Mass spectrometry-based proteomic analysis of biological stains identifies body fluids specific markers.

The identification of biological stains and their tissue resource is an important part of forensic research. Current methods suffer from several limitations including poor sensitivity and specificity, trace samples, and sample destruction. In this study, we profiled the proteomes of menstrual blood, peripheral blood, saliva, semen, and vaginal fluid with mass spectrometry technology. Tissue-enhanced and tissue-specific proteins of each group have been proposed as potential biomarkers. These candidate proteins were further annotated and screened through the combination with the Human Protein Atlas database. Our data not only validates the protein biomarkers reported in previous studies but also identifies novel candidate biomarkers for human body fluids. These candidates lay the foundation for the development of rapid and specific forensic examination methods.

The screening and validation process of miR-223-3p for saliva identification.

More accurate identification of the types of body fluids left at a crime scene is indispensable for improving the judicial chain of evidence. MicroRNAs (miRNAs) have become recognized as ideal molecular markers for the identification of body fluids in forensic science due to their short length, stability and high tissue specificity. In this study, small RNA sequencing was performed on 20 samples of five types of body fluids (peripheral blood, menstrual blood, saliva, semen, and vaginal secretions) with the BGISEQ-500 sequencing platform, and the specific miRNA markers of saliva and vaginal secretions were screened by bioinformatics methods, including differential expression analysis and significant enrichment analysis. Through RT-qPCR validation of 169 samples, we confirmed that miR-223-3p can be used as a saliva-specific marker. In addition, we considered miR-223-3p in combination with four other miRNA molecules (miR-451a, miR-891a-5p, miR-144-5p, miR-203a-3p) that had been previously screened and verified in our laboratory, and seven body fluid prediction models based on machine learning algorithms were constructed and verified. The results showed that a kernel density estimation (KDE) model based on the five miRNA markers for body fluid identification could achieve 100% accuracy in the samples tested in the present study.

Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples.

MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization.

Estimating bloodstain formation time by quantitative analysis of mtDNA degradation.

The estimation of bloodstain formation time is still an unsolved problem in forensic science and lacks accurate quantitative methods. Whether DNA can be adopted to estimate bloodstain formation time is still controversial, and there is no study to confirm the potential of mtDNA markers. To address these issues, a triple quantification method based on the ratio of mtDNA fragments of different lengths of COⅠ (mitochondrially encoded cytochrome c oxidase Ⅰ) for estimating bloodstain formation time was established. A total of 152 samples (140 old samples, 12 fresh samples) were collected and tested, and the absolute copies of different-sized fragments of COⅠ (304 bp, 120 bp, 41 bp) in all samples were quantified by SYBR Green real-time qPCR. The natural logarithms of two copy number ratios (304 bp/41 bp, 120 bp/41 bp) of COⅠ in old samples were calculated, which were used as degradation indexes to evaluate the degradation degree of mtDNA. The 140 old human blood samples from 1 to 14 years of storage were accumulated from casework of forensic practice to establish the method of estimating bloodstain formation time and used to analyze the impact of gender factors on the two degradation indexes, and 10 animal samples and 2 fresh human samples were collected to verify the human specificity of the method. There was a high correlation between degradation indexes and bloodstain formation time (the absolute values of correlation coefficients of these two degradation indexes were 0.901 and 0.758 respectively). A method with triple quantification and dual indexes estimating bloodstain formation timewas successfully established, which was highly human-specific. There was no statistically significant difference in degradation indexes between different gender samples (P > 0.05). This study confirmed that mtDNA can be utilized to estimate bloodstain formation time, which provides a new solution to the forensic problem of estimating the time of bloodstain formation.

Determination of extractable and non-extractable florfenicol residues as florfenicol amine in eggs by UPLC-MS/MS.

Some florfenicol (FF) metabolites have a strong binding affinity towards biomolecules in the edible tissues of some food animals. These bound FF residues cannot be extracted directly from edible tissues with organic solvents and are present in higher concentrations even than solvent extractable residues. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to detect the total residues of FF in eggs, by quantifying the metabolite florfenicol amine (FFA). The sample was hydrolyzed at 95-100 °C for 4 hours to release sample-matrix bound residues and convert them all into FFA. The hydrolyzed sample was washed with ethyl acetate to remove interfering substances, extracted with ethyl acetate under alkaline conditions, purified by solid phase extraction and quantified by UPLC-MS/MS. The recoveries of FFA in eggs ranged from 91.2 to 102.4%, with an RSD ≤ 10.9%. The LOD and LOQ were 0.5 and 1.0 µg/kg, respectively. This method can be applied to the quantification of total FF residues in eggs.

