Establishment And Validation Of Real-time Quantitative PCR System For Detection Of Drowning-related Plankton

Background:There were about 40,000 people died from drowning in 2019,making drowning the third leading cause of accidental death in China,only behind the falls and traffic accidents,according to the 2020 China Health Statistics Yearbook.Among those drowning crowds,56.6%were teenagers under the age of 14,ranking first.In terms of geographical distribution,rural areas with more water and a relative lack of infrastructure account for a greater proportion of drowning accidents.The vast majority of bodies in the water were accidental or suicides,while others were killed by drowning or dumped into the water.The cause,time,manner,place of death and identification of the corpse in water are all mandatory works that forensic experts have to perform.In particular,if the cause of death can be not determined in time,petitions,entanglements,speculation and even mass incidents might occur,such as "Guizhou Weng’an Incident",directly affecting the overall situation of social stability.Therefore,the identification of the cause of death timely and effective is crucial to the determination,investigation and resolution of the case.However,most of the corpses encountered in the actual work of forensic medicine have been highly corrupt,and the signs of drowning such as mushroom foam in the mouth and nose,foreign object in the respiratory tract and waterborne emphysema are often missing,so it is difficult to determine the cause of death according to observation and autopsy only.In recent years,diagnosing the cause of death of dead bodies in water has been a huge challenge.In recent years,there are many researches around the detection of cadaver ion components,virtual autopsy,diatom morphology,foreign objects,and target gene detection of algae and bacteria in water.However,most of the methods are limited to experimental research,classification and qualitative uncertainty,which might lead to misjudgment,and making it difficult to be applied in forensic practice.The forensic technology and equipment of diatom test developed by our team have achieved a great of innovation in the tissue digestion,enrichment of diatom,qualitative and quantitative analysis of diatom.And the method of diatom test we established has been transformed into China’s public security industry standards,which has been widely applied in the whole country and solved the major problems of diagnosis of cause of death among corruption bodies.However,this technique is based on morphological examination,which requires expensive electron microscope equipment and requires high requirements for operators,results in that it is difficult to be popularized in basic laboratories or economically underdeveloped areas.To address these deficiencies,our research embarks from the actual demands,on the basis of early researches,aiming at the diatom,cyanobacteria,green algae and Aeromonas,we have screened the specific genetic markers,designed specific primers,set up the fluorescent quantitative PCR(qPCR)system to diagnose drowning,and shows great application prospects.Method:Analyses the species and content of drowning related plankton comprehensively which had been reported,aiming at diatom,cyanobacteria,green algae and Aeromonas existed universally in the water,we have screened 4 specific primers,and detected those target genes by real-time fluorescent quantitative PCR(qPCR)SYBR Green I dye marker method,established a method to diagnose drowning.In accordance with the international SWGDAM standard,the species specificity,sensitivity,inhibition,animal model verification and actual case sample verification of 4 pairs of primers were carried out to test the accuracy and practicality of the qPCR diagnostic system in the diagnosis of drowning.Result:The qPCR drowning diagnosis system we have established was able to detect 12 diatoms,6 cyanobacteria,8 green algae,and 5 Aeromonas specifically,and there was no amplification products among 6 kinds of symbiotic bacteria,8 kinds of common human body non anthropogenic creature,and 2 kinds of human DNA standard(9947A and 9948).The DNA content can be detected in the system was lowest to 1 pg,and the period of detection was 3 hours.38 drowned animals and 120 actual cases were verified with accuracy of 94.74%and 93.33%,respectively.Conclusion:The qPCR method we have established for the diagnosis of drowning was of great species specificity,sensitivity and anti-inhibition.The characteristics of short detection period,high sensitivity,low cost and strong practicability make it easily to popularize,and can be applied in the basic DNA laboratory.If combined with diatom morphology test,the diagnostic accuracy and evidence of drowning would be improved greatly.