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The morphological changes based on deposition of secondary dentin and mineralization of the third molar have been proven to be related to chronological age. However, Kvaal’s method on the theory of deposition of secondary dentin was controversial with respect to dental age estimation in the recent research. The aim of this study was to combine the parameters of Kvaal’s method with relatively high correlation coefficients and mineralization stages of the third molar to improve the accuracy of predicting the dental age of subadults in northern China. A total of 340 digital orthopantomograms of subadults aged from 15 to 21 years were analysed. A training group was used to test the accuracy of the original Kvaal’s method and to establish novel methods for subadults in northern China. A testing group was used to compare the accuracy of the newly established methods with the Kvaal’s original method and with published method specifically used in northern China. To increase the feasibility of our estimation model, we combined the mineralization of the third molar to build a combined specific formula. The results showed that the combined specific model increased the coefficient of determination to 0.513, and the standard error of the estimate was reduced to 1.482 years. We concluded that the combined specific model based on the deposition of secondary dentin and mineralization of the third molar could improve the accuracy of dental age assessment of subadults in northern China. Key Points

The aim of this study was to evaluate the applicability of Cameriere’s European formula for age estimation in children in South China and to adapt the formula to establish a more suitable formula for these children. Moreover, the performance of dental age estimation based on Cameriere’s method combining the developmental information of permanent teeth (PT) and third molar (TM) was also analyzed. Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed. The samples of Group A were divided into training dataset 1 and test dataset 1, and the samples of Group B were also divided into training dataset 2 and test dataset 2. A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1. Additionally, a PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2. The Cameriere’s European formula underestimated chronological age with a mean difference (ME) of −0.47 ± 1.11 years in males and −0.69 ± 1.19 years in females. However, the South China-specific formula underestimated chronological age, with a mean difference (ME) of −0.02 ± 0.71 years in males and −0.14 ± 0.73 years in females. Compared with PT model and TM model, the PT and TM combined model obtained the smallest root mean square error (RMSE) of 1.29 years in males and 0.93 years in females. In conclusion, the South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children. Key points Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed. A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1. A PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2. The South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

In some criminal cases, the identity of suspect is unknown and there is no matching DNA profile in the DNA database. Forensic DNA Phenotyping can provide useful investigative information for these cases. Most forensic studies focus on visible characteristics rather than behavioral characteristics. However, smoking is prevalent in the Chinese population, and DNA methylation is the most promising biomarker for smoking. We collected 204 whole blood samples from the Chinese population and measured methylation levels of 9 smoking-related CpG loci using the methylation-sensitive single-nucleotide primer extension method (Ms-SnuPE). But the single-base extension primers of loci cg12803068 and cg21566642 contained other CpG sites, which may introduce bias, and only the other 7 CpG loci were included in subsequent statistical analysis. The methylation level of locus cg05575921 near the aromatic hydrocarbon receptor repressor (AHRR) gene was much lower in the current smoker group than in the never smoker group. To evaluate the ability of each of 7 CpG loci to predict smoking status, the logistic regression (LR) models were established separately, and locus cg05575921 had the best ability to predict smoking status compared with the other 6 loci. Then, combined (including loci cg19572487, cg05575921, cg23480021, cg23576855, cg21161138, cg01940273, and cg09935388) and stepwise (including loci cg05575921 and cg01940273) multinomial logistic regression (MLR) models were also established. Both combined and stepwise MLR models had good efficiencies in predicting smoking status, and outperformed the above 7 LR models. However, the accuracy, specificity and area under the curve (AUC) of stepwise MLR model in the testing dataset were slightly higher than those of combined MLR model, and the stepwise MLR model required less loci information. Therefore, the stepwise MLR model based on 2 significant CpG loci was more recommended model for predicting smoking status in the Chinese population, and the formula was as follow: P = 1/(1 +e

The AGCU Expressmarker 20 + 20Y Kit is a newly devised short tandem repeat (STR) multiplex system that simultaneously analyses a set of 19 autosomal STR loci, 20 Y chromosomal STR loci and the amelogenin locus with six-dye fluorescent labelling. Here, the AGCU Expressmarker 20 + 20Y system was validated following the guidelines published by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-condition, sensitivity, mixtures, species specificity, models of inhibition, precision, stutter percentage, concordance, population genetic studies, performance on three kinds of degraded DNA and a type of casework samples. The results indicated that the kit had high sensitivity when there was a small amount of DNA (0.0625 ng), more than one male (minor: major=1:19), or a mixture of males and females (male: female=1:32), models of inhibition (250 μM hematin, 1500 ng/μL humic acid and more than 100 ng/μL tannic acid) and degraded samples. The kit showed high precision level with standard deviation of allele size no more than 0.0930. Furthermore, this system was also tested in 444 random male samples of Chinese Han and Hui population, showing its high discrimination capability in Han and Hui population. Meanwhile, the system was applicable to the case of the AMELY abnormality. In short, the kit was verified and proved to be a robust, reliable and suitable tool for human identification and casework samples.

