DNA extracts;
(3) primer adopting step (1) to design and probe carry out real-time fluorescent PCR amplification.
In above-mentioned steps (1), the sequence of nucleic acid primer F, nucleic acid primer R and nucleic acid molecular probe P is respectively:
Nucleic acid primer F, i.e. upstream primer: 5 '-GAATCCCGCGAACCATCGAG-3 ' (SEQ ID No1) or its complementary strand,
Nucleic acid primer R, i.e. downstream primer: 5 '-TAAAGCGAGGTCGCCTACCA-3 ' (SEQ ID No2) or its complementary strand.
Nucleic acid molecular probe P:5 '-CACCCCACTAACTTGGTGT-3 ' (SEQ ID No3) or its complementary strand, this nucleic acid molecular probe P is Taqman fluorescence probe, its 5 ' end mark fluorescent group, 3 ' end mark quenching group.
Above-mentioned fluorophor can be: Fluoresceincarboxylic acid (FAM), and above-mentioned quenching group can be: carboxyl tetramethyl-falls rhodamine (TAMRA).
In above-mentioned steps (2): sample DNA extracts and adopts the ordinary method in this area to carry out.
In above-mentioned steps (3): real-time fluorescent PCR amplification reaction system is 25 μ l:2 × Premix ExTaqTM12.5 μ L, the each 1 μ L of nucleic acid primer F and nucleic acid primer R, nucleic acid molecular probe P1 μ L, DNA profiling 1 μ L, 50 × ROX Reference Dye II0.5 μ L, sterile purified water is mended to 25 μ L.Response procedures is: denaturation 95 DEG C of 10s; Then with 95 DEG C of 15s, 65 DEG C of 1min carry out 40 circulations.
The present invention is after the DNA extracting seed, nucleic acid primer F, nucleic acid primer R and nucleic acid molecular probe P is utilized to carry out real-time PCR detection, positive after pcr amplification can detect fluorescent signal, negative sample after pcr amplification and blank cannot detect the increase of fluorescent signal, can distinguish the sample of lolium temulentum and other lolium thus.
The conventional identification method of lolium temulentum utilizes morphological characteristics of seeds, cell biological characteristics etc. to identify, but mode of appearance qualification exists uncertain, and cell biology method not only needs the longer cycle, and is only still difficult to qualification according to chromosomal form.The present invention adopts the nucleic acid composition of nucleic acid primer F, nucleic acid primer R and nucleic acid molecular probe P and complementary nucleic acid chain formation thereof; real-time PCR detection technology is adopted to realize distinguishing the specificity of lolium temulentum; detection can be completed within half working days; substantially reduce detection time; and special sensitivity can identify lolium temulentum and allied species thereof quickly and accurately, be conducive to the fields such as Check and Examination of Port quarantine, agriculture production and plant protection to the Testing and appraisal fast and accurately of target sample.
Accompanying drawing explanation
Fig. 1 carries out real-time PCR detection result schematic diagram to the part in lolium temulentum in embodiment and approximate kind sample (see table 1) thereof by sample product for utilizing primer of the present invention and probe.
In figure, curve 0 is blank;
It is 21,23 that curve 1,2,3 is respectively lolium sequence number in embodiment sample table 1, the amplification curve of the lolium temulentum sample of 24;
Curve 4 is that in embodiment sample table 1, lolium sequence number is the amplification curve of the Itanlian rye of 1;
Curve 5 is that in embodiment sample table 1, lolium sequence number is the amplification curve of the English ryegrass of 2.
In Fig. 1, positive detection curve 1,2,3 increases with pcr amplification number of times, and the display of its fluorescence signal intensity obviously increases; And negative sample detection curve 4,5 and blank curve 0 do not show the increase of fluorescent signal.
Fig. 2 carries out real-time PCR detection result schematic diagram for utilizing primer of the present invention and probe another part to lolium temulentum in embodiment and approximate kind sample (see table 1) thereof by sample product.
In figure, curve 0 is blank;
Curve 1 is that in embodiment sample table 1, lolium sequence number is the amplification curve of the lolium temulentum sample of 21;
Curve 2 is that in embodiment sample table 1, lolium sequence number is the amplification curve of the English ryegrass of 10;
Curve 3 is that in embodiment sample table 1, lolium sequence number is the amplification curve of the Itanlian rye of 18;
Curve 4 is the amplification curve of the hard straight rye grass of 19 for lolium sequence number in embodiment sample table 1;
Curve 5 is that in embodiment sample table 1, festuca sequence number is the amplification curve of the alta fascue of 25.
In Fig. 2, positive detection curve 1 increases with pcr amplification number of times, and the display of its fluorescence signal intensity obviously increases; And negative sample detection curve 2,3,4,5 and blank curve 0 do not show the increase of fluorescent signal.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, embodiments of the present invention are explained in detail.
(1) design and synthesis primer and probe
Primers F, i.e. upstream primer: 5 '-GAATCCCGCGAACCATCGAG-3 ';
Primer R, i.e. downstream primer: 5 '-TAAAGCGAGGTCGCCTACCA-3 ';
Nucleic acid molecular probe P:5 '-FAM-CACCCCACTAACTTGGTGT-TAMRA-3 '.
