Study On Detection Of Plankton DNA By PCR-CE For Drowning Diagnosis

Diatom test is so far regarded as "the gold standard" for drowning diagnosis.In the forensic laboratory,observing the morphology of diatoms by microscope is often used,but there are many shortcomings,such as large amount of required materials,cumbersome operation,time-consuming,easy pollution and so on.With the rapid development of molecular biology,the method of PCR-CE is used to detect the plankton gene for diagnosing drowning accurately,which provides a new idea for the field of forensic medicine.Currently,related study has been focused on the detection of phytoplankton genes in river,while there is a rare study on planktonic bacteria.And there are some differences about the types and quantities of plankton in organs from different rivers.So it is found that only a single gene of a single species is detected,and there is a lack of test efficiency and possible missed detection.Therefore,To enhance the practical application effect of drowning diagnose by PCR-CE,it is necessary to find more reliable genetic markers of different plankton and establish a multiplex system for detecting multiple target genes of planktons.Based on that,a series of studies was carried out,and the contents are as follows:(1)Targeted to the conserved sequences of Aeromonas hly A,aer A and 16 SrRNA from GenBank,the specific primers(hly A-2,aer A-1 and P16s)were designed about them.And then,human being,3 species of human symbiotic bacteria,9 species of planktonic bacteria,and 38 species of planktonic algaes were detected to verify specificity and sensitivity of the primers.hly A-2 could amplify Aeromonas hydrophila and salmonicida specifically,of which PCR products were 150 bp and the sensitivity were 0.034 ng and 0.94 ng,respectively.aer-1 could amplify Aeromonas salmonicida and veronii specifically,of which PCR products were 180 bp and the sensitivity were 0.034 ng and 0.92 ng,respectively.P16 s could amplify Aeromonas salmonicida and veronii specifically,of which PCR products were 668 bp and the sensitivity were 0.90 ng and 0.013 ng,respectively.Besides,the model of experimental pig was established,and the organ of experimental pigs were detected by using the primers with high specificity and sensitivity.The positive rates of hly A-2,aer-1 and P16 s in drowning experimental pigs were 80 %,70 %,50 %,respectively.Furtherly,they were negative in the non-drowning tissues.(2)Based on the previous research,14 pairs of specific primers were designed and screened about homologous sequences of drowning-related plankton(diatom,cyanobacteria,green algae,dinoflagellate and planktonic bacteria),and the primers were labeled with 5-dye in multiplex system.This multiplex system could specifically amplify 35 species of planktonic algae and 3 species of planktonic bacteria.The preliminary positive rate of this system was investigated by 16 experimental pigs.For this multiplex system,the positive rate of drowning pigs was 90 %,and all the non-drowning pigs were negative.(3)57 cases of cadavers were detected by the related gene of Aeromonas by PCR-CE,the multiplex system and MD-VF-Auto SEM.The positive rates of hly A-2,aer-1 and P16 s in 57 cases of cadavers were 81.48 %,78.38 %,51.85 %,respectively.Related to the results of experimental pigs,hly A-2 was the best primer.As for the multiplex system and MD-VF-Auto SEM,the positive rates were 96.30 % and 98.15 %,respectively.Statistical analysis results show that there were no significant differences in the positive rate of the cadavers between multiplex system and MD-VF-Auto SEM(P > 0.05),but the positive rate of the cadavers by multiplex system was much higher than the related gene of Aeromonas by PCR-CE(P <0.05).On the study,based on the detection of hly A,aer A and 16 SrRNA gene in Aeromonas,the methods of PCR-CE were constructed,and this methods enriched the genetic markers of drowning diagnose in molecular biology and provided reliable target markers to establish a multiplex system about related genes of plankton.Besides,the established multiplex system,targeted to 14 different genes of drowning-related plankton,,had less tissue consumption,higher positive rate which increased the positve rates of liver and kidney significantly,and improved the detection efficiency.With a promising prospect for application in forensic drowning diagnosis,multiplex system is a better complementary to diatom tests.