Forensic Science International-Genetics最新文献

{"title":"Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model","authors":"","doi":"10.1016/j.fsigen.2024.103098","DOIUrl":"10.1016/j.fsigen.2024.103098","url":null,"abstract":"<div><p>A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (2⁷ dilution) or 0.0218 μL (2⁶ dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141850486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unique molecular identifier-based amplicon sequencing of microhaplotypes for background noise mitigation","authors":"","doi":"10.1016/j.fsigen.2024.103096","DOIUrl":"10.1016/j.fsigen.2024.103096","url":null,"abstract":"<div><p>Microhaplotypes (MHs), comprising two or more single-nucleotide polymorphisms in a short fragment, are promising forensic markers owing to their remarkable polymorphic nature. Several studies have demonstrated the utility of MHs through massively parallel sequencing (MPS). Nevertheless, the background noise level associated with MHs in MPS, which imposes a practical detection limit for the system, remains uninvestigated. Currently, unique molecular identifier (UMI) systems are known to effectively mitigate background noise by tracking original DNA molecules and facilitating PCR and MPS error corrections. Hence, this study aimed to design a UMI-based amplicon sequencing system, designated MH-UMIseq, which can amplify 46 MHs simultaneously and generate MPS libraries in four steps: barcoding PCR, nuclease reaction, boosting PCR, and indexing PCR. The performance of the MH-UMIseq system was evaluated using the Illumina NextSeq 550 and MiniSeq systems with 31 sets for 5 ng, 1 ng, and 200 pg of input DNA. The fgbio toolkit was used in conjunction with STRait Razor 3.0 and Visual Microhap to analyze the UMI data on MHs. The corresponding average <em>not suppressed noise</em> proportion of MH-UMIseq were 0.1 %, 0.3 %, and 0.7 % for 5 ng, 1 ng, and 200 pg of DNA, respectively, which substantially suppressed the background noise for more than 1 ng of DNA. Interestingly, the proportion of <em>not suppressed noise</em> in MH-UMIseq notably decreased as the amount of input DNA increased. The number of UMI families was proportional to the copy number of the template DNA and closely correlated with the system resolution. Therefore, the resolution of MH-UMIseq system is expected to be higher than that of conventional MPS for the deconvolution of mixtures containing more than 1 ng of DNA.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141728997","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Forensic Science International: Genetics is the leading journal in the field of legal medicine","authors":"","doi":"10.1016/j.fsigen.2024.103097","DOIUrl":"10.1016/j.fsigen.2024.103097","url":null,"abstract":"","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141725395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Timber DNA release using focused ultrasound extraction (FUSE) for genetic species identification","authors":"","doi":"10.1016/j.fsigen.2024.103094","DOIUrl":"10.1016/j.fsigen.2024.103094","url":null,"abstract":"<div><p>The use of genetic data for timber species and population assignment is a powerful tool for combating the illegal timber trade, but the challenges of extracting DNA from timber have prevented the routine use of genetics as a supply chain management tool. To overcome these challenges, we explored the feasibility of focused ultrasound extraction (FUSE) for rapid DNA release from timber. Using high-pressure ultrasound pulses, FUSE generates a cavitation bubble cloud that disintegrates samples into acellular debris, resulting in the mechanical release of DNA. In this work, FUSE was applied to white oak (<em>Quercus alba</em>) timber shavings to test the feasibility of using FUSE for timber DNA extraction for the first time. Results showed that FUSE processing disintegrated the tissue samples and released significant quantities of DNA. After five minutes of tissue processing DNA quantities of 0.21 ± 0.02 ng/mg, 0.99 ± 0.32 ng/mg, and 0.14 ± 0.01 ng/mg, were released from medium, coarse, and combination shaving groups, respectively. Amplification and sequencing of regions within the matK and rbcL chloroplast genes confirmed that the quality of DNA prepared with FUSE was suitable for PCR and short-read sequencing applications. Overall, these results show that FUSE can serve as a DNA sample preparation method capable of releasing high-quality DNA from timber in a fraction of the time required by conventional extraction methods. Based on the improved efficiency of DNA release with FUSE, ongoing work aims to develop this technology into portable systems that can be used to rapidly prepare timber samples for genetic species identification.