A novel multiplex PCR method for simultaneous identification of hypervirulent Listeria monocytogenes clonal complex 87 and CC88 strains in China.

Listeria monocytogenes is an important foodborne pathogen worldwide, with 20-30% fatality rate in vulnerable persons. The hypervirulent L. monocytogenes clonal complex (CC) 87 strains have emerging both in food production environments and clinic cases. The objective of this study was to develop a multiplex PCR to simultaneously detect L. monocytogenes CC87 and CC88 strains based on pan-genome analysis. A novel multiplex PCR comprised of genes A6K41_13255 (specific for CC87 and 88), BCW_4260_01987 group_8135 (specific for CC88) and 02-1103_01073 group_5869 (specific for L. monocytogenes) were designed. The specificity of this multiplex PCR was robust verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR for CC87 and CC88 were 1.7 × 104 cfu/mL and 2.1 × 104 cfu/mL, respectively. This multiplex PCR could accurately detect CC87 and CC88 strains with the interference of different ratios of L. monocytogenes CC8, CC9, CC121, CC155, and L. innocua strains. Furthermore, this multiplex PCR method could successfully detect 1.9 × 104 cfu/mL of L. monocytogenes CC87 and 1.7 × 104 cfu/mL CC88 strains in artificially contaminated milk after 9 h enrichment, respectively. In addition, this multiplex PCR could accurately detect CC87 isolates in food samples within 48 h, which was faster than the routine MLST analysis. In conclusion, this novel multiplex PCR offers a promising approach for accurate, inexpensive, and rapid detection of L. monocytogenes CC87 and CC88 strains simultaneously, which could apply to surveillance the prevalence of CC87 and CC88 strains in both food and food production environments and to evaluate the effect of disinfection measures for controlling the persistent L. monocytogenes contamination.

A new strategy for distinguishing menstrual blood from peripheral blood by the miR-451a/miR-21-5p ratio.

Distinction between menstrual blood and peripheral blood is vital for forensic casework, as it could provide strong evidence to figure out the nature of some criminal cases. However, to date no single blood-specific gene, including the most variable microRNAs (miRNAs) could work well in identification of blood source. In this study, we developed a new strategy for identification of human blood samples by using the copy number ratios of miR-451a to miR-21-5p based on 133 samples, including 56 menstrual blood and 47 peripheral blood, as well as 30 non-blood samples of saliva (10), semen (10) and vaginal secretion (10). The cut-off value and efficacy of the identification strategy were determined through receiver operating characteristic (ROC) analysis. Our results showed that when the miR-451a/miR-21-5p ratio below 0.929, the sample should be non-blood. In contrast, when the miR-451a/miR-21-5p ratio above 0.929 and below 10.201, the sample should be menstrual blood; and when this ratio above 10.201, the sample should be peripheral blood. External validation using 86 samples (62 menstrual blood and 24 peripheral blood samples) fully supported this strategy with the 100% sensitivity and 100% specificity. We confirmed that this result accuracy was not affected by various potential confounding factors of samples and different experimental platforms. We showed that 0.2 ng of total RNA from menstrual blood and peripheral blood was sufficient for qPCR quantification. In conclusion, our results provide an accurate reference to distinguish menstrual blood from peripheral blood for forensic authentication.

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification.

OBJECTIVES: To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification. METHODS: The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained. RESULTS: There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/µL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/µL, and the discrimination accuracy was 100%. CONCLUSIONS: In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Isolation and Characterization of a Novel Salmonella Phage vB_SalP_TR2.

Salmonella is a widely distributed foodborne pathogen. The use of Salmonella phages as biocontrol agents has recently gained significant interest. Because the Salmonella genus has high diversity, efforts are necessary to identify lytic Salmonella phages focusing on different serovars. Here, five Salmonella phages were isolated from soil samples, and vB_SalP_TR2 was selected as a novel phage with high lytic potential against the host Salmonella serovar Albany, as well as other tested serovars, including Corvallis, Newport, Kottbus, and Istanbul. Morphological analyses demonstrated that phage vB_SalP_TR2 belongs to the Podoviridae family, with an icosahedral head (62 ± 0.5 nm in diameter and 60 ± 1 nm in length) and a short tail (35 ± 1 nm in length). The latent period and burst size of phage vB_SalP_TR2 was 15 min and 211 PFU/cell, respectively. It contained a linear dsDNA of 71,453 bp, and G + C content was 40.64%. Among 96 putative open reading frames detected, only 35 gene products were found in database searches, with no virulence or antibiotic resistance genes being identified. As a biological control agent, phage vB_SalP_TR2 exhibited a high temperature and pH tolerance. In vitro, it lysed most S. Albany after 24 h at 37°C with multiplicities of infection of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. In food matrices (milk and chicken meat), treatment with phage vB_SalP_TR2 also reduced the number of S. Albany compared with that in controls. These findings highlighted phage vB_SalP_TR2 as a potential antibacterial agent for the control of Salmonella in food samples.

