CN113005216A - Specific genetic marker composition for identifying poppy and 3 allied species thereof - Google Patents
Specific genetic marker composition for identifying poppy and 3 allied species thereof Download PDFInfo
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Abstract
The invention discloses a primer pair composition, a PCR reagent, a PCR kit and a detection method for identifying poppy. The primer pair composition consists of 8 primer pairs, namely a primer pair P136, a primer pair P90, a primer pair BP13, a primer pair BP12, a primer pair P107, a primer pair P85, a primer pair BP14 and a primer pair P15. The primer pair composition can simultaneously amplify 8 SSR loci, is more accurate and quicker than the amplification of a single locus and the amplification of the prior 4 locus systems, and has higher species and interspecific specificity in the aspect of identifying poppy and 3 related plants (corn poppy, wild poppy and ghost poppy).
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a specific genetic marker composition for identifying poppy and 3 allied species thereof.
Background
Papaver (Papaver) is a plant belonging to the genus Papaveraceae (Papaveraceae) of the order ranuncules (ranunculus), and there are 120 or more species worldwide. The genus is mainly distributed in temperate regions of central europe, south europe to asia, and a few species are produced in america, oceania and south africa. In China, the poppy genus mainly includes 7 species of poppy (p.somniferum L.), poppy (p.rhoeas L.), poppy (p.rhoeas L.), poppy nodosa (p.nudicaule L.), poppy glauber (p.canescens L.), poppy (p.orientale L.), poppy nigre (p.pavoninum L.), and poppy longshanensis (p.radiata var. pseudo-oradiatum L.), which are mainly distributed in northeast, northwest, Ningxia, inner Mongolia and the like, wherein poppy, poppy nodosa and poppy are cultivated and distributed widely in many domestic areas because of their ornamental and medicinal values.
Poppy (Papaver somniferum L) is combined with hemp (Cannabis sativa L), erythrophyllum (Erythroxylumnovogranatum (Morris) Hier.) and called the world's three major narcotics. In China, illegal planting of the original drug plant poppy is one of the important sources of domestic drugs, and the poppy is also a main raw material for preparing some new drugs with strong concealment besides preparing common heroin and opium. Some illegal poppy planters cover the ear, and are purposely covered by planting closely related plants such as corn poppy and devil poppy in the poppy garden. The poppy has high morphological similarity with closely related species such as poppy, wild poppy, ghost poppy and other ornamental flowers and medicinal plants, and is difficult to identify only in appearance, which brings great difficulty to the distinction and identification of poppy in forensic science. The traditional identification method based on plant morphology and chemical physical and chemical analysis is very easily interfered by seasonality, material detection integrity and putrefaction degree. As an efficient molecular marker, the DNA molecular marker can be used for quickly, simply and conveniently identifying species and between species and is developed rapidly.
At present, molecular marker research carried out by poppy plants is few, and SSR molecular markers and systems which can be used for distinguishing and identifying poppy and related species thereof are rarely reported.
SSR sites have codominant inheritance, accord with Mendelian inheritance rule and have individual recognition capability; the amount of DNA required for detection is small, and degraded DNA can be identified; the polymorphism is high, the species specificity is strong, and the quantity is rich; the experimental repeatability is good, and the result is reliable; most SSRs have no functional effect, the increasing or decreasing frequency of repeated sequences is high, and the polymorphism among varieties is high. In addition, the SSR detection technology also has the advantages of simple experiment procedures, short experiment period, convenience in automatic detection and the like.
The patent application of the poppy species specific genetic marker detection system (CN105861711A) fills the blank state of the poppy SSR composite amplification system at home and abroad. However, the system only comprises 4 sites, only can distinguish and identify poppy related species corn poppy, and the detection capability of the system also has a great space.
Disclosure of Invention
The invention provides a primer pair composition for identifying poppy, which consists of 8 primer pairs, namely a primer pair P136, a primer pair P90, a primer pair BP13, a primer pair BP12, a primer pair P107, a primer pair P85, a primer pair BP14 and a primer pair P15.
The primer pair P136 consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
The primer pair P90 consists of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table.
The primer pair BP13 consists of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table.