Microhaplotype markers have become an important research focus in forensic genetics. However, many reported microhaplotype markers have limited polymorphisms. In this study, we developed a set of highly polymorphic microhaplotype markers based on tri-allelic single-nucleotide polymorphisms. Eleven newly discovered microhaplotypes along with nine previously identified in our laboratory were studied. The microhaplotype genotypes of unrelated individuals and familial samples were generated on the MiSeq PE300 platform. These 20 loci have an average greater than 3.5 effective number of alleles. Over the whole set, the cumulative power of discrimination was 1–3.3 × 10 −18 , the cumulative power of exclusion was 1–1.928 × 10 −7 and the theoretical probability of detecting a mixture was 1–1.427 × 10 −6 . Differentiation comparisons of 26 populations from the 1000 Genomes Project distinguished among East Asian, South Asian, African and European populations. Overall, these markers enrich the current microhaplotype marker databases and can be applied for individual identification, paternity testing and biogeographic ancestry distinction.

China has repeatedly been the subject of genetic studies to elucidate its prehistoric and historic demography. While some studies reported a genetic distinction between Northern and Southern Han Chinese, others showed a more clinal picture of small differences within China. Here, we investigated the distribution of Y chromosome variation along administrative as well as ethnic divisions in the mainland territory of the People's Republic of China, including 28 administrative regions and 19 recognized Chinese nationalities, to assess the impact of recent demographic processes. To this end, we analyzed 37,994 Y chromosomal 17-marker haplotype profiles from the YHRD database with respect to forensic diversity measures and genetic distance between groups defined by administrative boundaries and ethnic origin. We observed high diversity throughout all Chinese provinces and ethnicities. Some ethnicities, including most prominently Kazakhs and Tibetans, showed significant genetic differentiation from the Han and other groups. However, differences between provinces were, except for those located on the Tibetan plateau, less pronounced. This discrepancy is explicable by the sizeable presence of Han speakers, who showed high genetic homogeneity all across China, in nearly all studied provinces. Furthermore, we observed a continuous genetic North-South gradient in the Han, confirming previous reports of a clinal distribution of Y chromosome variation and being in notable concordance with the previously observed spatial distribution of autosomal variation. Our findings shed light on the demographic changes in China accrued by a fast-growing and increasingly mobile population.

The necrobiome is the postmortem community that includes bacteria, fungi, arthropods, and other cadaver-associated organisms. It has been suggested as biological evidence for forensic investigation. Fungi form distinctive mildew spots in colonizing decomposing bodies, converting them into moldy cadavers. However, the postmortem fungal community consists of more than these visible species. Characterizing the succession pattern of the fungal community during decomposition is valuable not only for understanding the ecosystem composition of the cadaver decomposition islands but also for contributing to forensic investigations. In the present study, the fungal composition of pig cadavers and succession patterns during decomposition were investigated with high-throughput sequencing. The succession patterns were easier to discern in outdoor cadavers, compared with those that were placed indoors. The metabarcoding approach revealed trends linking particular fungal taxa with specific postmortem intervals (PMIs). Dominant species increased notably in cadavers and soil. Furthermore, the succession of the soil community was driven by the cadaver decomposition. Significant mycoflora differences were observed between environmental and cadaveric soil. The results obtained suggested that postputrefaction mycoflora have considerable potential for PMI estimation, particularly in cases that involve heavily decomposed bodies. In addition, the diversity of fungal communities revealed by the metabarcoding approach allowed us to discriminate the sites of cadaver decomposition, implying that postputrefaction mycoflora may be helpful in identifying the environment in which a cadaver has been placed, or the original location from which a cadaver has been moved. Our results provide an important step towards developing fungal evidence for use in forensic science and add to the growing body of work on postmortem microbial communities.

The Han population accounts for the majority of the Chinese population. Hunan is a large province located at 108°47′–114°15′ E and 30°08′–24°38′ N in Southern China, specifically in the area in the...

