CN114381540B - Primer composition, kit and method for compound identification of polymorphic genetic markers of cannabis sativa - Google Patents

Primer composition, kit and method for compound identification of polymorphic genetic markers of cannabis sativa Download PDF

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CN114381540B
CN114381540B CN202111415390.5A CN202111415390A CN114381540B CN 114381540 B CN114381540 B CN 114381540B CN 202111415390 A CN202111415390 A CN 202111415390A CN 114381540 B CN114381540 B CN 114381540B
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张素华
李成涛
夏若成
于欢
陶瑞旸
陈安琪
刘希玲
阙庭志
林�源
赵珍敏
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Abstract

The invention provides a primer composition, a kit and a method capable of detecting 17 autosomal STR loci and 2 individual identification sites of a cannabis sample in parallel, wherein the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS 501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043,2 individual identification sites are DM029 and DM016; the primer composition comprises sequences described by SEQ ID NO. 1-38. The primer composition, the kit and the method for detecting 17 autosomes STR loci and 2 individual identification points of the cannabis sample containing fluorescent markers in parallel have the characteristics of high sensitivity, good specificity, strong stability, high accuracy and the like, and provide a brand-new detection means for the neutral identification, individual identification, source location deduction and STR database establishment in the forensic research of cannabis in China.

Description

Primer composition, kit and method for compound identification of polymorphic genetic markers of cannabis sativa
Technical Field
The invention relates to the technical field of molecular identification, in particular to a primer composition, a kit and a method for compound identification of polymorphic genetic markers of cannabis.
Background
Cannabis is a herb belonging to annual hermaphrodite strains of Cannabis (Cannabinae) belonging to the family Cannabis (Cannabinae), and is mainly distributed in areas such as Nepal, india, china, etc. in Europe, africa and Asia, and is one of the oldest cultivated crops on earth. Hemp fibers are good textile and papermaking raw materials, cannabinoids contained in plants are widely applied to the field of medicine and health, and seeds are rich in various nutritional ingredients such as proteins, amino acids, unsaturated fatty acids, trace elements and the like which are easy to be absorbed by human bodies, so that the hemp fibers become important economic crops in many countries. However, since Tetrahydrocannabinol (THC) contained in flowers and leaves has strong addiction and narcotics, cannabis has been listed by the united nations rule of forbidden drugs as one of three drugs juxtaposed with heroin, cocaine.
Cytological studies have shown that cannabis is a diploid plant containing 20 chromosomes (of which 18 are autosomes and 2 are sex chromosomes), the female chromosome is XX and the male chromosome is XY. The utility value of the female and male cannabis plants is different, the fiber quality of the male plants is obviously higher than that of the female plants, the male plants do not contain THC and Cannabidiol (CBD) or have low content, and the female plants have relatively high content, so the female plants have higher medicinal value and abuse potential.
In recent years, under the influence of legal hemp in some countries and regions internationally, the number of people who drink or abuse hemp in China continuously rises, and sales of hemp cases across countries are increasing. Therefore, rapid and accurate judicial identification and toxicity source tracking of cannabis have become urgent social demands. The current conventional cannabis identification method mainly performs morphological and chemical component analysis. However, in some cases, hemp is processed or mixed with tobacco leaves or the like, so that people cannot morphologically identify such specimens. For analysis of chemical components of cannabis sativa, a gas chromatography-mass spectrometry (GC-MS) method is mostly adopted to carry out qualitative and quantitative analysis on THC in biological detection materials; however, GC-MS analysis of chemical components of cannabis requires a large amount of fresh samples, because THC is easily oxidized in old samples and the THC content is affected by the developmental stage, location and planting environment of cannabis plants. In addition, sex differences have a large impact on cannabis THC content, however, older cannabis samples, processed cannabis samples, and cannabis prior to the flowering phase, do not allow for an accurate morphological or chemical judgment of cannabis plant sex. Finally, individual identification and source inference of cannabis is also of great importance in practical cases, which is precisely not done by morphological and chemical analysis. Scientists have attempted to find new ways to address these issues in order to better meet the needs of cannabis species identification, individual identification, and source-location inference in judicial identification.
With the rapid development of molecular biology technology, the identification of cannabis at the DNA level is becoming a research hotspot and new technological means. At present, research on cannabis DNA genetic markers is mainly focused on RAPD, AFLP, SCAR, DNA bar codes, STRs and the like. Study data showed that: RAPD, AFLP and SCAR genetic markers can be used for species and sex identification of cannabis; the DNA bar code can accurately identify the cannabis and the mixed and fake products thereof, but the genetic markers can not be used for carrying out individual identification and regional source inference on the cannabis.