The primer pair BP12 consists of a single-stranded DNA molecule shown in a sequence 7 in a sequence table and a single-stranded DNA molecule shown in a sequence 8 in the sequence table.
The primer pair P107 consists of a single-stranded DNA molecule shown in a sequence 9 in a sequence table and a single-stranded DNA molecule shown in a sequence 10 in the sequence table.
The primer pair P85 consists of a single-stranded DNA molecule shown in a sequence 11 in a sequence table and a single-stranded DNA molecule shown in a sequence 12 in the sequence table.
The primer pair BP14 consists of a single-stranded DNA molecule shown in a sequence 13 in a sequence table and a single-stranded DNA molecule shown in a sequence 14 in the sequence table.
The primer pair P15 consists of a single-stranded DNA molecule shown in a sequence 15 in a sequence table and a single-stranded DNA molecule shown in a sequence 16 in the sequence table.
Optionally, the molar ratio of the primer pair P136, the primer pair P90, the primer pair BP13, the primer pair BP12, the primer pair P107, the primer pair P85, the primer pair BP14 and the primer pair P15 is 3: 0.5: 6: 0.5: 2, and/or the molar ratio between the two primers in each primer pair is 1: 1.
Optionally, the 5' end of at least one primer of each of the 8 primer pairs is labeled with a fluorescent dye.
Optionally, the primers in the primer pair P136, the primer pair P90, the primer pair BP13, the primer pair BP12 are labeled with a fluorescent dye a; the primer pair P107, the primer pair P85 and the primer pair BP14 are labeled by a fluorescent dye B; the primer pair P15 is labeled with fluorescent dye C. The fluorescent dye A, the fluorescent dye B and the fluorescent dye C are fluorescent dyes with different colors. For example, the fluorescent dye is FAM, HEX, TAMRA. P136, P90, BP13 and BP12 are a group, and corresponding primers are marked by FAM; p107, P85 and BP14 are a group, and corresponding primers are marked by HEX; p15 is a group, and TAMRA is used as the corresponding primer.
The invention also provides a PCR reagent for identifying poppy, which comprises the primer pair composition.
In the above PCR reagents, the final concentration of each primer in the primer pair P136 in the PCR amplification system can be 0.3. mu.M, the final concentration of each primer in the primer pair P90 in the PCR amplification system can be 0.05. mu.M, the final concentration of each primer in the primer pair BP13 in the PCR amplification system can be 0.05. mu.M, the final concentration of each primer in the primer pair BP12 in the PCR amplification system can be 0.6. mu.M, the final concentration of each primer in the primer pair P107 in the PCR amplification system can be 0.05. mu.M, the final concentration of each primer in the primer pair P85 in the PCR amplification system can be 0.05. mu.M, the final concentration of each primer in the primer pair BP14 in the PCR amplification system can be 0.2. mu.M, and the final concentration of each primer in the primer pair P15 in the PCR amplification system can be 0.2. mu.M.
The invention also provides a PCR kit for identifying poppy, which comprises the primer pair composition or the PCR reagent.
The primer pair composition, the PCR reagent or the PCR kit are also used in the present invention.
The application is any one of A1) -A6) as follows:
A1) use in differentiating poppy and its allied species, the allied species being papaver rhoeas, poppy wildly and/or poppy ghost;
A2) use in the preparation of a product for differentiating poppy and its allied species, which are poppy, wild poppy and/or ghost poppy;
A3) use in identifying or assisting in identifying poppy;
A4) the application in preparing products for identifying or assisting in identifying poppy;
A5) the application in identifying or assisting in identifying whether the sample to be detected contains poppy or not;
A6) the application of the product in preparing and identifying or assisting in identifying whether the sample to be detected contains poppy or not.
