The Forensic Application Study On The Detection Of Algae 16SrDNA Gene In Diagnosis Of Drowning Based On PCR Method

Object:Drowning (drowning) is one of the mechanical asphyxia death, due to liquid medium (usually water) obstruct respiratory tract (nose and mouth) and cause the the body be short of oxygen, carbon dioxide retention, and lead to death. And in China, there are many river channel and corrupted bodies in river, which can reach to thousands of dead bodies in Guangzhou section. In this situation, the dead of bodies in water may be unexpected drowning deaths, but may also be suicide, homicide and dumped into the water after the death to forge the unexpected drowning and so on. Therefore, the key issue is to judge drowning or not drowning of the bodies found in water. The methods of judging drowning found bodies are often according to comprehensive analysis of autopsy, diatom test and experimental examination, In some cases, due to the combination of external factors lead to the body corruption and drowning signs disappeared, autopsy and laboratory examination for diagnosis of drowning often cannot work at same time, but diatom test may be a indispensable means of diagnosis of drowning. In present, there are so may methods of diatom test, including chemical digestion method, physical digestion method and the main steps of morphological observation for dealing with samples of water and body organs are centrifugal or membrane enrichment method to collect diatoms after different digestion, and by light or electron microscopy directly to observe the morphology of diatoms and species to assist in the diagnosis of drowning and drowning site. And one of most widely used method is strong acid digestion, but this method of diatoms damage is bigger, and lost some smaller diatoms in the process of centrifugal, besides, due to operating in open environment, so may lead to false negative and false positive results. Our group established a method in the early stage-Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method has greatly increased specificity and sensitivity of the diatom test, and can be quantitative and qualitative analysis of diatom test, this method has play an important role in diagnosis of drowning. But the method destroyed the cyanobacteria, green algae in the process of acid digestion, microwave digestion, which are related to drown algae. According to reports, green algae, cyanobacteria algae also exist in drowned body organs. In recent years, in order to provides a quick and sensitive method for drowning diagnosis, molecular biology method is applied to the of research of drowning diagnosis and become a research hotspot. Such as in1996, Kane, et al used specific primers for single category of picoplankton to diagnose drowning, and in 2003, Abe, et.al used specific primers of encoding diatom EG1, EG2, SK1, SK2 protein genes, in 2013 Tie J, et al. used 16SrDNA fragments specific primers for cyanobacteria to the diagnosis of drowning, Yu ZhengLiang, et al. used SSU gene specific primers to diagnosis of drowning in 2014. At present, the reports of using PCR technique to detect DNA segments are most of using specific primers for a category of algae to diagnose drowning, but cannot amplify other related to drowned related algae (such as algae, cyanobacteria, green algae, etc.). In order to detect a variety of drowned related algae, our study used specific primers for cyanobacteria algae, diatoms, green algae, to amplify V3-V4 fragment of 16SrDNA, aiming at providing a new method for the diagnosis of drowning.Methods:35 rabbits were divided into three groups randomly:drowning group(n=15), postmortem submission group (n=15), control group(n=5).7 species of algae as control algae (Melosira sp, Nitzschia sp, Synedra sp, Navicula sp, Microcystis sp, Cyclotella meneghiniana, Chlorella sp). Using PowerSoilTM DNA isolation Kit extracted experimental rabbit tissue and human case samples, and algae DNA, and using PCR-PAGE detected algae DNA of experimental rabbit organs. At the same time, using PCR-CE method was used to detect the drowning group of rabbits in the experimental, and compared two methods between PCR-PAGE and PCR-CE in detecting positive rate of product of drowned rabbits, process and the characteristics of each other.64 human samples found in water including 58 diatom positive samples and 6 diatom negative samples test by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy method (MD-VF-Auto SEM) and PCR-CE at the same time, and compare positive rate of two methods for drowning human organs.Results:Algae DNA was detected from most kinds of the tissues from drowning group of rabbits:lung(100%), liver(86%), kidney(86%); For postmortem submersion group, however, algae was detected only from two cases of lung (13%) and none from control group. The positive rate for algae detection in the drowning group was significantly higher than the postmortem submersion group and there has statistical significance (P<0.05).Preparation time before electrophoresis and analysis time of electrophoresis result significantly less than the PAGE electrophoresis (P<0.05), and the operation more convenient, concise, can deal with several samples at the same time, the result is easily to store.PCR-CE method to detect 20 cases samples test by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy method (MD-VF-Auto SEM) results:lung, liver, kidney, respectively 100%,60%,60%, positive rate between the two methods have statistical difference between kidney and liver(P< 0.05),and no statistical difference between lung(P>0.05).1 fresh samples of diatom negative samples was positive with CE electrophoresis.7 kinds of algae DNA of PCR product are positive and negative to amplify human and rabbits gene.Conclusion:Rabbit experimental results show that the method of PCR-PAGE can distinguish between drowning group and postmortem water immersion group. When containing algae in the lungs only should be cautious for diagnosis of drowning due to the water may through the respiratory into tract caused by water pressure from our data. The comparison of two method between PCR-PAGE and PCR-CE for detecting algae in drowning rabbits group have the same positive rate, but PCR-CE can be processed simultaneously, operate more convenient, and the results are easy to store and can detect more samples than PCR-PAGE in the process of detecting PCR products.The research results of detecting human samples with PCR-CE method show that the existence of the cadaver corruption has certainly influence on our PCR method due to algae DNA degradation. fresh cases material using the PCE-CE method have higher positive rate than corrupted material, and the positive rate of PCR-CE method is lower than MD-VF-Auto SEM method viscera samples of liver, kidney algae. In addition, when the numbers of diatom are less in some cases test by microwave digestion-vacuum suction filter-electron microscope scanning, the existence of other drowning related algae detecting by our PCR method at same time may be provide helpful information for the diagnosis of drowning. In this paper, the established PCR method can be to detect 16SrDNA fragment of several species of drowned related algae (cyanobacteria, green algae, diatoms, etc.), and has the good specificity and sensitivity. PCR-CE method is convenient, quick, can deal with many samples for PCR product at the same time. In addition, the results show that if diatom test results were negative or numbers of in the sample is small in some cases in this paper, the detection of other drowned related algae can provide helpful evidence for the diagnosis of drowning, so our method has promising application prospect.