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141714641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The advent of forensic DNA databases: It’s time to agree on some international governance principles!","authors":"","doi":"10.1016/j.fsigen.2024.103095","DOIUrl":"10.1016/j.fsigen.2024.103095","url":null,"abstract":"<div><p>National forensic DNA databases are a valuable investigative tool, that have the potential to increase the efficacy of criminal investigations. Their unfettered expansion in recent years raises unsettling ethical issues that require close attention. DNA database expansion threatens the rights to privacy, non-discrimination, and equality, and can undermine public trust in government. This perspective piece relies on data from an international mapping study of Forensic DNA Databases to document the expansion of these databases, highlight the ethical issues they raise, and propose key recommendations for more responsible use of this infrastructure.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1872497324000917/pdfft?md5=48d65edc4b93a6963787d424446ff01f&pid=1-s2.0-S1872497324000917-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141637409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An innovative approach for low input forensic DNA sample analysis using the GlobalFiler™ IQC PCR amplification Kit on the Magelia® platform","authors":"","doi":"10.1016/j.fsigen.2024.103093","DOIUrl":"10.1016/j.fsigen.2024.103093","url":null,"abstract":"<div><p>Short Tandem Repeat (STR) markers have been the gold standard for human identification testing in the forensic field for the last few decades. The GlobalFiler™ IQC PCR amplification Kit has shown sensitivity, high power of discrimination and is therefore widely used. Samples with limited DNA quantities remain a significant hurdle for streamlined human forensic identification. Reaction volume reduction in a closed system paired with automation can provide solutions to secure DNA profiles when routine methods fall short. We automated and optimized the GlobalFiler^{<strong>TM</strong>} IQC PCR Amplification Kit on the Magelia®, a closed molecular biology platform, to test whether reaction volume reduction in a confined automated system would improve signal and sensitivity. We evaluated the platform’s performance using reference and real casework samples (blood, cigarette butt, saliva and touch DNA) in the context of a 5-fold volume reduction when compared to the routine protocol. This strategy showed distinct advantages over standard treatment, notably increased signal for lower DNA inputs. Importantly, negative casework samples through routine treatment yielded “usable” DNA profiles after amplification using this strategy. This novel approach represents a first proof of concept for a method enabling users to treat limited samples, or to partition routine samples for multiple analyses.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141622466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pairwise kinship inference and pedigree reconstruction using 91 microhaplotypes","authors":"Yifan Wei ,&nbsp;Qiang Zhu ,&nbsp;Haoyu Wang,&nbsp;Yueyan Cao,&nbsp;Xi Li,&nbsp;Xiaokang Zhang,&nbsp;Yufang Wang,&nbsp;Ji Zhang","doi":"10.1016/j.fsigen.2024.103090","DOIUrl":"10.1016/j.fsigen.2024.103090","url":null,"abstract":"<div><p>Kinship inference has been a major issue in forensic genetics, and it remains to be solved when there is no prior hypothesis and the relationships between multiple individuals are unknown. In this study, we genotyped 91 microhaplotypes from 46 pedigree samples using massive parallel sequencing and inferred their relatedness by calculating the likelihood ratio (LR). Based on simulated and real data, different treatments were applied in the presence and absence of relatedness assumptions. The pedigree of multiple individuals was reconstructed by calculating pedigree likelihoods based on real pedigree samples. The results showed that the 91 MHs could discriminate pairs of second-degree relatives from unrelated individuals. And more highly polymorphic loci were needed to discriminate the pairs of second-degree or more distant relative from other degrees of relationship, but correct classification could be obtained by expanding the suspected relationship searched to other relationships with lower LR values. Multiple individuals with unknown relationships can be successfully reconstructed if they are closely related. Our study provides a solution for kinship inference when there are no prior assumptions, and explores the possibility of pedigree reconstruction when the relationships of multiple individuals are unknown.