The invention also provides a method for detecting or assisting in detecting whether a sample to be detected is poppy or contains poppy, which comprises the following steps:
(a1) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the 8 primer pairs in the primer pair composition to obtain an amplification product;
(b1) detecting the amplification product, and determining whether the sample to be detected is poppy or contains poppy according to the detection result as follows: if the amplification product meets the following 1) -8) all standards, the sample to be detected is or is candidate to be poppy, or contains or is candidate to contain poppy; if the amplification product does not meet all the following 1) -8), the sample to be tested is not or is not candidate to be poppy, or does not contain or is candidate not to contain poppy;
1) contains allele fragments with the size of 97-99bp (such as 98bp) obtained by carrying out PCR amplification on the primer pair P136;
2) contains two allele fragments with the size of 129-131bp (such as 130bp) and/or 133-135bp (such as 134bp) obtained by PCR amplification of the primer pair P90;
3) contains allele fragments with the size of 182-184BP (such as 183BP) obtained by PCR amplification of the primer pair BP 13;
4) contains allele fragments with the size of 242-244BP (such as 242BP) obtained by PCR amplification of the primer pair BP 12;
5) contains allele fragments with the size of 111-113bp (such as 112bp) or 115-117bp (such as 116bp) obtained by PCR amplification of the primer pair P107;
6) contains an allele fragment with the size of 147-149bp (such as 148bp) obtained by carrying out PCR amplification on the primer pair P85;
7) contains the allele fragment with the size of 210-212BP (such as 210BP) and the allele fragment with the size of 222-224BP (such as 222BP) obtained by amplifying the primer pair BP 14;
8) contains allele fragments with the size of 151-153bp (such as 152bp) or 154-156bp (such as 155bp) obtained by amplifying the primer pair P15.
The invention also provides a method for detecting or assisting in detecting whether a sample to be detected is poppy or contains poppy, which comprises the following steps:
(a2) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the 8 primer pairs in the primer pair composition to obtain an amplification product;
(b2) detecting the amplification product, and determining whether the sample to be detected is poppy or contains poppy according to the detection result as follows: if the amplification product meets the following 1) -8) all standards, the sample to be detected is or is candidate to be poppy, or contains or is candidate to contain poppy; if the amplification product does not meet all the following 1) -8), the sample to be tested is not or is not candidate to be poppy, or does not contain or is candidate not to contain poppy;
1) contains allele segment showing fluorescence color of the fluorescent dye A and having size of 97-99bp (such as 98 bp);
2) contains two allele fragments which show the fluorescence color of the fluorescent dye A and have the size of 129-131bp (such as 130bp) and/or 133-135bp (such as 134 bp);
3) contains allele segment which shows the fluorescence color of the fluorescent dye A and has the size of 182-184bp (such as 183 bp);
4) contains an allele fragment showing the fluorescence color of the fluorescent dye A and having a size of 241-243bp (e.g., 242 bp);
5) contains allele fragments which show the fluorescence color of the fluorescent dye B and have the size of 111-113bp (such as 112bp) or 115-117bp (such as 116 bp);
6) contains allele segment which shows the fluorescence color of the fluorescent dye B and has the size of 147-149bp (such as 148 bp);
7) contains two allele fragments which show the fluorescence color of the fluorescent dye B and have the size of 210-212bp (such as 210bp) and 222-224bp (such as 222 bp);
8) contains allele fragments which show the fluorescence color of the fluorescent dye C and have the size of 151-153bp (such as 152bp) or 154-156bp (such as 155 bp).
Specifically, in the above method, the PCR amplification procedure is: initial denaturation at 95 ℃ for 11 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 2min, extension at 72 ℃ for 30s, 28 cycles; final extension at 60 deg.C for 60 min; keeping the temperature at 25 ℃.
The invention also provides a poppy-specific genetic marker composition comprising SSR loci BP12, BP13, BP14, P15, P85, P90, P107 and P136. The BP12 is a fragment obtained by carrying out PCR amplification on BP12 by using poppy genomic DNA as a template. The BP13 is a fragment obtained by carrying out PCR amplification on BP13 by using poppy genomic DNA as a template. The BP14 is a fragment obtained by carrying out PCR amplification on BP14 by using poppy genomic DNA as a template. The P15 is a fragment obtained by using poppy genomic DNA as a template and carrying out PCR amplification on the P15 by adopting the primer pair. The P85 is a fragment obtained by using poppy genomic DNA as a template and carrying out PCR amplification on the P15 by adopting the primer pair. The P85 is a fragment obtained by using poppy genomic DNA as a template and carrying out PCR amplification on the P85 by adopting the primer pair. The P90 is a fragment obtained by using poppy genomic DNA as a template and carrying out PCR amplification on the P90 by adopting the primer pair. The P107 is a fragment obtained by carrying out PCR amplification on the P107 by using poppy genome DNA as a template and adopting the primer. The P136 is a fragment obtained by carrying out PCR amplification on the poppy genomic DNA by using the primer pair P90.