The short tandem repeat STR is an oligonucleotide sequence formed by connecting 2-6 bp core sequences in series, and is widely applied to individual identification, paternity test and population investigation in the field of judicial identification and becomes the most widely applied genetic marker in judicial identification due to the advantages of high sensitivity, high identification capability, gao Chongshu specificity, high accuracy of results, easiness in standardization and the like. Based on this, some forensic scientists have attempted to apply STR genetic markers to cannabis identification studies and demonstrated the potential of STR genetic markers in identifying cannabis, differentiating cannabis varieties and inferring cannabis origin, among others. At present, there are 28 hemp STR loci found and reported at home and abroad. In 2003, hsieh et al reported the first cannabis STR locus CS1 and observed that the number of repetitions of this locus varied from 3 to 40 in 108 cannabis samples, with heterozygosity of approximately 87.04%, demonstrating a high degree of polymorphism in the CS1 locus. More cannabis polymorphic STR loci were then developed, such as Alghanim et al, 11C 11-channel 1, B01-channel 1 and D02-channel 1, and the like, and Valverde et al, developed 6 four base repeat cannabis STR loci 5159, 4910 and 1528, which were also the first four base repeat cannabis STR loci reported. Based on the continuously developed hemp STR loci, research on a composite amplification system of the hemp STR loci is also developed. In 2008, howard et al successfully constructed a composite amplification system with 10 STR loci, and first performed verification studies on sensitivity, stability, species specificity and the like according to the DNA analysis method scientific working group (Scientific Working Group for DNA Analysis Methods, SWGDAM) verification guidelines, and the results indicate that the system can be used for building a hemp STR genetic database. Houston et al screen the hemp STR reported in the previous study and combine with 6 four-base repeated STRs developed by Valverde et al to successfully construct an STR composite amplification system of 13 loci, which is the system most studied and applied at present. The research of the STR gene locus of the hemp in China is just started. In 2008, ma Yuan and other genetic polymorphisms of cannabis sativa individuals and groups are investigated by selecting 3 loci ANUCS 301, ANUCS305 and CS1 with similar amplification conditions and higher heterozygosity, so that a theoretical basis is provided for deducing the variety and production place of the original drug plant cannabis sativa by using STR genetic information.
In summary, the current research on cannabis is mainly focused on identification of drug-type cannabis and non-drug-type cannabis in biochemical detection, and the research on cannabis DNA is less, so that the research on individual identification and source inference is very rare. Foreign researches on a hemp STR composite amplification system are in a primary stage, and few researches are performed in China.
Disclosure of Invention
In order to fill the blank of the research of a domestic hemp STR composite amplification system, provide a theoretical basis and a detection method for establishing a hemp STR database and develop and establish a primer composition, a kit and a method capable of detecting 17 autosomal STR loci and 2 individual identification sites of a hemp sample in parallel for realizing hemp sex identification, individual identification and source location inference.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a primer composition for the multiplex identification of polymorphic genetic markers of cannabis, comprising amplification primers for 17 autosomal STR loci of cannabis and amplification primers for 2 individual identification sites; wherein the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS 501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043,2, and the individuality identification points are DM029 and DM016.
Further, the sequences of the amplification primers of the 17 autosomal STR loci of cannabis sativa and the amplification primers of 2 individual identification sites are specifically as follows:
D02-CANN1：SEQ ID NO.1-2；C11-CANN1：SEQ ID NO.3-4；DM029：SEQ ID NO.5-6；DM016：SEQ ID NO.7-8；4910：SEQ ID NO.9-10；B01-CANN1：SEQ ID NO.11-12；E07-CANN1：SEQ ID NO.13-14；9269：SEQ ID NO.15-16；B05-CANN1：SEQ ID NO.17-18；H06-CANN2：SEQ ID NO.19-20；5159：SEQ ID NO.21-22；nH09：SEQ ID NO.23-24；ANUCS 501：SEQ ID NO.25-26；CS1：SEQ ID NO.27-28；ANUCS 305：SEQ ID NO.29-30；3735：SEQ ID NO.31-32；ANUCS 302：SEQ ID NO.33-34；1528：SEQ ID NO.35-36；9043：SEQ ID NO.37-38。
further, at least one primer of each pair of amplification primers is labeled with a fluorescent dye selected from one of FAM, HEX, TRMRA, ROX.