The flesh flies (Diptera: Sarcophagidae) are significant in forensic investigations. The mitochondrial genome (mitogeome) has been widely used as genetic markers for phylogenetic analysis and species identification. To further understand the mitogenome-level features in Sarcophagidae, the complete mitogenome of Sarcophaga formosensis (Kirneret Lopes, 1961) (Diptera: Sarcophagidae) and Sarcophaga misera (Walker, 1849) (Diptera: Sarcophagidae) was firstly sequenced, annotated, and compared with other 13 Sarcophagidae species. The result indicated that the gene arrangement, gene content, base composition, and codon usage were conserved in the ancestral arthropod. Evolutionary rate of the mitogenome fragments revealed that the nonsynonymous and synonymous substitution rates (Ka and Ks) ratio was less than 1.00, indicating these variable sites under strong purifying selection. Almost all transfer RNA genes (tRNAs) have typical clover-leaf structures within these sarcophagid mitogenomes, except tRNA-Ser (AGN) is lack of the dihydrouridine arm. This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the Sarcophagidae. Phylogenetic analyses containing the interspecific distances from different regions in these species provided us new insights into the application of these effective genetic markers for species identification of flesh flies.

Some representatives of flesh flies visiting/colonizing the decomposed remains demonstrated their values in estimating the minimal postmortem interval (PMImin) since death. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. Although mitochondrial genes have been proposed as a potential DNA barcode for the species-level identification of sarcophagids, some defects still remain such as the substantial memory and processing time taken for homologous comparisons online. Moreover, species identification is mainly achieved by Sanger sequencing based on PCR with genus-specific primers. In the present study we characterized 24 single nucleotide polymorphisms (SNPs) as robust markers of genetic variation for identifying different sarcophagids based on available cytochrome c oxidase I (COI) data and verified them through pyrosequencing (PSQ) technology to establish a SNP-based genotyping system. The system provides a preliminary foundation for developing a rapid, reliable, and high-throughput assay so as to efficiently and accurately identify the sarcophgid flies. Furthermore, the PSQ approach is proved to be faster, more cost-effective as well as more sensitive and specific than custom Sanger sequencing.

Body provides a home for diverse commensal microbiota. Microorganisms such as fungi have major roles in this microbial community stability. Postputrefaction fungi have been recorded in association with decomposed mammalian cadavers in disparate regions. The succession and diversity of these fungi are reviewed with a view to their potential as a forensic tool. The researches of mycology would be an interface to forensic investigation and may provide a means to estimate PMI within serious decomposition. To evaluate the use of succession and diversity of fungi species for PMI estimation, we investigate the Internal Transcribed Spacer (ITS) of fungi community on 3 points of time of rat carcasses conducted by Illumina MiSeq platform. Through high-throughput sequencing, several fungi genus such as Aspergillus , Ophiocordyceps are found in community succession. The community structure of each sample significantly differs in taxon richness and relative abundance patterns within decomposition progress. The succession and diversity of fungi community in corpse decomposition revealed by the indicator of ITS would be a potential forensic tool for PMI estimation.

Angioleiomyoma is a benign soft-tissue tumor originating from vascular smooth muscle, and is rare in the head and neck. The present study retrospectively examined a cohort of patients with head and neck angioleiomyoma treated at the West China Hospital of Stomatology, and also subjected archived tissues to modern immunohistochemical analysis. In total, 21 patients were treated for angioleiomyoma between 1978 and 2012 at the West China Hospital of Stomatology, Sichuan University (Chengdu, Sichuan, China). Medical records were examined and paraffin block sections were cut and stained with hematoxylin and eosin, Masson’s trichrome stain and Van Gieson stain, prior to being subjected to immunohistochemical analysis to re-evaluate and confirm the diagnoses. Angioleiomyomas were found to account for only 0.18% of the benign head and neck tumors in the patients presenting to the hospital over the past 34 years. The diagnosis was more common in males (male:female ratio, 1.625:1) and the mean age at diagnosis was 42.5 years. The most common sites were the buccal mucosa, parotid gland and palate. More than half of the tumors (61.9%) were >2 cm in diameter. Five tumors presented with pain and/or tenderness. The histological subtype was reported as solid in five cases, venous in six, cavernous in nine and venous-cavernous in one. Three tumors exhibited nerve neurofibrils. All tumors were excised with no subsequent recurrence. Cytological and imaging examinations were not useful for pre-operative diagnosis. Angioleiomyoma is a benign tumor that causes limited morbidity. Surgical excision is the only effective treatment and recurrence is rare. The present study revealed that nerves were present in a small proportion (14.3%) of tumors. It was hypothesized that the compression of nerves accompanying numerous blood vessels in the tumor may cause pain, particularly in venous- and cavernous-type angioleiomyomas.