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid.

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C10-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s-1 mM-1 to 119 s-1 mM-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L-1 day-1 and 160 g L-1 day-1, respectively.

A systematic analysis of miRNA markers and classification algorithms for forensic body fluid identification.

Identifying the types of body fluids left at the crime scene can be essential to reconstructing the crime scene and inferring criminal behavior. MicroRNA (miRNA) molecule extracted from the trace of body fluids is one of the most promising biomarkers for the identification due to its high expression, extreme stability and tissue specificity. However, the detection of miRNA markers is not the answer to a yes-no question but the probability of an assumption. Therefore, it is a crucial task to develop complicated methods combining multi-miRNAs as well as computational algorithms to achieve the goal. In this study, we systematically analyzed the expression of 10 most probable body fluid-specific miRNA markers (miR-451a, miR-205-5p, miR-203a-3p, miR-214-3p, miR-144-3p, miR-144-5p, miR-654-5p, miR-888-5p, miR-891a-5p and miR-124-3p) in 605 body fluids-related samples, including peripheral blood, menstrual blood, saliva, semen and vaginal secretion. We introduced the kernel density estimation (KDE) method and six well-established methods to classify the body fluids in order to find the most optimal combinations of miRNA markers as well as the corresponding classifying method. The results show that the combination of miR-451a, miR-891a-5p, miR-144-5p and miR-203a-3p together with KDE can achieve the most accurate and robust performance according to the cross-validation, independent tests and random perturbation tests. This systematic analysis suggests a reference scheme for the identification of body fluids in an accurate and stable manner.

Identification of five types of forensic body fluids based on stepwise discriminant analysis.

Peripheral blood, menstrual blood, semen, saliva and vaginal secretions are the five most common body fluids found at crime scenes, and the identification of these five body fluids is of great significance to the reconstruction of a crime scene and resolution of the case. However, accurate identification of these five body fluids is still a challenge. To address this problem, a mathematical model for differentiating five types of forensic body fluids based on the differential expression characteristics of multiple miRNAs in five body fluids (peripheral blood, menstrual blood, semen, saliva and vaginal secretions) was developed. A total of 350 forensic body fluids (70 of each type) were collected and tested, and relative expression of 10 miRNAs (miR-451a, miR-205-5p, miR-203-3p, miR-214-3p, miR-144-3p, miR-144-5p, miR-654-5p, miR-888-5p, miR-891a-5p, miR-124a-3p) in all samples was detected by SYBR Green real-time qPCR. Three hundred samples (60 samples of each body fluid) were used as the training set to screen meaningful identification markers by stepwise discriminant analysis, and a discriminant function was established. Fifty samples (10 samples of each body fluid) were used as a validation set to examine the accuracy of the model, and 25 samples (the types of samples were unknown to the experimenter) were used for a blind test. Except for miR-144-3p, the other miRNAs were selected to construct discriminant analysis models. The self-validation accuracy of the model was 99.7 %, cross-validation accuracy was 99.3 %, accuracy of the identification validation set was 100 %, and accuracy of the blind test result was 100 %. This study provides a reliable and accurate identification strategy for five common body fluids (peripheral blood, menstrual blood, semen, saliva, and vaginal secretions) in forensic medicine.

