黑龙江尚志地区莱姆病伯氏疏螺旋体的鉴定与遗传进化分析

自我国黑龙江省尚志市野外采集的蜱体内分离出1株莱姆病伯氏疏螺旋体,并对其进行了形态观察及分子生物学分析。利用BSK H分离培养法,经暗视野显微镜和姬姆萨染色观察,菌体呈螺旋状。根据已报道的伯氏疏螺旋体16S rRNA基因序列设计引物,结合参考文献设计的外膜蛋白A(OspA)基因引物经PCR扩增并测序,与GenBank中的各种伯氏疏螺旋体基因型建立系统发生树并进行同源性分析。结果表明,16S rRNA基因核苷酸序列与GenBank中的伽氏疏螺旋体(Borrelia garinii)DK27(X85193)具有较高的同源性,可达99.5%;OspA基因核苷酸序列与报道的Borrelia gariniiKhab(AY502600)有较高的同源性,为93.6%。根据序列分析结果,认为该分离株属于Borrelia garinii基因型,命名为SZ,该菌株16SrRNA基因和OspA基因核酸序列已提交到GenBank(登录号分别为HM007279和HM007278)。     

Mitochondrial DNA Extraction from Burial Soil Samples at Incremental Distances: A Preliminary Study

Preservation variance of soil DNA is neglected in the literature, and exceptional cases exaggerate amplification capabilities. This study sought to amplify a short mitochondrial fragment (212 bp) specific to Sus scrofa domesticus from the soil surrounding decomposing pig remains from an open‐air locale. Samples collected above the body at incremental distances after 145 days of initial placement yielded pig DNA. A secondary sampling was collected in 2017, approximately 768 days after burial. Inhibition tests corroborated that pig DNA was no longer present in the soil resulting in a loss of original DNA between 145 and 768 days. The results provide evidence that genetic material leaches out radially from the source and DNA fragments longer than 200 bp do not persist in soil for a relatively short timeframe in western Montana. The conclusions support the collection of soil in crime scene investigation procedures within the first few months of decomposition.     

Familial DNA searching- an emerging forensic investigative tool

In recent years, jurisdictions across the United States have expressed a growing interest in aiding criminal investigations through the use of familial DNA searching (FDS)- a forensic technique to identify family members through DNA databases. The National Survey of CODIS Laboratories surveyed U.S. CODIS laboratories about their perceptions, policies, and practices related to FDS. In total, 103 crime labs completed the survey (77% response rate). Labs in 11 states reported using FDS, while labs in 24 states reported using a similar-but distinct- practice of partial matching. Although the majority of labs had positive perceptions about the ability of FDS to assist investigations, labs also reported a number of concerns and challenges with implementing FDS. Respondents reported using either practice a limited amount with modest numbers of convictions resulting from both FDS and partial matching. The article reports on varying practices related to official policies, training, eligibility, the software search, lineage testing, requirements for releasing information, and subsequent investigative work. Finally, the article discusses what can be learned from this survey, accompanying limitations, and implications for decision-makers considering using FDS.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Determination of DNA yield rates in six different skeletal elements in ancient bones

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18^th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.     

How to avoid driving DNA caseworkers crazy: CaseSolver,an expert system to investigate complex crime scenes

DNA analyses can be used for both investigative (crime scene-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, low template LT-DNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches of national DNA databases is problematic because it is realistically restricted to clear major contributors and the occurrence of masking and drop-out means that there will not be a definitive DNA profile to perform the search with.CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio (LR) based on a qualitative model (forensim) c) LR based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the LRs, which can be exported as a report. The software can also identify contributors from small or large databases (e.g., staff database or 1 mill. individuals). In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we describe recent improvements made to the software in version v1.5.0, made in response to user requirements during intensive casework usage.     

Development of a new 17 Y-STRs system using fluorescent-labelled universal primers and its application in Shanxi population in China

Y-chromosomal short tandem repeats (Y-STRs) polymorphisms are useful in forensic identification, population genetics and constructing of human structures. Increasing the number of Y-STRs and their polymorphism will drastically narrow down the matching number of genealogy populations or pedigrees when searching against a forensic DNA databank. In this study, we develop a system containing 17 complementary Y-STRs that are compatible and reinforce the current commercially available Y-STR kits. This system was constructed by multiplex PCR with expected size of 126bp-400bp using home-made universal primers labeled by different fluorescence (DYS715, DYS709, DYS716, DYS713, DYS607, DYS718, DYS723, DYS708, DYS714, DYS712, DYS717, DYS721, DYS605, DYS719, DYS726, DYS598 and DYS722). The genetic data were obtained from 394 individuals in Shanxi province, China. The Y-STR system has 131 haplotypes and high discrimination power is 1. In conclusion, our study provides a robust, sensitive and cost-effective genotyping method for human identification, which is beneficial for narrowing the searching scope when applying to the genealogy searching with Y-STR DNA databank.     

The best possible result from the minimum available

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory.To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination.Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.