Further, amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910 and BO1-CANN1 are marked by FAM; amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; amplification primers of ANUCS 501, CS1 and ANUCS305 are marked by TRMRA; 3735. amplification primers of ANUCS 302, 1528 and 9043 were labeled with ROX.
In a second aspect, the invention also provides a kit for complex identification of polymorphic genetic markers of cannabis comprising the above primer composition, which further comprises 4 XPCR reaction premix VII [50mM Tris-HCl (pH 8.3), 50mM KCl,1.5mM MgCl 2 ,0.2mM dNTPs,0.08mg/mL bovine serum albumin(BSA)]And deionized water.
In a third aspect, the invention also provides a method for multiplex identification of polymorphic genetic markers of cannabis, in particular for cannabis sex identification, individual identification and source inference, using the above primer composition or kit; the method comprises the following steps:
step one, carrying out PCR multiplex amplification on a genomic DNA sample of a sample to be detected by adopting an amplification primer with a sequence shown in SEQ ID NO. 1-38;
step two, uniformly mixing the PCR amplification product with a proper amount of molecular weight internal standard and formamide; denaturing and cooling the mixture;
and thirdly, adopting a genetic analyzer to carry out typing detection.
Further, the total volume of the amplification system for PCR multiplex amplification was 10. Mu.L, comprising: 4 XPCR reaction premix VII 2.5. Mu.L, primer mix (10. Mu.L) 1. Mu.L, deionized water 5.5. Mu.L, 1 ng/. Mu.L genomic DNA 1. Mu.L.
Further, the concentrations of the amplification primers in the amplification system are as follows: D02-CANN1:0.03 μm; C11-CANN1:0.03 μm; DM029: 0.05. Mu.M; DM016:0.03 μm;4910: 0.04. Mu.M; B01-CANN1:0.06 μm; E07-CANN1: 0.05. Mu.M; 9269: 0.04. Mu.M; B05-CANN1:0.03 μm; H06-CANN2: 0.04. Mu.M; 5159: 0.04. Mu.M; nH09: 0.04. Mu.M; ANUCS 501: 0.05. Mu.M; CS1: 0.05. Mu.M; ANUCS 305: 0.05. Mu.M; 3735: 0.05. Mu.M; ANUCS 302: 0.04. Mu.M; 1528: 0.05. Mu.M; 9043: 0.05. Mu.M.
Further, the PCR multiplex amplification procedure is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95℃for 5 seconds, annealing at 56℃for 1 minute, extension at 60℃for 30 seconds, 28 cycles total; final extension at 60 ℃ for 5 min; preserving heat at 15 ℃.
Further, the PCR multiplex amplification adopts a reaction thermal cycler; the thermal reaction cycler is one selected from ABI9700, ABI 9600, ABI2720, bio-Rad iCycler and Bio-Rad C1000.
Further, the molecular weight internal Standard adopts a T500Size Standard and adopts LIZ fluorescence labeling; fluorescence correction was used with 5-Dye Matrix Standards.
Further, in the second step, the volume ratio of the PCR amplification product, the molecular weight internal standard and the formamide is 1:8.5:0.5.
Further, the genetic analyzer is one selected from 3100 series, 3130 series and 3500 series genetic analyzers.
Further, the first step further comprises DNA extraction of flowers, stems, leaves and/or seeds of the sample to be tested to prepare a genomic DNA sample.
Further, the denaturation condition in the second step is 95 ℃ for 3 minutes; the cooling conditions were ice-cooled for 3 minutes.
Compared with the prior art, the invention has the following technical effects:
1. the invention simultaneously expands 17 autosomal STR loci and 2 individual identification loci of the hemp, which is superior to a compound amplification system containing 15 loci at most in earlier stage research; and the present invention first attempted to add sex identification sites to the hemp STR composite amplification system.
2. The invention provides an effective method for further research of the hemp STR gene locus, establishment of a hemp STR database and detection and identification of hemp in a case.
3. The invention establishes a forensic detection kit with high sensitivity, good specificity and strong stability, provides an effective detection tool for sex identification, individual identification, source location inference and the like of cannabis, and has important significance for meeting the requirements of cannabis judicial identification.
