Poppy Species Specific Genetic Marker Detection System
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A poppy, target sequence technology, applied in the biological field, achieves high sensitivity and stability, high accuracy, and accurate amplification
Active Publication Date: 2019-09-24
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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The advantage of STR technology is that the loci are rich, the polymorphism is high, the result is stable, and the detection can be automated, saving time and manpower. However, the research on the STR multiplex amplification system of poppy at home and abroad is still blank
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Embodiment 1
[0053] Embodiment 1, STR composite amplification primer design
[0054] 1. Design principles
[0055] Suitable poppy STR loci were screened according to the following principles:
[0056] (1) The repeating sequence of the locus is simple, and the repeating unit is a locus with two, three, four or five nucleotides;
[0057] The size of the amplified fragment of the gene locus is between 90 and 400 bp. In order to prevent overlapping between the loci, there should be at least 9 to 10 bp interval between the loci of the same fluorescent combination;
[0058] (2) The locus mutation rate is low and the polymorphism is good, ensuring the stability and reliability of the test results;
[0059] (3) The primers of each locus must be compatible, that is, the primers do not interact, especially the complementary sequences cannot appear at the 3' end of the primers; because the special sequences between the primers of the loci interact, such as primer dimers and hairpin structures. Som...
Embodiment 2
[0068] Embodiment 2, the application of STR composite amplification primers in the specific identification of poppy species
[0069] 1. Genomic DNA extraction
[0070] Genomic DNA was extracted separately from the flowers, stems, leaves and seeds of the poppy samples.
[0071] 2. Optimization of STR multiplex amplification system
[0072] Using the genomic DNA of poppy leaves as a template, the primer pairs P5, P12, P13, and P14 were mixed for PCR amplification, and the amplification system and reaction conditions were optimized, as follows:
[0073] The amplification system before optimization is shown in Table 2:
[0074] Table 2 Amplification system before optimization
[0075]
[0076] The STR multiplex amplification reaction conditions before optimization are as follows:
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Abstract
The invention discloses a poppy species specificity genetic marker detecting system. A primer pair set for identifying poppy is provided and is composed of a primer pair P12 (sequence 1 and 2), a primer pair P13 (sequence 3 and 4), a primer pair P5 (sequence 5 and 6) and a primer pair P14 (sequence 7 and 8). Four STR loci of poppy are amplified at the same time, and the amplification is more accurate and rapid compared with amplification of a single locus; the system has higher species specificity when identifying poppy and affinis plants, and an effective method is provided for further studying poppy STR loci and inspecting and identifying the poppy species in a case; in an optimized poppy STR composite amplification system, amplification of all loci can be balanced, sensitivity is high, stability is high, and frequently-seen corrosion check materials in a drug-related case can be easily inspected; accuracy is high, parting of four loci of each of poppy samples from different producing areas can be achieved through detection and data statistic analysis.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a poppy species-specific genetic marker detection system. Background technique [0002] Poppy (Papaver somniferum L.) is an annual herb with a stem height of 30-80 cm, branched, alternate leaves, pinnately parted, lobes lanceolate or strip-lanceolate, with rough hairs on both sides, native to West Asia , as early as the Six Dynasties, it was introduced into China and planted, because it is the main raw material for making opium, and its extract is also a variety of sedatives such as morphine, thebaine, codeine, papaverine, and narcotin Therefore, it is listed as one of the world's three major drug original plants by the United Nations Convention Against Narcotics. In forensic science, the traditional test for it is mainly through biomorphological and chemical methods. Although these methods can detect alkaloid residues in poppy shell foods and have a wide range of applications, the de...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 裴黎李元元徐小玉王瑛张颖高珊叶健刘开会王白石谷建平刘京
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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