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141539124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Saliva-derived secondary DNA transfer on fabric: The impact of varying conditions","authors":"Melanie S. Gegar ,&nbsp;German A. Cisneros ,&nbsp;Joanne Cox ,&nbsp;Melanie Richard ,&nbsp;Krista A. Currie","doi":"10.1016/j.fsigen.2024.103092","DOIUrl":"https://doi.org/10.1016/j.fsigen.2024.103092","url":null,"abstract":"<div><p>This study explored secondary DNA transfer involving saliva, a body fluid often encountered in forensic investigations. Various factors were examined to investigate their potential impact on the transfer of DNA from saliva stains deposited onto common types of fabric (cotton, nylon, and towel). We examined varying types of saliva moisture (wet, dry, and rehydrated) and different types of contact (controlled pressure and active/friction pressure) to quantitatively evaluate how such variables could impact transfer and possible conclusions surrounding saliva-derived DNA deposits. The transfer of DNA was generally least pronounced with more absorbent primary fabrics (cotton and towel materials) while a less absorbent primary fabric (nylon) exhibited a greater propensity for DNA transfer. There were significantly higher amounts of transferred DNA (p &lt; 0.05) observed in wet saliva samples compared to dry and rehydrated saliva samples. Further, the use of active pressure (friction) appeared to result in more DNA transfer overall as compared to controlled pressure contact. Experiments conducted with wet saliva and active pressure (friction) demonstrated the highest likelihood of transfer, with the primary nylon and secondary towel fabric combination demonstrating the greatest average transfer percentage of 94.74 %. The variables explored in this study presented multiple combinations wherein a sufficient amount of DNA (≥ 240 pg total) was transferred to the secondary fabric, making it potentially suitable for STR-PCR amplification in our laboratory. The findings from this study indicate that the type of primary fabric receiving the saliva deposit, the type of saliva moisture, the type of secondary fabric and its moisture type, and the type of contact all have the potential to affect the quantity of DNA transferred and recovered. This study provides empirical data on the ease, and to what extent, DNA from saliva transfers between fabrics and aids DNA activity level evaluations. The significance of this research lies in its contribution to expanding our current understanding of DNA transfer involving saliva within forensic science and criminal investigations.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141541359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of a newly constructed NGS panel with 45 X-linked microhaplotypes demonstrates the unique value of X-MH for kinship testing and mixture analysis","authors":"Guanju Ma ,&nbsp;Kailiang Liu ,&nbsp;Chaolong Lu ,&nbsp;Qingqing Du ,&nbsp;Mengjie Zhang ,&nbsp;Qian Wang ,&nbsp;Guangping Fu ,&nbsp;Junyan Wang ,&nbsp;Chunling Ma ,&nbsp;Bin Cong ,&nbsp;Shujin Li ,&nbsp;Lihong Fu","doi":"10.1016/j.fsigen.2024.103091","DOIUrl":"https://doi.org/10.1016/j.fsigen.2024.103091","url":null,"abstract":"<div><p>X-linked microhaplotypes (X-MHs) have the potential to be a valuable supplementary tool in complex kinship identification or the resolution of DNA mixtures, because they bring together the distinctive genetic pattern of X chromosomal markers and the benefits of microhaplotypes (MHs). In this study, we used the 1000 Genome database to screen and select 63 X-MHs; 18 MHs were filtered out though a batch sequencing assessment of the DNA samples collected from 112 unrelated Chinese Han individuals. The resulting 45-plex panel performed well in comprehensive assessments including repeatability, sensitivity, species specificity, resistance to PCR inhibitors or degradation, mutation rate, and accuracy in detecting DNA mixture samples. The minimum amount of DNA template that can be tested with this panel is 0.5 ng. Additionally, the alleles of the minor contributor can be accurately detected when the mixture rate is larger than 1:9 in female-male mixture or 1:19 in male-male mixture. Then, we calculated population parameters on each MH based on the allele frequency data obtained from the sequence results of the aforementioned 112 unrelated samples. Combining these parameters on each MH, it can be calculated that <span><math><msub><mrow><mi>TDP</mi></mrow><mrow><mi>m</mi></mrow></msub></math></span>, <span><math><msub><mrow><mi>TDP</mi></mrow><mrow><mi>f</mi></mrow></msub></math></span>, CPET, <span><math><msub><mrow><mi>CPED</mi></mrow><mrow><mi>FM</mi></mrow></msub></math></span>, <span><math><msub><mrow><mi>CPED</mi></mrow><mrow><mi>FF</mi></mrow></msub></math></span> and <span><math><msub><mrow><mi>CNCEP</mi></mrow><mrow><mn>3</mn></mrow></msub></math></span> of the 45-plex system were 1–8.99×10⁻¹³, 1–1.62×10⁻¹⁹, 