Yadong Guo

Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing

Decomposition is a complex process involving the interaction of both biotic and abiotic factors. Microbes play a critical role in the process of carrion decomposition. In this study, we analysed bacterial communities from live rats and rat remains decomposed under natural conditions, or excluding sarcosaphagous insect interference, in China using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 1,394,842 high-quality sequences and 1,938 singleton operational taxonomic units were obtained. Bacterial communities showed notable variation in relative abundance and became more similar to each other across body sites during the decomposition process. As decomposition progressed, Proteobacteria (mostly Gammaproteobacteria) became the predominant phylum in both the buccal cavity and rectum, while Firmicutes and Bacteroidetes in the mouth and rectum, respectively, gradually decreased. In particular, the arrival and oviposition of sarcosaphagous insects had no obvious influence on bacterial taxa composition, but accelerated the loss of biomass. In contrast to the rectum, the microbial community structure in the buccal cavity of live rats differed considerably from that of rats immediately after death. Although this research indicates that bacterial communities can be used as a “microbial clock” for the estimation of post-mortem interval, further work is required to better understand this concept.

Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China based on COI and period gene

Unequivocal identification of insect specimens is an essential requirement in forensic entomology. With the development of molecular identification, spate of discussions about the feature of the DNA fragments have been raised. Relying solely on single DNA fragment for delimiting closely related species is supposed to be dangerous. Aiming at obtaining more reliable markers that might be universally used, we explore the utility of 700-bp COI fragment and 678-bp period gene fragment in the identification of Sarcophagidae (Diptera). Thirty-six sarcophagid fly specimens were collected from 19 locations in 11 Chinese provinces. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into nine species with relatively strong supporting values, which indicated the possibility of separation congeneric species with COI and period gene fragments. The difference between intraspecific threshold and interspecific divergence confirmed that the combination of nuclear and mitochondrial genes for species identification is much more accurate. The results of this research will be instrumental for implementation of the Chinese Sarcophagidae database.

Genetic polymorphism of 21 non-CODIS STR loci in the Chinese Mongolian ethnic minority.

In this research, we investigated the allele frequencies and forensic parameters of 21 non-, CODIS short tandem repeat (STR) loci (D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435 and D5S2500) among 523 unrelated, Chinese Mongolians in the city of Tongliao, Horqin district, Inner Mongolia Autonomous Region.

Genetic polymorphism of 17 STR loci in Chinese population from Hunan province in Central south China

We selected 586 unrelated healthy individuals (338 males and 248 females) whose ancestors lived in Hunan Province for at least three generations. After informed consent in compliance with the ethical norms set by Chinese legislation was acquired, we obtained bloodstains from each of the subjects. The study was approved by the ethics committee of the Third Xiangya Hospital of Central South University, and was carried out following the guidelines for publication of population data required by the journal [1] and according to the International Society of Forensic Genetics (ISFG) recommendations [2]. The laboratory has passed through proficiency testing in the field of forensic science by the Institute of Forensic Science of Chinese Ministry of Justice. Genomic DNA was extracted from the bloodstains using the Chelex-100 and proteinase K protocol [3]. The quantity of the extracted DNA was determined spectrophotometrically. The 17 short tandem repeat (STR) loci included in the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, USA) and the PowerPlex 16 Monoplex System Kit (Promega, USA) were respectively co-amplified according to the manufacturer’s instruction [4]. Amplification reactions were carried out using a GeneAmp PCR system 9700 (Applied Biosystems). Laboratory internal control standards and kit controls (negative control and 9947A) were used for quality control. The amplified products were separated by capillary electrophoresis on ABI PRISM 3130 Genetic Analyzers (Applied

Identification of forensically significant calliphorids based on mitochondrial DNA cytochrome oxidase I (COI) gene in China