Heterogeneity, Characteristics, and Public Health Implications of Listeria monocytogenes in Ready-to-Eat Foods and Pasteurized Milk in China.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in humans. This study aimed to identify the pathogenic potential of L. monocytogenes isolated from ready-to-eat (RTE) foods and pasteurized milk in China on the basis of its phenotypic and genotypic characteristics. Approximately 7.7% (44/570) samples tested positive for L. monocytogenes among 10.8% (39/360) RTE and 2.4% (5/210) pasteurized milk samples, of which 77.3% (34/44) had < 10 MPN/g, 18.2% (8/44) had 10-110 MPN/g, and 4.5% (2/44) had > 110 MPN/g. A total of 48 strains (43 from RTE foods and five from milk samples) of L. monocytogenes were isolated from 44 positive samples. PCR-serogroup analysis revealed that the most prevalent serogroup was II.2 (1/2b-3b-7), accounting for 52.1% (25/48) of the total, followed by serogroup I.1 (1/2a-3a) accounting for 33.3% (16/48), serogroup I.2 (1/2c-3c) accounting for 12.5% (6/48), and serogroup II.1 (4b-4d-4e) accounting for 2.1%. All isolates were grouped into 11 sequence types (STs) belonging to 10 clonal complexes (CCs) and one singleton (ST619) via multi-locus sequence typing. The most prevalent ST was ST87 (29.2%), followed by ST8 (22.9%), and ST9 (12.5%). Virulence genes determination showed that all isolates harbored eight virulence genes belonging to Listeria pathogenicity islands 1 (LIPI-1) (prfA, actA, hly, mpl, plcA, plcB, and iap) and inlB. Approximately 85.4% isolates carried full-length inlA, whereas seven isolates had premature stop codons in inlA, six of which belonged to ST9 and one to ST5. Furthermore, LLS (encoded by llsX gene, representing LIPI-3) displays bactericidal activity and modifies the host microbiota during infection. LIPI-4 enhances neural and placental tropisms of L. monocytogenes. Results showed that six (12.5%) isolates harbored the llsX gene, and they belonged to ST1/CC1, ST3/CC3, and ST619. Approximately 31.3% (15/48) isolates (belonging to ST87/CC87 and ST619) harbored ptsA (representing LIPI-4), indicating the potential risk of this pathogen. Antimicrobial susceptibility tests revealed that > 95% isolates were susceptible to 16 antimicrobials; however, 60.4 and 22.9% isolates were intermediately resistant to streptomycin and ciprofloxacin, respectively. The results show that several isolates harbor LIPI-3 and LIPI-4 genes, which may be a possible transmission route for Listeria infections in consumers.

A stepwise strategy to distinguish menstrual blood from peripheral blood by Fisher's discriminant function.

Blood samples are the most common and important biological samples found at crime scenes, and distinguishing peripheral blood and menstrual blood samples is crucial for solving criminal cases. MicroRNAs (miRNAs) are important molecules with strong tissue specificity that can be used in forensic fields to identify the tissue properties of body fluid samples. In this study, the relative expression levels of four different miRNAs (miR-451, miR-205, miR-214 and miR-203) were analysed by real-time PCR, with 200 samples from 5 different body fluids, including two kinds of blood samples (peripheral blood and menstrual blood) and three kinds of non-blood samples (saliva, semen and vaginal secretion). Then, a strategy for identifying menstrual and peripheral blood based on Fisher's discriminant function and the relative expression of multiple miRNAs was established. Two sets of functions were used: Z1 and Z2 were used to distinguish blood samples from non-blood samples, and Y1 and Y2 were used to distinguish peripheral blood from menstrual blood. A 100% accuracy rate was achieved when 50 test samples were used. Ten samples were used to test the sensitivity of the method, and 10 ng or more of total RNA from peripheral blood samples and 10 pg or more of total RNA from menstrual blood samples were sufficient for this method. The results provide a scientific reference to address the difficult forensic problem of distinguishing menstrual blood from peripheral blood.

Comparative studies of interaction of ß-lactoglobulin with three polyphenols.

To investigate the interaction mechanism between bovine protein ß-lactoglobulin (ß-LG) and theaflavin (TA), chlorogenic acid (CA) and delphinidin-3-O-glucoside (D3G), multi-spectrometry analytical methods and molecular modeling were applied. Fluorescence experiments proved that polyphenols strongly quenched the intrinsic fluorescence of ß-LG mainly through static quenching and the main interaction force was hydrophobic interaction. Moreover, Fourier transform infrared (FTIR) and circular dichroism (CD) indicated that polyphenols changed ß-LG secondary and tertiary structure. Enzyme-linked immunosorbent assay and molecular modeling study manifested that complex of ß-LG with polyphenols could significantly reduce the IgE-binding capacity of ß-LG due to the polyphenol binding site directly obscures the IgE linear epitopes. In conclusion, polyphenols had impact on the structure and potential functionality of ß-LG, which would be valuable in dairy processing industry and food nutrition security.

Massively parallel sequencing of microRNA in bloodstains and evaluation of environmental influences on miRNA candidates using realtime polymerase chain reaction.