The invention establishes a fluorescence composite amplification detection system containing 8 sites, which can simultaneously distinguish poppy and its related species corn poppy, wild poppy and ghost poppy.
The primer pair composition, the PCR reagent and the PCR kit can simultaneously amplify 8 SSR gene loci, are more accurate and quicker than the amplification of a single gene locus and the amplification of the prior 4 gene locus systems, have higher species and interspecific specificity in the aspect of identifying poppy and 3 closely related plants (corn poppy, wild poppy and ghost poppy), and provide an effective method for the detection and identification of poppy interspecific in related cases. The invention has important application value.
The optimized amplification system can realize balanced amplification, has high sensitivity and stability, and is favorable for detecting putrefactive detection materials frequently encountered in drug-related cases.
The primer pair composition, the PCR reagent and the PCR kit have high detection accuracy. The examples demonstrate that poppy samples taken from different origins, both tested and subjected to data analysis, can be genotyped at 8 loci each.
Drawings
FIG. 1 shows the results of the measurement of Papaver somniferum L.from Asian origin.
FIG. 2 shows the results of Wuwei-derived poppy assay.
FIG. 3 shows the results of the Wuhan-derived poppy assay.
Fig. 4 shows the measurement results of poppy from shaoguan.
FIG. 5 shows the results of detection of Linzhi-derived poppy.
FIG. 6 shows the results of the detection of Hangjin derived poppy.
FIG. 7 shows the measurement results of the Papaver somniferum L.from south China.
FIG. 8 shows the results of the measurement of poppy from Fuyang.
FIG. 9 shows the results of the wild poppy test.
FIG. 10 shows the results of the detection of Argemone pauciflorum.
FIG. 11 shows the result of the Papaver rhoeas assay.
FIG. 12 shows the results of rice testing.
FIG. 13 shows the measurement results of Ephedra.
FIG. 14 shows the results of hemp detection.
In FIGS. 1 to 14, the upper panel shows the result of FAM, the middle panel shows the result of HEX, and the lower panel shows the result of TAMRA.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 SSR primer preparation and detection methods
Preparation of SSR primer
The poppy genome was screened for interspecific specific and appropriately long loci as shown in table 1 below.
TABLE 1 selected poppy loci
According to the primers in table 1, the 5' end of the forward primer is labeled with a fluorescent dye to prepare the SSR primer, and the specific fluorescent dye used is shown in table 2.
TABLE 2 primer modifications
Second, detection method
1. Extracting the genome DNA of a sample to be detected;
2. and carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the SSR primer prepared in the step one. The PCR amplification system is specifically shown in tables 3 and 4.
TABLE 3 PCR amplification System
TABLE 4 concentration of each primer in PCR amplification System
Amplification cycle procedure and parameters:
initial denaturation conditions: at 95 ℃ for 11 min;
and (3) denaturation conditions: 30s at 94 ℃;
annealing conditions: at 59 ℃ for 2 min;
and (3) extension conditions: 72 ℃ for 30 s;
circulation conditions are as follows: 28 times of
Final extension conditions: 60min at 60 ℃;
and (3) heat preservation: maintaining at 25 ℃.