Above-mentioned primer pair and probe entrust Shanghai Sheng Gong biotechnology company and Ji Kang biotech firm to synthesize respectively.
(2). sample DNA extracts
3 seed liquid nitrogen grind, and extract DNA with RNA isolation kit (the DP320 plant DNA extraction kit of Tian Gen company).
(3). real-time PCR detection
Real-time fluorescent PCR amplification system 25 μ l:Premix Ex TaqTM (2 ×) 12.5 μ L, upstream and downstream primer (5 μm of ol/L) each 1 μ L, TaqMan nucleic acid molecular probe P (5 μm of ol/L) 1 μ L, DNA (10 ~ 20ng/ μ L) template 1 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, sterile purified water is mended to 25 μ L, and ABI7500 type Fast Real-Time PCR System carries out real-time fluorescent PCR amplification.Response procedures is: denaturation 95 DEG C of 10s; Then with 95 DEG C of 15s, 65 DEG C of 1min carry out 40 circulations.
Adopt above-mentioned identical detecting step, detect the sample (see table 1) of the lolium temulentum and approximate kind thereof that come from different areas, detected result is in table 2:
Table 1 pcr amplification material therefor and source thereof
Table 2 PCR detects lolium temulentum and allied species result thereof
"+" represents: positive, and along with the increase of PCR cycle index, the fluorescent signal in sample is also in increase;
"-" represents: negative, along with the increase of PCR cycle index, the fluorescent signal in sample no longer increases, identical with initial conditions.
In addition, also show the amplification curve being partly subject to sample product in sample table in accompanying drawing 1 and accompanying drawing 2.
Detected result shows, detected result conforms to completely with experiment material, lolium temulentum and field lolium temulentum (positive) sample have amplification, fluorescent signal can be detected, lolium temulentum is similar to kind Itanlian rye, English ryegrass, hard straight rye grass and alta fascue (feminine gender) sample and blank do not have the increase of fluorescent signal.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above realize specific embodiments of the invention, and in actual applications, various change can be done to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (8)
1. for detecting a primer pair for lolium temulentum, it is characterized in that, its sequence is:
Nucleic acid primer F:5 '-GAATCCCGCGAACCATCGAG-3 ' (SEQ ID No 1);
Nucleic acid primer R:5 '-TAAAGCGAGGTCGCCTACCA-3 ' (SEQ ID No 2).
2. for detecting a probe for lolium temulentum, it is characterized in that, its sequence is:
Nucleic acid molecular probe P:5 '-CACCCCACTAACTTGGTGT-3 ' (SEQ ID No 3), its 5 ' end mark fluorescent group, 3 ' end mark quenching group.
3. the probe for detecting lolium temulentum according to claim 2, is characterized in that, described fluorophor is Fluoresceincarboxylic acid.
4. for detecting a nucleic acid composition for lolium temulentum, it is characterized in that, comprising nucleic acid primer F according to claim 1, nucleic acid primer R and nucleic acid molecular probe P according to claim 2.
5. nucleic acid composition according to claim 4 is carrying out the purposes in real-time PCR detection to lolium temulentum.
6. for detecting a real-time fluorescence PCR detection method for lolium temulentum, it is characterized in that, comprising the following steps:
(1) design and synthesis primer and probe: described primer is nucleic acid primer F according to claim 1 and nucleic acid primer R, described probe is nucleic acid molecular probe P according to claim 2;
(2) sample DNA extracts;
(3) primer adopting step (1) to design and probe carry out real-time fluorescent PCR amplification.
7. real-time fluorescence PCR detection method according to claim 6, it is characterized in that, described pcr amplification reaction system is 25 μ l:2 × Premix Ex TaqTM 12.5 μ L, the each 1 μ L of nucleic acid primer F and nucleic acid primer R, nucleic acid molecular probe P 1 μ L, DNA profiling 1 μ L, 50 × ROX Reference DyeII 0.5 μ L, sterile purified water is mended to 25 μ L.
8. real-time fluorescence PCR detection method according to claim 6, is characterized in that, described pcr amplification reaction program is: denaturation 95 DEG C of 10s; Then with 95 DEG C of 15s, 65 DEG C of 1min carry out 40 circulations.
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| CN104178579A (en) * | 2014-09-12 | 2014-12-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Primer pair, probe, nucleic acid composition and method for detecting Sorghum halepense |
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| CN1696642A (en) * | 2005-05-16 | 2005-11-16 | 王有福 | Verification sample of identification ability for inspecting darnel, and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1696642A (en) * | 2005-05-16 | 2005-11-16 | 王有福 | Verification sample of identification ability for inspecting darnel, and preparation method |
Non-Patent Citations (2)
| Title |
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| 毒麦及其近似种rDNA ITS-RFLP初步分析;张姮 等;《植物检疫》;20081231(第2期);全文 * |
| 毒麦属6个种的rDNA ITS序列测定及分析;张姮 等;《湖南农业大学学报(自然科学版)》;20091231(第1期);全文 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104178579A (en) * | 2014-09-12 | 2014-12-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Primer pair, probe, nucleic acid composition and method for detecting Sorghum halepense |
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