MicroRNAs (miRNA) are small (22-24 nucleotides) non-coding RNAs with potential application in forensic science because of their anti-degradation property and tissue specificity. Recent studies on the use of miRNA in forensic applications have mainly focused on body fluid identification using realtime polymerase chain reaction or microarray analysis. However, the exploration of miRNA in bloodstains, which are the most valuable source of biological evidence during case investigations, is currently lacking, particularly for aged and environmentally compromised forensic samples. Recent developments in massively parallel sequencing (MPS) technology provide the opportunity to establish a whole-genome miRNA profile with high throughput and efficiency. However, MPS analysis of genome-wide miRNA profiles from bloodstains has not been reported to date. In this study, the whole-genome miRNA profiles of bloodstains were examined using MPS, revealing 633 known miRNAs and 266 novel miRNAs. To further explore the stability of miRNAs in bloodstains under various circumstances, the expression levels of six miRNAs (miR-16-5p, miR-20a-5p, miR-486-5p, miR-148a-3p, miR-151a-3p, and miR-451a) that were abundant in blood/bloodstains were examined. The results showed that freezing/thawing and a high concentration of oxidant solution affects the absolute expression of miRNA significantly, while storage for up to 5 months and a temperature of 37 °C did not have any observed effects. This study not only provides a novel method to explore miRNA profiles in bloodstains using MPS, but also points to the circumstantial influences on miRNA expression, which are an important consideration for practical application. Collectively, our work may shed light on MPS-based approaches with miRNA analysis of bloodstains in forensics.

Twenty-seven continental ancestry-informative SNP analysis of bone remains to resolve a forensic case.

We employed our previously developed 27-plex ancestry-informative single nucleotide polymorphism (SNP) panel to infer the ancestral components of bone remains of a possible foreign pilot found in south-western China. For ancestry assignment of this unknown individual, we first obtained the 27-SNP genotype of the individual. Then, based on a reference database of 3081 individuals from 33 populations, we calculated the match probability and likelihood ratio using the self-developed software program Forensic Intelligence. Inferred ancestral components of this individual were calculated by structure at K = 3. A complete profile was obtained for the individual using our multiplexed SNP assay. The European population was within one order of magnitude of the highest likelihood. The major ancestral component of this individual was 97.6% European.

Assessment of Binding Interaction between Bovine Lactoferrin and Tetracycline Hydrochloride: Multi-Spectroscopic Analyses and Molecular Modeling.

In this paper, the interaction between bovine lactoferrin (bLf) and tetracycline hydrochloride (TCH) was researched by microscale thermophoresis (MST), multi-spectroscopic methods, and molecular docking techniques. Normal fluorescence results showed that TCH effectively quenched the intrinsic fluorescence of bLf via static quenching. Moreover, MST confirmed that the combination force between bLf and TCH was very strong. Thermodynamic parameters and molecular docking further revealed that electrostatic forces, van der Waals, and hydrogen bonding forces played vital roles in the interaction between bLf and TCH. The binding distance and energy transfer efficiency between TCH and bLf were 2.81 nm and 0.053, respectively. Moreover, the results of circular dichroism spectra (CD), ultraviolet visible (UV-vis) absorption spectra, fluorescence Excitation-Emission Matrix (EEM) spectra, and molecular docking verified bLf indeed combined with TCH, and caused the changes of conformation of bLf. The influence of TCH on the functional changes of the protein was studied through the analysis of the change of the bLf surface hydrophobicity and research of the binding forces between bLf and iron ion. These results indicated that change in the structure and function of bLf were due to the interaction between bLf and TCH.

Comparison of preprocessing methods and storage times for touch DNA samples.

AIM: To select appropriate preprocessing methods for different substrates by comparing the effects of four different preprocessing methods on touch DNA samples and to determine the effect of various storage times on the results of touch DNA sample analysis. METHOD: Hand touch DNA samples were used to investigate the detection and inspection results of DNA on different substrates. Four preprocessing methods, including the direct cutting method, stubbing procedure, double swab technique, and vacuum cleaner method, were used in this study. DNA was extracted from mock samples with four different preprocessing methods. The best preprocess protocol determined from the study was further used to compare performance after various storage times. DNA extracted from all samples was quantified and amplified using standard procedures. RESULTS: The amounts of DNA and the number of alleles detected on the porous substrates were greater than those on the non-porous substrates. The performances of the four preprocessing methods varied with different substrates. The direct cutting method displayed advantages for porous substrates, and the vacuum cleaner method was advantageous for non-porous substrates. No significant degradation trend was observed as the storage times increased. CONCLUSION: Different substrates require the use of different preprocessing method in order to obtain the highest DNA amount and allele number from touch DNA samples. This study provides a theoretical basis for explorations of touch DNA samples and may be used as a reference when dealing with touch DNA samples in case work.