3. Performing fluorescence detection on the amplification product obtained in the step 2 on a genetic analyzer;
4. carrying out typing analysis on the fluorescence detection result, and if the amplification product meets the following 1) -8) all standards, determining that the sample to be detected is or is candidate to be poppy, or contains or is candidate to contain poppy; if the amplification product does not meet all the following 1) -8), the sample to be tested is not or is not candidate to be poppy, or does not contain or is candidate not to contain poppy;
1) contains allele fragments which display the fluorescence color of the fluorescent dye FAM (blue is displayed by an instrument) and have the size of 97-99 bp;
2) two allele fragments which show the fluorescence color (blue) of the fluorescent dye FAM and have the size of 129-131bp and/or 133-135 bp;
3) contains an allele fragment showing the fluorescence color (blue) of the fluorescent dye FAM and having the size of 182-184 bp;
4) contains an allele fragment showing the fluorescence color (blue) of the fluorescent dye FAM and having a size of 241-243 bp;
5) contains an allele fragment which shows the fluorescence color of the fluorescent dye HEX (the instrument shows green) and has the size of 111-113bp or 115-117 bp;
6) contains allele segment which shows the fluorescence color (green) of the fluorescent dye HEX and has the size of 147-149 bp;
7) contains two allele fragments which show the fluorescence color (green) of the fluorescent dye HEX and have the size of 210-212bp and 222-224 bp;
8) contains an allele fragment which shows the fluorescence color of the fluorescent dye TAMRA (the instrument shows black) and has the size of 151-153bp or 154-156 bp.
The SSR primers prepared above are adopted to detect poppy, corn poppy, ghost poppy and wild poppy respectively by the detection method. The results of capillary electrophoresis are shown in Table 5.
TABLE 5 amplified fragment sizes at each site
Wherein "-" indicates that there is no specifically amplified fragment.
Example 2 detection of poppy in different places of origin
Using the SSR primers prepared in example 1, the method of example 1 was used to detect the DNA content of poppy from Ardong, Wuwei, Wuhan, Shaoguan, Linzhi, Hangjin, Nanning and Fuyang, respectively, and the amount of poppy was 1-4 ng.
The detection results are shown in figures 1-8, and the amplification products of poppy from yadong, wuwei, wuhan, shaoguan, linzhi, hangjin, nanning and funyang all meet all the standards 1) -8) in the detection method of the above example 1, and the accuracy is high.
Example 3 detection of specificity between species
Using the SSR primers prepared in example 1, wild poppy, ghost poppy and corn poppy, which are related species of poppy, were detected by the detection method in example 1, and the amount of the genome of the sample to be detected was 1-4 ng.
As shown in FIGS. 9 to 11, the results of the detection were all that was not satisfied with the above detection methods 1) to 8), in which the amplified product of Argemone mexicana contained an allele fragment (BP14) exhibiting a color of HEX group and having a size of 208 BP; the amplified product of the poppy foenum-graecum contains an allele fragment (BP14) which shows the color of the HEX group and has the size of 205 BP; the corn poppy amplification product contains two allelic fragments with FAM group color and size of 180BP (BP13) and TAMRA group color and size of 205BP (BP 14).
Example 4 intergeneric specificity assays
In the botanical classification, the species Papaver (Papaver somniferum L.) belonging to the genus Papaver of the genus papaveraceae of the order Papaver of the phylum angiospermae is not in the same genus as rice, ephedra herb and hemp, but it is difficult to identify the species in terms of morphology, and thus identification is also required.
The SSR primers prepared in the embodiment 1 are used for detecting rice, ephedra herb and hemp by the detection method in the embodiment 1, and the genome consumption of the sample to be detected is 1-4 ng.
As shown in FIGS. 12 to 14, the results of the tests showed that the rice, the ephedra herb and the hemp did not satisfy all the criteria 1) to 8) of the above-mentioned test methods, and the corresponding gene fragments did not show the specific gene fragments belonging to the poppy in these non-poppy plants.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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Claims (10)
1. A primer pair composition for identifying poppy, comprising: the primer pair consists of 8 primer pairs, namely a primer pair P136, a primer pair P90, a primer pair BP13, a primer pair BP12, a primer pair P107, a primer pair P85, a primer pair BP14 and a primer pair P15;
the primer pair P136 consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table;
the primer pair P90 consists of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
the primer pair BP13 consists of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table;
the primer pair BP12 consists of a single-stranded DNA molecule shown in a sequence 7 in a sequence table and a single-stranded DNA molecule shown in a sequence 8 in the sequence table;
the primer pair P107 consists of a single-stranded DNA molecule shown in a sequence 9 in a sequence table and a single-stranded DNA molecule shown in a sequence 10 in the sequence table;
the primer pair P85 consists of a single-stranded DNA molecule shown in a sequence 11 in a sequence table and a single-stranded DNA molecule shown in a sequence 12 in the sequence table;
the primer pair BP14 consists of a single-stranded DNA molecule shown in a sequence 13 in a sequence table and a single-stranded DNA molecule shown in a sequence 14 in the sequence table;
the primer pair P15 consists of a single-stranded DNA molecule shown in a sequence 15 in a sequence table and a single-stranded DNA molecule shown in a sequence 16 in the sequence table.
