Study On Drowning Related Plankton DNA Detection Technology

At present,deaths caused by drowning are the second most common cause of abnormal deaths after traffic accidents in the world.Most of them are caused by accidental drowning,but there are also individual cases of suicides or homicides.Therefore,scientific and accurate forensic methods,used to identify the cause of death in water bodies,have positive social significance for determining the cause of death and eliminating social impact and influence.However,drowning diagnosis still has difficulties existed to be eagerly sloved by scientific researches,such as easy-polluting sampling tools,scare diagnostic technology and few evaluation methods,which exert negative impacts on the stability of diagnosis.This study integrated a series technologies including electronic circuits design,mechanical manufacturing,modern molecular biological technology and Scanning Electron Microscope(SEM)technology,and tried to establish the invention of skeletal sampling tool and to enrich the practical methods for drowning diagnosis,by means of investigation of sampling tool design and manufacture,and methods setup and eveluation.1.based on the principle of vertical drilling machinery,to design and manufacture a processing equipment of mechanical fixture and electric control component,which includes skeletal 4 function modules:1,the special power supply,suporting 4 branch power circuit for controling lighting,UV lighting,vertical movement of the drill,the horizontal movement of fixture;2,the sampling chamber,with metal mechanical clamp,stepper motor,removable drill to fulfil the sampling instead of manual sampling;3,the sample collecting chamber,including the collecting funnel(30°)and shifting gear for supporting continuous sample collection 4,all components made of 304 stainless steel and shelled with the bearing(>50 kg)ensured the equipment operation stability.20 cadaver bone accumulated by routine cases examination were selected to study as the materials for the sampling tool.The samples obtained from the equipment and hand sampling had no significant effect on the results of DNA test(p>0.05).No pollution was detected in the sampling process of the equipment invented,but 3 samples were suspected to be contaminated by manual sampling.The time consumption of the equipment sampling is only 1/2 of the manual sampling.In positive detection rates,there is no significant difference between the manual sampling and the equipment sampling.No significant difference in species and quantity of diatoms was found in the bone marrow extracted from the drowned corpse between machinery extraction and manual extraction.The results showed the bone marrow of the corpse contained drowning related diatoms,which could meet the diagnostic requirements of drowning.2.to establish Polymerase Chained Reaction-Capillary Eletropheris(PCR-CE)technology for drowning diagnosis,the procedures involved screening the high sensitive and specific primers targeted to the biological standard strains involve in drowning process,and constructing PCR-CE detection method validation to test drowning bodies,so as to lay a foundation for the research on PCR multiplex setup.1)By Primer premier 5.0 design,Oligo 7.0 evaluation and PAGE validation,3 pairs of primers(rbcL197,UPA159,A457)targeted to rbcL gene,UPA gene and 18S rDNA gene respectively,were successfully screened for high specificity,sensitivity to diatoms,respectively.The primer specificity were tested by 16 species of algae strains,3 species of aquatic bacteria strains,3 human commensal bacteria strains and human DNA.The results showed rbcL197 and UPA 159,from the chloroplast gene fragments,were specific for diatoms such as:Syndra,Navicula,Melosira,Cyclotella sp.,Nitzschia,fragilaria.A457 is comprehensively specific to Nostoc,Synedra radians,Navicula sp.,Melosira varian,Chlorella pyrenoidosa,Chlorella vulgaris,Scenedesmus obliquus,Ankistrodesmus falcatus,Cyclotella sp.,Nitzschia sp.,Fragilaria sp.,the products amplified were 197bp,159bp,655bp.PCR-CE methods established with 3 primers was used for the cases validation(30 drowned corpses,3 land death as blanks and 2 cases of postmortem drowned).The total detection rates of PCR-CE methods constructed by primers rbcL197,UPA 159 and A457 were 85.9%,83.7%,52.8%,respectively.The detection rate of detection systems constructed with primers rbcL197 and UPA159 were higher than the primer A457.As to compare diatoms DNA detection rates in the various organs(lung,haper and kidney)from drowned corpses and water samples,there was no significant difference(p>0.05)between the PCR-CE test methods constructed by primers rbcL197 and UPA159.2)By Primer premier 5.0 design,Oligo 7.0 evaluation and PAGE validation,4 pairs of primers(AH,Ah,AHH,AER)were studied,targeted to gyrB gene,16S rDNA gene,hly gene and aer gene gene in Aeromonas hydrophila respectively,were successfully screened for high specificity,sensitivity to Aeromonas hydrophila,respectively.The primer specificity were tested by 16 species of algae strains and 1 species of aquatic bacteria strain,1 species of human commensal bacteria strain and human DNA.4 primers(AH,Ah,AHH,AER)showed high specificity and sensitity for Aeromonas hydrophila,which enable to the PCR-CE establishment.PCR-CE methods established with 4 primers was used for the cases validation(36 drowned corpses,4 land death as blanks and 1 cases of postmortem drowned).The total detection rates of PCR-CE methods constructed by primers AH,Ah,AHH and AER were 86.1%,52.8%,83.3%,44.4%,respectively.The detection rate of detection system constructed with primers AH and AHH were higher than the primer Ah and AER.As to compare Aeromonas hydrophila DNA detection rates in the various organs(lung,haper and kidney)from drowned corpses and water samples,there was no significant difference(p>0.05)between the PCR-CE test methods constructed by primer AH and AHH.3.The main separation and enrichment methods for diatoms were compared in the drowning diagnosis,and provide the preference for diatom test in forensics,Then,with which,the PCR-CE detection techniques established were evaluated.1)With 60 drowning rabbits,3 methods mainly used in the current forensic laboratory were compared as following:(A)Microwave acid digestion-nylon NM filtration-automated scanning electron microscopy(SEM)detection,(B)Microwave acid digestion-PES NM filtration-SEM detection and(C)Strong acid digestion method-centrifugation-SEM detection.(traditional acid digestion centrifugal enrichment).The results show that method(A)was the optimum for the total recovery rate in diatoms(76.9 ± 20.2%)and less damage rate of diatoms(0.9 ± 1.1%),the longest acidity duration;method(B)had no significant difference from(A)on the total recovery rate of diatoms.Though inferior to method(A)on acid resistance,but the transparent PES film can be applied to optical inspection with lower costs and more promotion of the feasibility.2)The evaluation of the PCR-CE methods of planktonic diatoms and bacteria(Aeromonas hydrophila)DNA test were conducted with method(A).The total positive rate 85.9%,83.7%by the PCR-CE methods constructed by primers UPA159 and rbcI.197 of planktonic diatom DNA respectively,had no statistically difference from the positive rate 97.2%with MD-VF-Auto SEM method in drowning diagnosis.But primer A457 had statistical difference.The total positive rate 86.1%,83.3%,by the PCR-CE methods constructed by primers AH and AHH of Aeromonas hydrophila DNA respectively,had no statistical difference from the positive rate 97.2%with MD-VF-Auto SEM method in drowning diagnosis.Primer Ah and AER(52.8%,44.4%)had statistical difference.