Drawings
FIG. 1 is a Ladder typing map of multiplex amplification for identifying genetic markers for polymorphisms of cannabis in one embodiment of the present invention;
FIG. 2 is a typing pattern of female sample DNA using a multiplex amplification method for multiplex identification of cannabis sativa comprising 17 autosomal STR loci and 2 unique identification points in accordance with an embodiment of the invention;
FIG. 3 is a pattern diagram of male sample DNA using a multiplex amplification method for multiplex identification of cannabis sativa containing 17 autosomal STR loci and 2 individual identification sites in accordance with an embodiment of the invention.
FIG. 4 is a typing pattern of a compound amplification method identity study for compound identification of cannabis sativa comprising 17 autosomal STR loci and 2 individual identification sites in an embodiment of the invention, wherein FIGS. A-C show the typing patterns of genomic DNA of flowers, leaves, stems, respectively;
FIG. 5 is a graph showing the results of a sensitivity study of a multiplex amplification method for multiplex identification of cannabis sativa containing 17 autosomal STR loci and 2 individual identification sites in accordance with an embodiment of the invention;
FIG. 6 is a typing pattern of a composite amplification method species specificity study for composite identification of cannabis sativa containing 17 autosomal STR loci and 2 individual identification sites in an embodiment of the invention.
Detailed Description
The invention provides a primer composition, a kit and a method for the complex identification of polymorphic genetic markers of cannabis, in particular for the sex identification, individual identification and source inference of cannabis. The primer composition comprises amplification primers of 17 autosomal STR loci of cannabis sativa and amplification primers of 2 individual identification points; wherein the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS 501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043,2, and the individuality identification points are DM029 and DM016.
In a preferred embodiment of the invention, the sequences of the amplification primers for 17 autosomal STR loci and the amplification primers for 2 individual identification sites of cannabis sativa are as follows:
D02-CANN1：SEQ ID NO.1-2；C11-CANN1：SEQ ID NO.3-4；DM029：SEQ ID NO.5-6；DM016：SEQ ID NO.7-8；4910：SEQ ID NO.9-10；B01-CANN1：SEQ ID NO.11-12；E07-CANN1：SEQ ID NO.13-14；9269：SEQ ID NO.15-16；B05-CANN1：SEQ ID NO.17-18；H06-CANN2：SEQ ID NO.19-20；5159：SEQ ID NO.21-22；nH09：SEQ ID NO.23-24；ANUCS 501：SEQ ID NO.25-26；CS1：SEQ ID NO.27-28；ANUCS 305：SEQ ID NO.29-30；3735：SEQ ID NO.31-32；ANUCS 302：SEQ ID NO.33-34；1528：SEQ ID NO.35-36；9043：SEQ ID NO.37-38。
in a preferred embodiment of the invention, at least one primer of each pair of amplification primers is labeled with a fluorescent dye selected from one of FAM, HEX, TRMRA, ROX.
In a preferred embodiment of the present invention, the amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910 and BO1-CANN1 are FAM labeled; amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; amplification primers of ANUCS 501, CS1 and ANUCS305 are marked by TRMRA; 3735. amplification primers of ANUCS 302, 1528 and 9043 were labeled with ROX.
In addition, the kit comprises the primer composition for the cannabis sativa compound identification polymorphism genetic marker, 4X PCR reaction premix VII and deionized water.
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
The embodiment provides a method for the compound identification of polymorphic genetic markers of cannabis, comprising the following steps:
1. screening of hemp STR loci suitable for forensic applications;
according to literature reports and gene databases, the currently developed hemp STR loci are searched, but not all of these STR loci are suitable for research of hemp multiplex amplification systems. Polymorphic STR loci are screened for, and loci in which dinucleotide repeats are knocked out. Finally, 17 optimal autosomal STR loci and 2 individual identification sites are selected for kit development, wherein the loci are D02-CANN1, C11-CANN1, DM029, DM016, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS 501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043. Wherein sex determination site DM029 is associated with the X chromosome, all samples show a single peak, sex determination site DM016 is associated with the Y chromosome, all male samples show a single peak, and female samples do not show a peak.
2. Designing an amplification primer component;
a composite amplification detection kit containing five-color fluorescent markers (FAM, HEX, TAMRA, ROX and LIZ) is established, and LIZ fluorescent markers (T500) are adopted as the molecular weight internal standards.
The genetic markers described in this example and their corresponding amplification primer sequences, marker fluorescence and final reaction concentrations are shown in Table 1.