2. The primer pair composition of claim 1, wherein: the molar ratio of the primer pair P136, the primer pair P90, the primer pair BP13, the primer pair BP12, the primer pair P107, the primer pair P85, the primer pair BP14 and the primer pair P15 is 3: 0.5: 6: 0.5: 2, and/or the molar ratio between the two primers in each primer pair is 1: 1.
3. Primer-pair composition according to claim 1 or 2, characterized in that: the 5' end of at least one primer in each of the 8 primer pairs is labeled with a fluorescent dye.
4. The primer pair composition of claim 3, wherein: the primers in the primer pair P136, the primer pair P90, the primer pair BP13 and the primer pair BP12 are labeled by a fluorescent dye A; the primer pair P107, the primer pair P85 and the primer pair BP14 are labeled by a fluorescent dye B; the primer pair P15 is labeled by a fluorescent dye C, and the fluorescent dye A, the fluorescent dye B and the fluorescent dye C are fluorescent dyes with different colors.
5. A PCR reagent for identifying poppy, characterized in that: comprising the primer set composition of any one of claims 1-4.
6. A PCR kit for identifying poppy, characterized in that: comprising the primer set composition of any one of claims 1 to 4 or the PCR reagent of claim 5.
7. Use of the primer set composition of any one of claims 1 to 4, the PCR reagent of claim 5 or the PCR kit of claim 6) in any one of a1) -a6) below;
A1) use in differentiating poppy and its allied species, the allied species being papaver rhoeas, poppy wildly and/or poppy ghost;
A2) use in the preparation of a product for differentiating poppy and its allied species, which are poppy, wild poppy and/or ghost poppy;
A3) use in identifying or assisting in identifying poppy;
A4) the application in preparing products for identifying or assisting in identifying poppy;
A5) the application in identifying or assisting in identifying whether the sample to be detected contains poppy or not;
A6) the application of the product in preparing and identifying or assisting in identifying whether the sample to be detected contains poppy or not.
8. A method for detecting or assisting in detecting whether a sample to be detected is poppy or contains poppy, which is characterized in that: the method comprises the following steps:
(a1) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the 8 primer pairs in the primer pair composition according to claim 1 to obtain an amplification product;
(b1) detecting the amplification product, and determining whether the sample to be detected is poppy or contains poppy according to the detection result as follows: if the amplification product meets the following 1) -8) all standards, the sample to be detected is or is candidate to be poppy, or contains or is candidate to contain poppy; if the amplification product does not meet all the following 1) -8), the sample to be tested is not or is not candidate to be poppy, or does not contain or is candidate not to contain poppy;
1) contains allele segments with the size of 97-99bp obtained by PCR amplification of the primer pair P136;
2) contains two allele fragments with the size of 129-131bp and/or 133-135bp obtained by PCR amplification of the primer pair P90;
3) contains an allele fragment with the size of 182-184BP obtained by carrying out PCR amplification on the primer pair BP 13;
4) contains an allele fragment with the size of 242-244BP obtained by PCR amplification of the primer pair BP 12;
5) contains an allele fragment with the size of 111-113bp or 115-117bp obtained by carrying out PCR amplification on the primer pair P107;
6) contains an allele fragment with the size of 147-149bp obtained by carrying out PCR amplification on the primer pair P85;
7) contains an allele fragment with the size of 210-212BP and an allele fragment with the size of 222-224BP, which are obtained by amplifying the primer pair BP 14;
8) contains an allele fragment with the size of 151-153bp or 154-156bp obtained by amplifying the primer pair P15.
