Determination the Presence of Amplification Products of 16S Rrna Microcystis Aeruginosa As a Biomarker of Drowning
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Rom J Leg Med [27] 16-21 [2019] DOI: 10.4323/rjlm.2019.16 © 2019 Romanian Society of Legal Medicine FORENSIC PATHOLOGY Determination the presence of amplification products of 16s rRNA microcystis aeruginosa as a biomarker of drowning Volodymyr M. Voloshynovych1,*, Roman O. Kasala2, Uliana Ya. Stambulska3, Marian S. Voloshynovych4 _________________________________________________________________________________________ Abstract: Forensic medical diagnostics of drowning now is a difficult issue to resolve. Determination of diatom plankton with light microscopy is one of the supplementary methods for diagnostics of drowning. The disadvantage of this method is the use of concentrated acids to destroy the tissues of the organs, which greatly complicates, and sometimes precludes the detection of diatom plankton. In this case, the detection of other phytoplankton species in internal organs is treated as pseudoplankton, but does not have a diagnostic value. We have developed a sensitive and specific method of drowning diagnostics using a pair of specific oligonucleotide primers by the polymerase chain reaction (PCR) method to determine the presence of DNA of Cyanobacteria of the genus Microcystis, namely a fragment of the 16S rRNA gene in the tissues of mice and water samples in order to establish the fact and place of drowning. In order to evaluate the diagnostic value of this method, we conducted an experimental study to detect fragments of the 16S rRNA gene in mice tissues during drowning and post-mortem immersion. The amplification products were found in the tissues of heart, kidneys, liver, spleen, bone tissue, brain tissue, and lungs in case of drowning. During post-mortem immersion products of amplification are detected only in the tissues of lungs. The results indicate that the proposed PCR method is a potentially useful tool for diagnosing of mechanical asphyxia as a result of drowning. Key Words: Drowning, Forensic pathology, DNA, Polymerase chain reaction, Cyanobacteria. INTRODUCTION asphyxia as a result of drowning is currently a difficult issue for resolution [3-8]. Drowning is a leading cause According to a global report by the World of unintentional deaths [9]. A number of methods have Health Organization, drowning occurs annually with been developed over the years to determine mechanical 360,000 people worldwide dying as a result of drowning, asphyxia as a result of drowning [10]. accounting for more than 9% of global mortality [1]. More Determination of the presence of diatom algae is than 90% of deaths due to drowning are recorded in low one of the supplementary methods of diagnosis, not only and middle-income countries. Mechanical asphyxia as a drowning but sometimes the place of drowning [11-13]. result of drowning in Ivano-Frankivsk region amounted The scientific community is constantly discussing the to 354 cases for the period from 2012 to 2016. This is 4,5% authenticity and reliability of the diatom test at drowning of all types of expertise conducted by the Ivano-Frankivsk [14]. There are even several cases where the expert's Regional Bureau of Forensic Medical Examination [2]. findings obtained using diatoms have been challenged Forensic medical diagnostics mechanical and sometimes canceled in court [15]. 1) Ivano-Frankivsk National Medical University, Department of Pathomorphology and Forensic Medicine, Ivano- Frankivsk, Ukraine * Corresponding author: Tel: +380509569868, E-mail: [email protected] 2) Kharkiv Sate Zoo Veterinary Academy, Mala Danylivka, Ukraine 3) Vasyl Stefanyk Precarpathian National University, Department of Biochemistry and Biotechnology, Ivano-Frankivsk, Ukraine 4) Ivano-Frankivsk National Medical University, Department of Dermatology and Venereology, Ivano-Frankivsk, Ukraine 16 Romanian Journal of Legal Medicine Vol. XXVII, No 1(2019) In some cases, the presence of diatom algae and biological base (vivarium) of the Ivano-Frankivsk during drowning is so small that it is not possible to National Medical University were used during the detect them by microscopy [16]. Picoplankton, for scientific experiment procedure. All animals were example, could not be morphologically discriminated randomly divided into three groups: animals that were from other bacteria such as Proteobacteria and Bacillus drowned (n = 6), animals that were immersed in water by microscopy [17-19]. In such cases, the presence of after death (n = 6) and control group (n = 6). All mice phytoplanktonic DNA detected by PCR in tissues during were derived from the experiment by human methods drowning is perhaps the only method for diagnosis of of killing in accordance with Article 2 of the European drowning [20-25]. Convention for the Protection of Vertebrate Animals Cyanobacteria is one of the most common used for Experimental and other Scientific Purpose [28]. phytoplankton groups that can be found not only The experiment was conducted in September 2017. in standing water of lakes but also in seawater [26]. Microcystis aeruginosa is one of the most common types Experimental drowning of cyanobacteria. These are the simplest single-celled For experimental drowning, was selected the algae, in which there is no nucleus and mitochondria. town lake of Tysmenytsia, Ivano-Frankivsk region, Microscopic colonies of spherical and carnal forms Ukraine, which is characterized by a slow movement of develop in fresh water with standing and slowly flowing surface waters, standing water (lentil conditions of water water. Cyanobacteria that produce hepatotoxins can be movement). Drowning was carried out at the coordinates harmful to human and animal health, especially when of point 1: latitude 48°54'4.5 "N longitude 24°50'32.93"E. they have a high concentration of water and cause the Height above sea level is 245 m. formation of blooms [27]. The group of animals subjected to drowning The development of criteria for determination (n = 6) was placed in cagesand immersed in water, at a of drowning, using experimental drowning of mice, depth of 30 cm, for 1 minute, and then removed from the satisfies the statistical requirements necessary for water. 30 seconds after the animals were taken, out they argumentation. When experimental drowning water were again immersed in water at the same depth until with available phytoplankton in it penetrates into closed the animal died [29]. For post-mortem immersion in organs, as in the case of lethal drowning in humans, the water (n = 6) animals were used, which had previously differences between experimental and real drowning will been intraperitoneally injected with a lethal dose of be minimal. anesthetic. The animals in the cages were immersed in water at the same depth as during drowning. Duration MATERIALS AND METHODS of the immersion was 6 hours. During the autopsy of the mice, a collection of tissues was performed, namely: Sample collection of water lung, heart with blood, kidney, liver, spleen, femoral For the research, a water sample from the town bone, and brain tissue removed from the cranial cavity. lake of Tysmenytsia, Ivano-Frankivsk region, Ukraine Several sets of sterilized instruments and distilled water was conducted. This lake is characterized by lentil were used during autopsy to exclude phytoplankton conditions, namely the lack of significant movement of from outside of the samples under investigation. water. Water was taken from five different places in a Samples from each type of tissue were crushed and then sterile plastic tube with a volume of 10 mL. Coordinates homogenized with a small amount of non-planktonic of water collection points: point number 1 - latitude water by a homogenizer. 48°54'4.51"N longitude 24°50'32.93"E; point number 2 - latitude 48°54'5.05"N longitude 24°50'30.49"E; A pure culture of cyanobacteria point number 3 - latitude 48°54'8.51"N longitude Samples of water obtained from the reservoir of 24°50'31.06"E; point number 4 - latitude 48°54'10.87"N the town lake Tysmenytsia were cultivated on a Fitzgerald’s longitude 24°50'33.80"E; point number 5 - latitude liquid medium modified by Zender and Gorham (№ 11) 48°54'7.85"N longitude 24°50'34.20"E. at a temperature of 22-25°C and i artificial llumination Before the sampling of water samples with of 3000 lux of daylight lamps with alternating light and phytoplankton, the temperature of air and water was darkness of 16-8 hours [30-31]. When growing colonies, determined. The temperature was measured using a agar was added to the medium and a series of transplants mercury thermometer with a graduation scale of 0,5° was made in glass biological test tubes. Thus, we managed C. The water temperature at the place of drowning was to obtain a purely cyanobacterial culture of Microcystis 16°C. All water sampling was carried out between 11 and aeruginosa Kütz. emend Elenk. Pure culture served as 13 hours of PM. a marker to denote the differences between products of PCR and reservoirs, as there are different types of Experimental animals cyanobacteria in reservoirs. Eighteen white mice of any sex from the clinical 17 Voloshynovych V.M. et al. Determination the presence of amplification products of 16s rrna microcystis aeruginosa Removal of cyanobacteria from the test 1. Denaturation of nucleic acid, which consisted substances of 30 cycles of denaturation at 95°C for 30 s; Plastic tubes of 10 mL with samples of water from 2. Annealing - 30s at 57°C; the lake were centrifuged at 3000 rpm for 10 minutes. 3. Extension (or elongation) at 72°C for 30 s, and The top layer of water was removed using a pipette. The PCR was conducted using a thermal cycler. remainder of the water in the test tubes was transferred Electrophoretic separation of PCR products to Eppendorf tubes of 2 mL volume, and they were to be in a 6% polyacrylamide gel for 30 min allows a clear frozen. determination of the presence of DNA of the Microcystis Removal of cyanobacteria from the test aeruginosa species at the appropriate size of the amplified specimens was carried out in accordance with the fragment in the biological tissues of the mice that were extraction procedure described by Terazawa and drowned in the experiment.