Construction And Application Of 3 STR Loci Fluorescent Multiplex Amplification System

Objective:Opium poppy(Papaver somniferum L.),a annual herbaceous plant,is one of the original plants of three hard drugs in the worlds.Because of its extract can be made of opiate drugs(including opium,morphine,heroin addiction,etc.)has strong toxicity and cause serious harm to individuals and society,so the sale of illicit cultivation of poppy and add in food are prohibited by law(the countries which is now more poppy into food seasoning,but events)in the high profit,worldwide illicit poppy activities are rampant,means more subtle and sophisticated,because of poppy petals,color and fruit and ornamental plants(Lichun fruit)poppy and other closely related species are very similar,increases the difficulty of poppy identification,drug control challenges to shovel poison.In forensic science,traditional test is mainly by biological morphology and chemical methods.These methods although applicable scope broad,but not of poppy seedlings,seeds and plant species source of clearly defined,in order to better reach poppy species identification purpose,to try to find new and better ways to solve this problem.Microsatellite loci is composed of 1-6 nucleotides as the repeating unit of the tandem repeat sequence,which is widely distributed in the genome of human,animals and plants,is the most commonly used molecular marker in human DNA analysis.The STR marker of the plant is called the SSR marker,there are many applications in plant genetic diversity analysis,species identification,individual identification,genetic map construction,etc.Because of the limited access to the opium poppy,the research on genetic marker of DNA molecular biology is not much at home and abroad.The aim of thisstudy was to develop and construct the poppy three SSR gene a fluorescent multiplex PCR system,Using this system,it can distinguish the plant from the poppy and its closely related species.Provide a simple,rapid and accurate method when the public security organ for drug-related cases.Methods:1)By using two kinds of plant DNA extraction methods(the optimization of the silica bead extraction method and the commercial kit)respectively,the DNA of the known poppy plant samples were extracted.2)Screen EST-SSR and genomic SSR primers designed in the early stage of our laboratory through repeated amplification experiment3)Construct a composite amplification system containing 3 SSR loci.Optimize composite amplification system by regulating enzyme concentration,primer concentration,Mg2+ concentration,DNTP concentration,annealing temperature,cycle times.4)Vetify the sensitivity,tissue identity,species specificity and repeatability of the fluorescent composite amplification system.5)The composite amplification system was used in practical cases.Further verify the effectiveness of the system,by testing the sample of 5poppy samples in drug-related cases of suspected.Results:1)The two plant DNA extraction methods were used to extract the DNA.2 primers P12 and P13 were screened out from the primers designed by ourselves.2 poppy specific SSR loci were developed.Combined with the previous 1 SSR loci,A fluorescent multiplex PCR system with 3 SSR loci was successfully constructed.2)In species of the genus specific study,The multiplex PCR system to detect poppy,Chelidonium majus,Oriental Poppy,Papaver rhoeas,Iceland poppy,marijuana and other common plants more than 10 kinds of plant samples,respectively.The results showed that poppy amplification products containing the size of 243bp(±1bp),183bp(±1bp),211bp(±1bp)\ 223bp(±1bp)gene fragment;Oriental Poppy amplification products containing thesize of 205bp(±1bp)gene fragment;poppy Papaver Iceland amplification products containing the size of 208bp(±1bp)gene fragment;hemp,tobacco and other common plant samples without any amplification products3)In the sensitivity test,the minimum detectable amount of this system is0.1 ng/uL4)The fluorescent composite amplification system was used to detect the samples from 5 different sources in the actual cases,and the results were consistent with the original identification results.The specificity detection in the molecular biology level was realized.Conclusions: In this study,we successfully constructed a fluorescent compound amplification system with 3 SSR loci,The system is easy to be optimized,high sensitivity and stable result.Effective differentiation of poppy and its related species of plants,hemp and other common plants.It provides a new method for the identification and identification of the opium poppy.It is a rich and supplement of the traditional chemical methods.it is of great significance to investigate and deal with the illegal cultivation of opium poppy in the public security organs and to combat drug crime.