China has repeatedly been the subject of genetic studies to elucidate its prehistoric and historic demography. While some studies reported a genetic distinction between Northern and Southern Han Chinese, others showed a more clinal picture of small differences within China. Here, we investigated the distribution of Y chromosome variation along administrative as well as ethnic divisions in the mainland territory of the People's Republic of China, including 28 administrative regions and 19 recognized Chinese nationalities, to assess the impact of recent demographic processes. To this end, we analyzed 37,994 Y chromosomal 17-marker haplotype profiles from the YHRD database with respect to forensic diversity measures and genetic distance between groups defined by administrative boundaries and ethnic origin. We observed high diversity throughout all Chinese provinces and ethnicities. Some ethnicities, including most prominently Kazakhs and Tibetans, showed significant genetic differentiation from the Han and other groups. However, differences between provinces were, except for those located on the Tibetan plateau, less pronounced. This discrepancy is explicable by the sizeable presence of Han speakers, who showed high genetic homogeneity all across China, in nearly all studied provinces. Furthermore, we observed a continuous genetic North-South gradient in the Han, confirming previous reports of a clinal distribution of Y chromosome variation and being in notable concordance with the previously observed spatial distribution of autosomal variation. Our findings shed light on the demographic changes in China accrued by a fast-growing and increasingly mobile population.

p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.

The field of microbial forensics focuses on the attribution of numerous microbes used by perpetrators inbiocrime and bioattack, including many foodborne pathogens such as Escherichia coli O157:H7 caused serious zoonotic disease. Recent source tracing analysis with the 16S rRNA gene, even genome-wide variations of a pathogen, is increasing as sequencing technologies advance. By using whole genome sequence analysis, more SNPs have been used to create a very finely resolved phylogenetic tree for discriminating close evolutionary relationship among strains. In our study, 1401bp of 16S rRNA gene in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains were sequenced. The result showed that nucleotide transition and nucleotide transversion happened at 14 positions of sequence in the research strains, and variation of seven positions was located at one of nine separate hypervariable regions (V7). Another SNP within the constant region of 16S rRNA gene was also observed as the previous studies. Through comparing the available sequences obtained from the NCBI database, SNP was the only 16S rRNA polymorphism detected among the closely related EHEC O157:H7 stains. Our investigation demonstrates that unique SNPs in 16S rRNA gene have potential discriminatory power as other species-/strain-specific informative markers for attribution purpose.

Oxidative stress is significant in numerous types of cancer. Tobacco smoke, an important risk factor for oral squamous cell carcinoma (OSCC), is able to generate reactive oxygen species (ROS) and cause oxidative DNA damage. Superoxide dismutase (SOD) is an endogenous antioxidant enzyme that is critical in limiting the oxidative burden effectively. The purpose of this study was to investigate the effects of the mitochondrial SOD2 and Cu/Zn enzyme SOD1 gene polymorphisms on the susceptibility to and clinicopathological characteristics of OSCC, as well as the synergistic effect between these gene polymorphisms and the well-known risk factor of tobacco consumption. Patients with clinically diagnosed OSCC (n=362) and healthy normal individuals (n=358) were investigated for four single nucleotide polymorphisms (SNPs; rs4880, rs5746136, rs1804450 and rs11556620) by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. Following adjustment for other confounders, no significant difference was observed in the rs5746136 SOD2 SNPs between the patients and controls. However, the incidence of the CT genotype of SOD2 SNP rs4880 was higher in the patients than in normal subjects in the additive model [CT vs. TT; P=0.045; adjusted odds ratio (AOR)=1.484; 95% confidence interval (CI), 1.009-2.182] and in the dominant model (CT/CC vs. TT; P=0.022; AOR=1.559; 95% CI, 1.067-2.278). For those who smoked, the incidence of the CT genotype of rs4880 increased markedly in the patients compared with the controls in the additive model (CT vs. TT; P=0.003; AOR=2.325; 95% CI, 1.330-4.064) and in the dominant model (CT/CC vs. TT; P=0.001; AOR=2.448; 95% CI, 1.417-4.230). For SOD1, polymorphisms at rs1804450 and rs11556620 were not present in any of the OSCC or control subjects. The results suggest that SOD2 rs4880 may be involved in the tumorigenesis of OSCC and may be useful as a genetic susceptibility marker for OSCC.