TABLE 1 primer sequences, final reaction concentrations and labeling fluorescence used for multiplex amplification
3. Constructing and optimizing a multiplex PCR amplification system;
and (3) carrying out adjustment and optimization on PCR reaction conditions including primer proportioning concentration, DNA template quantity, annealing temperature, cycle number and the like on the constructed multiplex amplification parting system to obtain balanced and stable PCR product parting results, and realizing multiplex amplification of 19 genetic markers.
The multiplex PCR amplification system comprising 17 autosomal STR loci and 2 individual identification sites of cannabis described in this example is shown in table 2, wherein DNA is extracted from cannabis samples such as flowers, stems, leaves, seeds.
Table 2 multiplex PCR amplification System comprising 17 autosomal STR loci and 2 individual identification sites of Cannabis sativa
The reaction system can obtain good results by adopting the following procedures on various thermal reaction cyclers (such as ABI9700, ABI 9600, ABI2720, bio-Rad iCycler, etc.): pre-denaturation at 95 ℃ for 2 min; denaturation at 95℃for 5 seconds, annealing at 56℃for 1 minute, extension at 60℃for 30 seconds, 28 cycles total; final extension at 60 ℃ for 5 min; preserving heat at 15 ℃.
4. Establishing an analysis method and detecting a composite amplification product;
a 3100 series, 3130 series, or 3500 series genetic analyzer spectral correction (Matrix) file was created. When capillary electrophoresis was used, 1. Mu.L of the PCR amplification product was mixed with 8.5. Mu.L of formamide and 0.5. Mu.L of a molecular weight internal Standard (T500 Size Standard); the mixture was denatured at 95 ℃ for 3 min, then placed on ice for 3 min; the typing detection is carried out on 19 genetic markers by adopting the model genetic analyzer. Electrophoresis migration parameters of different alleles of each marker were obtained by capillary electrophoresis and based thereon according to GeneMapperID v3.2.1 and respectivelyThe format of v1.5 software requires writing the corresponding Bin file and Panel file, creating an electrophoretic analysis method.
Wherein, the Ladder typing map adopting the method is shown in figure 1, the typing map adopting the method for female sample DNA is shown in figure 2, and the typing map for male sample DNA is shown in figure 3.
Example 2
This example is a test of cannabis sample DNA using the method provided in example 1.
A sample of cannabis was collected from various tissues (flowers, stems, leaves and seeds) for DNA extraction and quantification. The above DNA samples were subjected to multiplex amplification of 19 genetic markers using the kit constructed in example 1. All samples gave efficient amplification products on the genetic markers.
Example 3
In this example, forensic verification (identity, sensitivity, species specificity, and forensic parameter calculation) was performed on the method provided in example 1 according to SWGDAM requirements, and the superiority of the method was further determined, and the specific operation was as follows:
(1) Identity study: the genomic DNA of flowers, leaves and stems of the same plants was quantified to 1 ng/. Mu.L and separately examined using the method provided in example 1.
The results show (fig. 4) that the kit constructed in example 1 has good tissue identity: flowers, leaves and stems of the same plant have the same genotyping.
(2) Sensitivity study: hemp genomic DNA was diluted to 2 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.25 ng/. Mu.L, 0.125 ng/. Mu.L, 0.0625 ng/. Mu.L, 0.03125 ng/. Mu.L and 0.015625 ng/. Mu.L in a double ratio, and the above concentrations of hemp genomic DNA were detected separately using the method provided in example 1, and the detection was repeated 3 times for each template amount sample.
The results show (fig. 5) that the kit constructed in example 1 has high sensitivity: complete genotyping of the genetic markers was obtained at DNA template amounts as low as 0.125 ng.
(3) Species specificity study: the genomic DNA of dogs, cats, mice, sheep, pigs, cattle, rabbits, chickens, ducks, monkeys, nightshade, corn poppy, mulberry leaves, poppy, sage and humulus scandens was quantified to 5 ng/. Mu.L, and the non-cannabis-derived genomic DNA was separately detected using the method provided in example 1.
The results show (fig. 6) that the kit constructed in example 1 has species specificity: no product peaks were detected in DNA from species other than sage and humulus, although some were observed in the sage and humulus samples, most were not at the allele position and were also different from the normal peak pattern of cannabis, and did not affect the interpretation of the results.