9. A method for detecting or assisting in detecting whether a sample to be detected is poppy or contains poppy, which is characterized in that: the method comprises the following steps:
(a2) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the 8 primer pairs in the primer pair composition according to claim 4 to obtain an amplification product;
(b2) detecting the amplification product, and determining whether the sample to be detected is poppy or contains poppy according to the detection result as follows: if the amplification product meets the following 1) -8) all standards, the sample to be detected is or is candidate to be poppy, or contains or is candidate to contain poppy; if the amplification product does not meet all the following 1) -8), the sample to be tested is not or is not candidate to be poppy, or does not contain or is candidate not to contain poppy;
1) contains allele segment which shows the fluorescence color of the fluorescent dye A and has the size of 97-99 bp;
2) contains two allele fragments which show the fluorescence color of the fluorescent dye A and have the size of 129-131bp and/or 133-135 bp;
3) contains an allele fragment which shows the fluorescence color of the fluorescent dye A and has the size of 182-184 bp;
41 contains an allele fragment which shows the fluorescence color of the fluorescent dye A and has the size of 241-243 bp;
5) contains an allele fragment which shows the fluorescence color of the fluorescent dye B and has the size of 111-113bp or 115-117 bp;
6) contains allele segment which shows the fluorescence color of the fluorescent dye B and has the size of 147-149 bp;
7) contains two allele fragments which show the fluorescence color of the fluorescent dye B and have the sizes of 210-212bp and 222-224 bp;
8) contains allele segments which show the fluorescence color of the fluorescent dye C and have the size of 151-153bp or 154-156 bp.
10. A poppy-specific genetic marker composition characterized by: comprising SSR loci BP12, BP13, BP14, P15, P85, P90, P107 and P136, wherein the BP12 is a fragment obtained by using poppy genomic DNA as a template and performing PCR amplification on the BP12 by using the primers of any one of claims 1 to 4, the BP13 is a fragment obtained by using the poppy genomic DNA as a template and performing PCR amplification on the BP13 by using the primers of any one of claims 1 to 4, the BP14 is a fragment obtained by using the poppy genomic DNA as a template and performing PCR amplification on the BP14 by using the primers of any one of claims 1 to 4, the P15 is a fragment obtained by using the poppy genomic DNA as a template and performing PCR amplification on the P15 by using the primers of any one of claims 1 to 4, the P85 is a fragment obtained by using the poppy genomic DNA as a template and performing PCR amplification on the P15 by using the primers of any one of claims 1 to 4, and the P85 is a fragment obtained by using the poppy genomic DNA as a template, the fragment obtained by PCR amplification using the primer pair P85 according to any one of claims 1 to 4, wherein P90 is a fragment obtained by PCR amplification using the genomic DNA of Papaveris as a template and the primer pair P90 according to any one of claims 1 to 4, P107 is a fragment obtained by PCR amplification using the genomic DNA of Papaveris as a template and the primer pair P107 according to any one of claims 1 to 4, and P136 is a fragment obtained by PCR amplification using the genomic DNA of Papaveris as a template and the primer pair P90 according to any one of claims 1 to 4.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861711A (en) * | 2016-05-23 | 2016-08-17 | 公安部物证鉴定中心 | Poppy species specificity genetic marker detecting system |
| CN108754007A (en) * | 2018-06-01 | 2018-11-06 | 中国科学院昆明植物研究所 | Using SSR molecular marker to the identification method of opium poppy |
| CN110004247A (en) * | 2019-04-30 | 2019-07-12 | 中国科学院武汉植物园 | A kind of SSR kit of Rapid identification opium poppy |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861711A (en) * | 2016-05-23 | 2016-08-17 | 公安部物证鉴定中心 | Poppy species specificity genetic marker detecting system |
| CN108754007A (en) * | 2018-06-01 | 2018-11-06 | 中国科学院昆明植物研究所 | Using SSR molecular marker to the identification method of opium poppy |
| CN110004247A (en) * | 2019-04-30 | 2019-07-12 | 中国科学院武汉植物园 | A kind of SSR kit of Rapid identification opium poppy |
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| JAKUB VAŠEK等: "New EST-SSR Markers for Individual Genotyping of Opium Poppy Cultivars ( Papaver somniferum L.)", 《PLANTS》 * |
| 王白石等: "罂粟分子遗传标记研究现状及开发前景", 《中国法医学杂志》 * |
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