Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death

Abstract

Diatoms are unicellular microalgae with cell wall made up of rigid silica found in all open water bodies. They thus resist degradation and hence are important tool to diagnose cause of death in drowned bodies. The nitric acid digestion method practiced conventionally in forensic science laboratories has limitations due to manual error. Plant chloroplast genes found in diatoms such as ribulose‐1,5‐bisphosphate carboxylase oxygenase (rbcL‐3P) and rbcL, universal plastidic amplicon (UPA), and photosynthesis I P700 apoprotein chlorophyll Al (psaA), which play an important role in photosystems I and II of photosynthesis, are tested to diagnose drowning in experimental mice. It was seen that psaA‐2 showed amplification at 150 bp in all biological samples. The sequences of psaA‐2 gene marker showed 100% proximity to Thalassiosira weissflogii and rbcL‐3P showed 99% resemblance to Pseudo‐nitzschia multiseries. On the other hand, in postmortem drowned biological samples, the chloroplast‐based gene marker failed to show any amplification.

Photosystem I P700 chlorophyll a apoprotein A1 as PCR marker to identify diatoms and their associated lineage

“…The molecular study also revealed diversity in PCR amplicons for 13 fresh water diatoms (Table 1) using about 22 pairs of gene based PCR markers (Table ; Vinayak, 2020) run against standard DNA ladder of 100 kb size. It was observed that primers specific for gene locus DRR3 ‐1‐6, ScfcpA , Lhcf11 , SIT1 , LOC101218388 , SIT3 , SIT4 ‐1‐4, COI ‐ 5P 1‐6, and 18S rRNA 3‐4 did not amplify the diatoms at same PCR conditions.…”

“…This demonstrates that sequencing lineage trees not only identifies the diatom correctly but also gives information about its ancestorial origin from fresh water or marine water. This has wide application in phylogenetic studies, screening diatoms for biofuel (Khan et al, 2020; Khan et al, 2021; Kumar et al, 2018b; Kumar et al, 2018a; Vinayak et al, 2014; Vinayak et al, 2015), and use of diatoms in forensic science (Vinayak, 2020; Vinayak & Gautam, 2019).…”

“…The diatom monocultures after morphological screening were washed with double distilled water and centrifuged at 174 g. The supernatant was discarded and pellet stored at −20℃ for DNA extraction by CTAB method for further PCR and downstream processing (Doyle & Doyle, 1990;Li et al, 2013). The molecular identification was processed using PCR markers designed using primer 3 software (Rozen & Skaletsky, 1999;Untergasser et al, 2012) specific for gene locus DRR3, scfcpA, Lhcf11, SIT1, SIT3, SIT4, LOC101218388, COI-5P, rbcL, rbcL-3P, LSU D2/D3, UPA, psaA, and 18S rRNA used in earlier studies from this laboratory and others (Vinayak, 2020; Table S2). Thereafter, the PCR amplicons were purified by PCR purification kit (Qiagen™).…”