(4) And (3) forensic parameter calculation: 126 parts of cannabis genomic DNA were quantitated to 1 ng/. Mu.L and separately tested using the method provided in example 1. The heterozygosity, polymorphism information content, individual identification probability, non-father exclusion probability, cumulative individual identification probability, cumulative non-father exclusion probability, and individual sample identity information for 17 autosomal STR loci are counted and calculated.
The results show (table 3) that the kit constructed in example 1 has better system performance: the heterozygosity range of 17 autosomal STR loci in 126 hemp samples is 0.2381-0.7937, the polymorphism information content range is 0.2754-0.9419, the individual identification probability range is 0.4624-0.9855, the non-father exclusion probability range is 0.0410-0.5873, and the cumulative individual identification probability is 1-3.0X10 -15 The cumulative non-father exclusion probability is 1-7.4X10 -3 The method comprises the steps of carrying out a first treatment on the surface of the Both the hemp sex determination sites DM029 and DM016 can meet the requirement of sex determination in the detection samples, and both DM029 and DM016 peaks are detected in all male samples, and only a single DM029 peak is detected in the female samples.
TABLE 3 forensic parameters for 17 autosomal STR loci of Cannabis sativa
According to the embodiment, the primer composition, the kit and the method for detecting 17 autosomes STR loci and 2 individual identification sites of the cannabis sample in parallel with fluorescent markers provide a brand-new detection means for the neutral identification, individual identification, source location inference and STR database establishment in the forensic research of cannabis in China.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Sequence listing
<110> scientific institute of judicial identification
<120> a primer composition, kit and method for complex identification of polymorphic genetic markers of cannabis
<150> 2021102127562
<151> 2021-02-25
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> primer for amplifying D02-CANN1 (Artificial Sequence)
<400> 1
ggttgggatg ttgttgttgt g 21
<210> 2
<211> 21
<212> DNA
<213> primer for amplifying D02-CANN1 (Artificial Sequence)
<400> 2
agaaatccaa ggtcctgatg g 21
<210> 3
<211> 22
<212> DNA
<213> primer for amplifying C11-CANN1 (Artificial Sequence)
<400> 3
gtggtggtga tgatgataat gg 22
<210> 4
<211> 19
<212> DNA
<213> primer for amplifying C11-CANN1 (Artificial Sequence)
<400> 4
tgaattggtt acgatggcg 19
<210> 5
<211> 20
<212> DNA
<213> primer for amplifying DM029 (Artificial Sequence)
<400> 5
gatgacagac ttcctgattg 20
<210> 6
<211> 20
<212> DNA
<213> primer for amplifying DM029 (Artificial Sequence)
<400> 6
gtctaagagt gggaatgcta 20
<210> 7
<211> 18
<212> DNA
<213> primer for amplifying DM016 (Artificial Sequence)
<400> 7
gcccaagttg ctgctgag 18
<210> 8
<211> 18
<212> DNA
<213> primer for amplifying DM016 (Artificial Sequence)
<400> 8
cccaccgttt agggagca 18
<210> 9
<211> 19
<212> DNA
<213> primer for amplification 4910 (Artificial Sequence)
<400> 9
agattcccaa gatgagcaa 19
<210> 10
<211> 20
<212> DNA
<213> primer for amplification 4910 (Artificial Sequence)
<400> 10
acaaactggt atcaagagcc 20
<210> 11
<211> 22
<212> DNA
<213> primer for amplification of B01-CANN1 (Artificial Sequence)
<400> 11
atgacatacc agacagaaac tc 22
<210> 12
<211> 21
<212> DNA
<213> primer for amplification of B01-CANN1 (Artificial Sequence)
<400> 12
catccatagc attatcccac t 21
<210> 13
<211> 19
<212> DNA
<213> primer for amplifying E07-CANN1 (Artificial Sequence)
<400> 13
caaatgccac accaccttc 19
<210> 14
<211> 21
<212> DNA
<213> primer for amplifying E07-CANN1 (Artificial Sequence)
<400> 14
gtggtagcca ggtataggta g 21
<210> 15
<211> 20
<212> DNA
<213> primer for amplification 9269 (Artificial Sequence)
<400> 15
cccaaactac tgtttgtgcc 20
<210> 16
<211> 22
<212> DNA
<213> primer for amplification 9269 (Artificial Sequence)
