Yunfeng Chang

Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China, based on COI and 16S rDNA gene sequences.

Abstract:  Insects attracted to cadavers may provide important indications of the postmortem interval (PMI). However, use of the flesh flies (Diptera: Sarcophagidae) for PMI estimation is limited as the species are often not morphologically distinct, especially as immatures. In this study, 23 forensically important flesh flies were collected from 13 locations in 10 Chinese provinces. Then, a 278‐bp segment of the cytochrome oxidase subunits one (COI) gene and a 289‐bp segment of the 16S rDNA gene of all specimens were successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into four species (Boerttcherisca peregrina [Robineau‐Desvoidy, 1830], Helicophagella melanura [Meigen, 1826], Parasarcophaga albiceps [Meigen, 1826], and Parasarcophaga dux [Thompson, 1869]) with relatively strong supporting values, thus indicating that the COI and 16S rDNA regions are suitable for identification of sarcophagid species. The difference between intraspecific threshold and interspecific divergence confirmed the potential of the two regions for sarcophagid species identification.

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 flesh flies based on a shorter fragment of the cytochrome oxidase subunit I gene in China

With the development of molecular identification, there has been a great deal of discussion about the feature of the mitochondrial DNA (mtDNA) fragments. Although longer fragments may minimize stochastic variation across taxa and be more likely to reflect broader patterns of nucleotide divergence, shorter fragments have many advantages, such as quick, easy and economical. Extensive application of long mtDNA segments for species identification cannot always be achieved as a result of constraints in time and money. In the present study, a molecular identification method involving the sequencing of a 272‐bp ‘barcode’ fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 55 specimens, representing 7 Chinese sarcophagid species from varying populations, was evaluated. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into seven species, which indicated the possibility of separation congeneric species with the short fragments. The results of this research will be instrumental for the implementation of the Chinese Sarcophagidae database.

An investigation and pathological analysis of two fatal cases of cadmium poisoning

Pollution associated with population growth, and with industrial and urban development has led to a serious decline in the water quality of Chinese rivers. Cadmium (Cd) is recognized as one of the most toxic metals and is strongly accumulated by organisms. Humans are exposed to cadmium originating from the environment and from industrial pollution. In spite of thousands of published studies on Cd, there is little information on its pathological features seen in human autopsy. The gross and pathological findings of forensic autopsies of two case of cadmium poisoning are presented and related to an epidemiological investigation. In both cases, multiple organ damage was observed, involving brain, lung, liver, kidney, red blood cells, and platelets, which is consistent with reports in the literature. In particular, in both cases, transmission electron microscopy revealed a large number of dense lysosomal and phagocytic particles in the cytoplasm near the nucleus, indicating the need for a genotoxic study of cadmium. Our observations provide new clues for the future recognition and prevention of Cd poisoning.

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 important blow fly species (Diptera: Calliphoridae) in China by mitochondrial cytochrome oxidase I gene differentiation

Abstract  Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases. However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278‐bp segment of the cytochrome oxidase subunit I gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278‐bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification, especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata (Meigen, 1826) and Lucilia caesar (Linnaeus, 1758).

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.

Trimethylamine in postmortem tissues as a predictor of postmortem interval estimation using the GC method

Trimethylamine (TMA), a simple amine in volatile compounds, indicating the spoilage degree of meat, related with the late postmortem interval (PMI) during the cadaver decomposition process. In this paper, a headspace gas chromatography (HS-GC) method has been successfully built to analyze the content of TMA in postmortem rat tissues (liver, myocardial and skeletal muscle tissues), which is used for PMI estimation with the wide range of PMI (0-720 h postmortem) at 16 °C ± 1 °C. Correspondingly, three equations about the relationship between the PMI and TMA content were established, in which they showed that the TMA content in all three tissues increased from the firstly detected time to 192 h and gradually decreased after 384 h. Furthermore, the TMA measurement in five human samples was carried out and the results (<35 h PMI) showed a good agreement about the change trend of TMA content between human and rat, which elucidated that this research might provide a new insight for the forensic scientist to band HS-GC technology together with TMA determination in the PMI estimation.

Rapid Determination of H2S Poisoning in a Forensic Study Using a Novel Fluorescence Assay Based on Zn/Cu@BSA Nanoclusters

The quantitative determination of H2S in the blood can provide valid evidence for H2S poisoning through occupational exposure. However, known traditional methods for the detection of H2S in blood are time consuming, require complicated pretreatments, and have low sensitivity. In this paper, a new fluorescence sensing assay is proposed for the rapid detection of H2S poisoning in forensic cases based on bovine serum albumin (BSA)-stabilised zinc/copper (Zn/Cu) bi-metal nanoclusters (Zn/Cu@BSA NCs). The as-prepared Zn/Cu@BSA NCs probes have been characterised by UV-vis absorption and fluorescence spectroscopy. The fluorescence of Zn/Cu@BSA NCs can be quenched through specific interactions between HS−/S2− and the Zn2+/Cu2+ bi-metal ions. Under optimised conditions, the fluorescence sensing method was linear in the concentration range of 2.5 nM to 30 mM with 0.69 nM as the limit of detection. Moreover, the practical feasibility of this fluorescence sensing method has also been demonstrated by the analysis of mice blood samples containing different levels of sulfide and human blood samples from forensic cases of H2S poisoning. Compared with gas chromatography/mass spectrometry (GC/MS), this fluorescence sensing method is quite simple, straightforward, and can be accurate for the quantitative determination of H2S poisoning in a few minutes for forensic analysis. Overall, this is the first report of a bi-metal fluorescence sensing assay for detecting H2S poisoning directly in blood. This research may provide a new approach for forensic toxicologists to monitor poisoning by H2S using a fluorescence-sensing method.

A novel aptasensor for malathion blood samples detection based on DNA-silver nanocluster

Pesticides have drawn great attention because of their bad effects on health and the environment. Among these, organophosphates were the most widely used in China and malathion is the most common. Direct or indirect contact with malathion does harm to our nervous, respiratory and digestive systems. Thus, accurate detection of malathion in biological samples is very useful to ensure the safety of human health, especially in agricultural countries. In this paper, a sensitive and selective aptasensor based on DNA–silver nanocluster probes has been proposed for the simple and efficient detection of malathion in biological samples. First its feasibility was verified when the UV-vis absorbance apparently decreased with inclusion of malathion and the response differed from that with other chemicals. Then under optimized conditions, a concurrent decrease in absorbance with the addition of malathion from 0 μg mL−1 to 25 μg mL−1 was achieved with a limit of detection of 25 pg mL−1. The linear equations between the UV-vis absorbance spectrum and the concentration of malathion were y = 10.167x + 1.051, R2 = 0.986 (range from 0.000025 μg mL−1 to 0.025 μg mL−1) and y = 0.0136x + 1.431, R2 = 0.995 (range from 0.25 μg mL−1 to 25 μg mL−1). Moreover, investigations in blood samples were conducted to test the biocompatibility of the aptasensors for the sake of practical applications in forensic research. The detection results of the proposed method were almost consistent with those of conventional high-performance liquid chromatography (HPLC). In view of its simplicity, low cost, selectivity, sensitivity and reliability, the aptasensor introduced in our work may potentially be a better way to detect malathion in forensic toxicology examination.