Precise species identification of every insect sample collected from criminal scenes play an essential role in the accurate estimation of postmortem interval (PMI) [1]. The morphological similarity poses a great challenge for forensic entomologists. DNA-based method can be used as a supplemental means of morphological method. In previous studies, various segments of the COI region had been used to discriminate forensically important blowfly species based largely on monophyly [2–6]. Even partial sequences of this COI gene have been proven to have sufficient discrimination power. The composition of each species has its own local characteristics [7]. To the best of our knowledge, there were no large-scale analyzing sequences of the COI gene of blowflies form China. We collected 70 forensically important blow flies from 25 locations in 19 provinces of China. Then A portion of 272 bp fragment of the mitochondrial COI gene was amplified (F: 50-CAGATCGAAATTTAAATAC-TTC-30, R: 50-GTATCAACATCTATTCCTAC-30) and sequenced. All sequences obtained in this study were aligned using ClustalW (http://www.ddbj.nig.ac.Jp/E-mail/clastalw-e.html) and then had been deposited in GenBank by Sequin (http://www.ncbi.nlm.nih.Gov/equin/index.html). Phylogenetic analyses were conducted in MEGA4 [8]. We assessed the 272 bp fragment of COI gene as a potential marker for the identification of Calliphoridae family flies from China. The monophyletic branches of the phylogenetic tree reveal that this marker is suitable for discrimination between these 12 species of three genera of Calliphoridae, especially between some morphologically similar sister species pairs (Lucilia sericata/Lucilia cuprina, Lucilia illustris/Lucilia caesar, Calliphora vomitoria/Calliphora vicina, Chrysomya rufifacies/Chrysomya albiceps). The clear defined levels of intra versus interspecific variation confirmed the ability of this COI gene in species identification. No evident geographical pattern was observed within each blowfly species here, although a few of inter-cladistic specific variations were showed between mitotypes in two distinct populations of P. terraenovae and Ch. megacephala respectively. At the species level, each species formed their own distinct conspecific and monophyletic cluster with high bootstrap values (395%), except for L. cuprina at 76%. High support was obtained for L. illustris as distinct from L. caesar. The relatively high sequence homology of the paired species (interspecific variation is 2.5%) suggests that they were diverged very recently. Although the immature stage of species L. cuprina and H. ligurriens, in particular their third instars, are generally similar in morphological appearance [9], the two species were well distinguished here, and the result is meaningful for further forensic caseworks. H. ligurriens

Genetic polymorphic investigation of 21 autosomal short tandem repeat loci in the Chinese Li ethnic group

Abstract Population genetic data of 21 autosomal short tandem repeats (STRs) were obtained in a sample of 106 unrelated healthy individuals of Bai ethnic minority born in the Dali Bai Autonomous Prefecture in Yunnan Province. We observed 138 alleles with corresponding allelic frequencies ranging from 0.005 to 0.575. The genotypic frequency distributions at those STR loci were consistent with Hardy–Weinberg equilibrium (Bonferronis correction was used for Hardy–Weinberg equilibrium tests). The combined probability of exclusion, power of discrimination, probability of matching value for all 21 STR loci were 0.9999975729, 0.999999999999999999872 and 1.28×10 −19 , respectively. The population data in this study showed significant differences from the previously published population data of Tibetan and Salar groups in some loci.

Estimation of postmortem interval by vitreous potassium evaluation with a novel fluorescence aptasensor

Estimation of postmortem interval (PMI) is a central role in medico-legal identification. Analysis of vitreous potassium ions (K+) concentration is frequently used by forensic workers to estimate PMI. This paper describes interdisciplinary research to introduce fluorescence sensing techniques into forensic medicine. On the basis of silver nanoclusters (AgNCs) probe stabilized by DNA, a simple and highly sensitive fluorescence aptasensor has been proposed to selectively detect K+ ions. The linear range for K+ ions was found to be 0.1 nM-1 mM, with limit of detection of 0.06 nM. Moreover, 63 vitreous humour cases within 36 h after death were further studied to verify the utility of K+ ions in estimating the PMI. By the fluorescence aptasensor method, a new formula was built to determine the postmortem interval based on K+ ions concentration: PMI(h) = −0.55 + 1.66 × CK+(r = 0.791). And the real significance of this research was demonstrated by additional 6 cases with known PMIs. In comparison with the conventional method, the presented aptasensor strategy is cost-effective and easy in measuring vitreous K+, which may be potentially a better way for estimation of PMI in medico-legal practice.