<400> 16
acttgcacgt gatgttagat cc 22
<210> 17
<211> 19
<212> DNA
<213> primer for amplifying B05-CANN1 (Artificial Sequence)
<400> 17
ttgatggtgg tgaaacggc 19
<210> 18
<211> 21
<212> DNA
<213> primer for amplifying B05-CANN1 (Artificial Sequence)
<400> 18
ccccaatctc aatctcaacc c 21
<210> 19
<211> 19
<212> DNA
<213> primer for amplifying H06-CANN2 (Artificial Sequence)
<400> 19
tggtttcagt ggtcctctc 19
<210> 20
<211> 19
<212> DNA
<213> primer for amplifying H06-CANN2 (Artificial Sequence)
<400> 20
acgtgagtga tgacacgag 19
<210> 21
<211> 20
<212> DNA
<213> primer for amplification 5159 (Artificial Sequence)
<400> 21
ccagagcttg tggatctcct 20
<210> 22
<211> 20
<212> DNA
<213> primer for amplification 5159 (Artificial Sequence)
<400> 22
agtacgaaag ggcactgagg 20
<210> 23
<211> 21
<212> DNA
<213> primer for amplifying nH09 (Artificial Sequence)
<400> 23
ccaacatttt ctcagaaccc a 21
<210> 24
<211> 21
<212> DNA
<213> primer for amplifying nH09 (Artificial Sequence)
<400> 24
tcttgactgt agtaatccag c 21
<210> 25
<211> 22
<212> DNA
<213> primer for amplifying ANUCS 501 (Artificial Sequence)
<400> 25
agcaataatg gagtgagtga ac 22
<210> 26
<211> 23
<212> DNA
<213> primer for amplifying ANUCS 501 (Artificial Sequence)
<400> 26
agagatcaag aaattgagat tcc 23
<210> 27
<211> 20
<212> DNA
<213> primer for amplifying CS1 (Artificial Sequence)
<400> 27
aagcaactcc aattccagcc 20
<210> 28
<211> 23
<212> DNA
<213> primer for amplifying CS1 (Artificial Sequence)
<400> 28
taatgatgag acgagtgaga acg 23
<210> 29
<211> 16
<212> DNA
<213> primer for amplifying ANUCS305 (Artificial Sequence)
<400> 29
agcccgaccg tgaaga 16
<210> 30
<211> 17
<212> DNA
<213> primer for amplifying ANUCS305 (Artificial Sequence)
<400> 30
tgaagccgat gccctat 17
<210> 31
<211> 22
<212> DNA
<213> primer for amplifying 3735 (Artificial Sequence)
<400> 31
tgattctgtg tttgtgtgca at 22
<210> 32
<211> 20
<212> DNA
<213> primer for amplifying 3735 (Artificial Sequence)
<400> 32
catcgcaccc acaggttagt 20
<210> 33
<211> 21
<212> DNA
<213> primer for amplifying ANUCS 302 (Artificial Sequence)
<400> 33
aacataaaca ccaacaactg c 21
<210> 34
<211> 20
<212> DNA
<213> primer for amplifying ANUCS 302 (Artificial Sequence)
<400> 34
atggttgatg ttttgatggt 20
<210> 35
<211> 21
<212> DNA
<213> primer for amplification 1528 (Artificial Sequence)
<400> 35
ggactttgtc tagtgccttt g 21
<210> 36
<211> 20
<212> DNA
<213> primer for amplification 1528 (Artificial Sequence)
<400> 36
gagtacttgg ctgatgatgg 20
<210> 37
<211> 21
<212> DNA
<213> primer for amplification 9043 (Artificial Sequence)
<400> 37
aggtctgcgt tgtgcattat t 21
<210> 38
<211> 19
<212> DNA
<213> primer for amplification 9043 (Artificial Sequence)
<400> 38
agggctggtt tcagtttcg 19

Claims (13)

1. A primer combination for the compound identification of polymorphic genetic markers of cannabis, which is characterized by comprising amplification primers of 17 autosomal STR loci of cannabis and amplification primers of 2 individual identification sites; wherein the 17 autosomal STR loci consist of D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS 501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043, and the 2 individual identification sites are DM029 and DM016;
the sequences of the amplification primers of the 17 autosomal STR loci of the cannabis sativa and the amplification primers of the 2 individual identification sites are specifically as follows:
D02-CANN1：SEQ ID NO. 1-2；C11-CANN1：SEQ ID NO. 3-4；DM029：SEQ ID NO. 5-6；DM016：SEQ ID NO. 7-8；4910：SEQ ID NO. 9-10；B01-CANN1：SEQ ID NO. 11-12；E07-CANN1：SEQ ID NO. 13-14；9269：SEQ ID NO. 15-16；B05-CANN1：SEQ ID NO. 17-18；H06-CANN2：SEQ ID NO. 19-20；5159：SEQ ID NO. 21-22；nH09：SEQ ID NO. 23-24；ANUCS 501：SEQ ID NO. 25-26；CS1：SEQ ID NO. 27-28；ANUCS 305：SEQ ID NO. 29-30；3735：SEQ ID NO. 31-32；ANUCS 302：SEQ ID NO. 33-34；1528：SEQ ID NO. 35-36；9043：SEQ ID NO. 37-38。
2. the primer composition of claim 1, wherein at least one primer of each pair of amplification primers is labeled with a fluorescent dye selected from one of FAM, HEX, TRMRA, ROX.