0.9999999995, 0.9999981, 0.9955, 0.9999971 and 0.99940, respectively, indicating that the panel is capable in personal identification and parentage testing. To reveal the unique advantage of X-MHs in the analyses of complex kinship and male DNA mixture, further assessments were made. For complex kinship identification, 22 types of individual pairs with different second-degree kinship were simulated and different types of likelihood ratios (LR) were calculated for each. The results revealed that the panel can achieve accuracy of approximately 70 %∼80 % when dividing each of the three types of second-degree kinships into three or four groups. Theoretically, such sub-division cannot be done by using independent autosomal markers. For male DNA mixture analysis without suspects, the maximum likelihood ratio strategy was derived and employed in the estimation of the number of male contributors (NOMC). Simulations were conducted to verify the efficacy of the 45-plex panel in the field and to compare it with autosomal markers by assuming the 45 MHs as autosomal ones. The results showed that X-MHs can achieve higher accuracy in the estimation of NOMC than autosomal ones when the mixed males were unrelated. The results high","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141485509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Who bit the boat? New DNA collection and genomic methods enable species identification in suspected shark-related incidents","authors":"Belinda Martin ,&nbsp;Michael P. Doane ,&nbsp;Jessica Henkens ,&nbsp;Jess A.T. Morgan ,&nbsp;Laura Inglis ,&nbsp;Victor M. Peddemors ,&nbsp;Elizabeth A. Dinsdale ,&nbsp;Charlie Huveneers ,&nbsp;Lauren Meyer","doi":"10.1016/j.fsigen.2024.103087","DOIUrl":"https://doi.org/10.1016/j.fsigen.2024.103087","url":null,"abstract":"<div><p>Species identification following shark-related incidents is critical for effective incident management and for collecting data to inform shark-bite mitigation strategies. Witness statements are not always reliable, and species identification is often ambiguous or missing. Alternative methods for species identification include morphological assessments of bite marks, analysis of collected teeth at the scene of the incident, and genetic approaches. However, access to appropriate collection media and robust genetic assays have limited the use of genetic technologies. Here, we present a case study that facilitated a unique opportunity to compare the effectiveness of medical gauze readily available in first-aid kits, and forensic-grade swabs in collecting genetic material for shark-species identification. Sterile medical gauze and forensic-grade swabs were used to collect transfer DNA from the bite margins on a bitten surf ski which were compared to a piece of shark tissue embedded along the bite margin. Witness accounts and the characteristics of the bite mark impressions inferred the involvement of a <em>Carcharodon carcharias</em> (white shark). The morphology of a tooth found on the boat that picked up the surf ski, however, suggested it belonged to an <em>Orectolobus spp.</em> (wobbegong). Genetic analysis of DNA transferred from the shark to the surf ski included the application of a broad-target nested PCR assay followed by Sanger sequencing, with white shark contribution to the ‘total sample DNA’ determined with a species-specific qPCR assay. The results of the genetic analyses were congruent between sampling methods with respect to species identification and the level of activity inferred by the donor-specific DNA contribution. These data also supported the inferences drawn from the bite mark morphology. DNA from the recovered tooth was PCR amplified with a wobbegong-specific primer pair designed for this study to corroborate the tooth’s morphological identification. Following the confirmation of gauze used for sampling in the case study event, two additional isolated incidents occurred and were sampled <em>in situ</em> using gauze, as typically found in a first-aid kit, by external personnel. DNA extracted from these gauze samples resulted in the identification of a white shark as the donor of the DNA collected from the bite marks in both instances. This study, involving three incidents separated by time and location, represents the seminal application of gauze as a sampling media after critical human-shark interactions and strongly supports the practical implementation of these methods in the field.</p></div>","PeriodicalId":50435,"journal":{"name":"Forensic Science International-Genetics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1872497324000838/pdfft?md5=3e18921d6a5688e4dec6273eee63c254&pid=1-s2.0-S1872497324000838-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141596806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}