Screening and confirmation of microRNA markers for distinguishing between menstrual and peripheral blood

The identification of menstrual blood (MB) and peripheral blood (PB) left at a crime scene is crucial for crime reconstruction, especially in sexual assaults. MicroRNAs (miRNAs), a class of non-protein coding molecules, have been demonstrated to be a viable tool for body fluid identification in forensic casework. Several groups have searched for miRNAs that are specific to different body fluids. Blood has been studied the most extensively. However, menstrual blood was only involved in five studies, and the results confirming the presence of specific miRNAs could not be reproduced in other studies. In this study, we attempted to screen new markers that can differentiate between menstrual blood and peripheral blood by using Exiqons miRCURY™ LNA Array. Five miRNAs were selected based on the microarray results, namely, miR-141-3p, miR-373-3p, miR-497-5p, miR-143-5p, and miR-136-5p, whose expression levels exhibited 27.95-, 17.96-, 16.74-, 10.14-, and 9.21-fold changes, respectively, compared to the level in peripheral blood. Two classic quantitative methods, TaqMan hydrolysis probes (TaqMan for short) and SYBR Green fluorochrome (SYBR Green for short), were applied in the confirmation step to study the impact of different quantitative methods on the results. Three miRNAs (miR-141-3p, miR-497-5p, and miR-143-5p) were confirmed by TaqMan and one (miR-141-3p) by SYBR Green. Furthermore, bioinformatic methods were applied to interpret the candidate miRNAs. Our results established a multi-step procedure for body fluid identification and showed that the choice of quantitative method is important when miRNAs are used to identify the origin of blood samples.

One-step, visual and sensitive detection of phorate in blood based on a DNA–AgNC aptasensor

The ingestion of organophosphate (Op) pesticides for attempting suicide has been a serious health issue recently. Phorate, a class of Op, has attracted attention in this regard and so the rapid diagnosis and detection of phorate is crucial for human health. In this study, a one-step and visual method for the rapid determination of phorate via an aptasensor based on DNA-templated silver nanoclusters (DNA–AgNCs) has been presented. The designed DNA–AgNC nanoprobe, containing a special structure with an intercalated aptamer, shows excellent stability. Introducing phorate to the system easily triggers the aggregation of DNA–AgNCs, producing a brown-to-colorless color change in the solution in only 6 minutes. Under the optimal conditions, the aptasensor can allow for the determination of phorate in the concentration range of 0–25 μg mL−1, using two linear equations: y = −2.308x + 0.996 (R2 = 0.977) for 0–0.125 μg mL−1 and y = −0.020x + 0.664 (R2 = 0.971) for 0.125–25 μg mL−1. The estimated detection limit of phorate was 0.012 ng mL−1. Moreover, the practicability of this proposed method was also validated by analyzing phorate-spiked human blood samples and the results were in agreement with those from gas chromatography/mass spectrometry (GC/MS) analysis. Therefore, this fabricated aptasensor is suitable for sensing phorate in complex biological samples, and shows high potential for clinical diagnosis or forensic toxicology analysis.

A brief review of forensically important flesh flies (Diptera: Sarcophagidae)

ABSTRACT Forensic entomology could provide valuable data for the minimum postmortem interval (PMImin) estimation and other relevant information, such as causes and circumstances of death. Some representatives of flesh flies are one of the dominant necrophagous insects during early stages of decomposition, demonstrating unique biological characteristics compared with other necrophagous flies. Moreover, they lead to global health concerns as carriers of various pathogenic micro-organisms, and dominantly result in the traumatic myiasis. Thus, sarcophagid flies are considered important in decomposition processes for PMImin estimation. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. The aim of this paper is to provide a brief review on the species, distribution and biological habit of forensically important sarcophagid flies. In addition, the relation between traumatic myiasis and flesh flies, molecular identification methods and developmental pattern of flesh flies are summarized.