3. The primer composition of claim 2, wherein amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910, B01-CANN1 are labeled with FAM; amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; amplification primers of ANUCS 501, CS1 and ANUCS305 are marked by TRMRA; 3735. amplification primers of ANUCS 302, 1528 and 9043 were labeled with ROX.
4. A kit for multiplex identification of polymorphic genetic markers comprising the primer composition according to any one of claims 1-3, further comprising 4 x PCR reaction premix vii, deionized water.
5. A method for multiplex identification of polymorphic genetic markers using the primer composition according to any one of claims 1 to 3 or the kit according to claim 4, wherein the method is used for sex identification, individual identification and source inference of cannabis; the method comprises the following steps:
step one, carrying out PCR multiplex amplification on a genomic DNA sample of a sample to be detected by adopting an amplification primer with a sequence shown in SEQ ID NO. 1-38;
step two, uniformly mixing the PCR amplification product with a proper amount of molecular weight internal standard and formamide; denaturing and cooling the mixture;
and thirdly, adopting a genetic analyzer to carry out typing detection.
6. The method of claim 5, wherein the total volume of the PCR multiplex amplification system is 10 μl, comprising: 4×PCR reaction premix liquid VII 2.5 mu L,10×primer mixture 1 mu L, deionized water 5.5 mu L,1 ng/mu L genome DNA1 mu L.
7. The method according to claim 5, wherein the concentration of the amplification primers in the amplification system is as follows: D02-CANN1:0.03 [ mu ] M; C11-CANN1:0.03 A [ mu ] M; DM029:0.05 A [ mu ] M; DM016:0.03 A [ mu ] M;4910:0.04 A [ mu ] M; B01-CANN1:0.06 A [ mu ] M; E07-CANN1:0.05 A [ mu ] M;9269:0.04 A [ mu ] M; B05-CANN1:0.03 A [ mu ] M; H06-CANN2:0.04 A [ mu ] M;5159:0.04 A [ mu ] M; nH09:0.04 A [ mu ] M; ANUCS 501:0.05 A [ mu ] M; CS1:0.05 A [ mu ] M; ANUCS 305:0.05 A [ mu ] M;3735:0.05 A [ mu ] M; ANUCS 302:0.04 A [ mu ] M;1528:0.05 A [ mu ] M;9043:0.05 And [ mu ] M.
8. The method of claim 5, wherein the PCR multiplex amplification procedure is: pre-denaturation at 95 ℃ for 2 min; denaturation at 95℃for 5 seconds, annealing at 56℃for 1 minute, extension at 60℃for 30 seconds, 28 cycles total; final extension at 60 ℃ for 5 min; preserving heat at 15 ℃.
9. The method of claim 5, wherein the PCR multiplex amplification employs a thermal cycler; the thermal reaction cycler is selected from one of ABI9700, ABI 9600, ABI2720, bio-Rad iCycler and Bio-Rad C1000.
10. The method of claim 5, wherein the internal molecular weight Standard is T500Size Standard and is fluorescently labeled with LIZ; fluorescence correction was used with 5-Dye Matrix Standards.
11. The method according to claim 5, wherein in the second step, the volume ratio of the PCR amplification product, the internal molecular weight standard and the formamide is 1:8.5:0.5.
12. The method of claim 5, wherein the genetic analyzer is selected from one of the 3100 series, 3130 series, and 3500 series genetic analyzers.
13. The method of claim 5, wherein step one further comprises DNA extracting flowers, stems, leaves and/or seeds of the sample to be tested to prepare a genomic DNA sample.
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