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.

SNPs, abundant in human genome with lower mutation rate, are attractive to genetic application like forensic, anthropological and evolutionary studies. Universal SNPs showing little allelic frequency variation among populations while remaining highly informative for human identification were obtained from previous studies. However, genotyping tools target only dozens of markers simultaneously, limiting their applications. Here, 124 SNPs were simultaneous tested using Ampliseq technology with Ion Torrent PGM platform. Concordance study was performed with 2 reference samples of 9947A and 9948 between NGS and Sanger sequencing. Full concordance were obtained except genotype of rs576261 with 9947A. Parameter of FMAR (%) was introduced for NGS data analysis for the first time, evaluating allelic performance, sensitivity testing and mixture testing. FMAR values for accurate heterozygotes should be range from 50% to 60%, for homozygotes or Y-SNP should be above 90%. SNPs of rs7520386, rs4530059, rs214955, rs1523537, rs2342747, rs576261 and rs12997453 were recognized as poorly performing loci, either with allelic imbalance or with lower coverage. Sensitivity testing demonstrated that with DNA range from 10 ng-0.5 ng, all correct genotypes were obtained. For mixture testing, a clear linear correlation (R2 = 0.9429) between the excepted FMAR and observed FMAR values of mixtures was observed.

As a multi-ethnic country, China has some indigenous population groups which vary in culture and social customs, perhaps as a result of geographic isolation and different traditions. However, upon close interactions and intermarriage, admixture of different gene pools among these ethnic groups may occur. In order to gain more insight on the genetic background of X-Chromosome from these ethnic groups, a set of X-markers (18 X-STRs and 16 X-Indels) was genotyped in 5 main ethnic groups of China (HAN, HUI, Uygur, Mongolian, Tibetan). Twenty-three private alleles were detected in HAN, Uygur, Tibetan and Mongolian. Significant differences (p

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.

The estimation of postmortem interval (PMI) is one of the most difficult tasks in forensic practice, especially in putrefied bodies. After death, organisms are decomposed by a variety of enzymes and microorganisms. To investigate the succession of bacterial community in the decomposition process, rat remains were placed outside to decompose under natural conditions in Changsha city, China. Bacterial communities from two regions (buccal cavity and rectum) were sampled when experiment animals were alive, soon after they died and at various time span after death. Bacterial samples were analyzed by high throughput metagenomic sequencing of 16S rRNA gene conducted on an Illumina MiSeq platform. Our data showed that several bacteria genera were potentially useful for estimating the PMI, such as Streptococcus , Ignatzschineria , Acinetobacter , Aggregatibacter , Prevotella and Proteus . There were significant bacterial community structure differences in taxon richness and relative abundance patterns through the decomposition process and across different body sites. As decomposition progressed, a negative linear relationship for taxon richness was found along with a shift from aerobic bacteria to anaerobic bacteria. We first reported the bacterial biodiversity in the decomposition process in Chinese terrestrial scenarios and climatic conditions. Bacteria have a remarkable potential for estimate the PMI and next generation sequencing is a novel method to support the application of bacteria in forensic science.

Bacterial genotyping can be used for crime scene investigations and contribute to the attribution of biological attacks for microbial forensics. PyroMark ID Pyrosequencer as an accurate detection platform for single nucleotide polymorphisms (SNPs) has been applied to identify and resolve microorganisms involved in closely Escherichia coli O157:H7 (E. coli O157:H7). To explore more applications and improve the efficiency for pyrosequencing in this field, we developed a method integrated multiplex pyrosequencing with a universal primer. Two multiplex pyrosequencing assays with a universal biotinylated primer were designed to analyze five SNPs located in four gene of E. coli O157:H7 strain. The accuracy of the established assays was validated by genotyping reference strain E. coli O157:H7 EDL933 and E. coli K-12. We also demonstrated that two multiplex pyrosequencing assays were specific and sensitive for genotyping closely related E. coli O157 strains. Reproducibility of results and multiplexing capability were evaluated by a comparison of this method with the monoplex pyrosequencing. Furthermore, these two multiplex pyrosequencing assays have been successfully applied to detect 11 E. coli O157 strains isolated from 1504 Chinese livestock samples. This method reduces costs and time consumption in the process of pyrosequencing analysis, and potentially serves as a rapid tool and reliable candidate strategy for the microbial identification and other genotyping application.