Species identification from dried snake venom

Illegal trade in snake parts has increased enormously. In spite of strict protection under wildlife act, a large number of snakes are being killed ruthlessly in India for venom and skin. Here, an interesting case involving confiscation of crystallized dried snake venom and subsequent DNA-based species identification is reported. The analysis using the universal primers for cytochrome b region of the mitochondrial DNA revealed that the venom was extracted from an Indian cobra (Naja naja). On the basis of this report, the forwarding authority booked a case in the court of law against the accused for illegal hunting of an endangered venomous snake and smuggling of snake venom. This approach thus has immense potential for rapid identification of snake species facing endangerment because of illegal trade. This is also the first report of DNA isolation from dried snake venom for species identification.     

A DNA-based approach for the forensic identification of Asiatic black bear (Ursus thibetanus) in a traditional Asian medicine

Attempts to prevent illegal trade in bile and gallbladders from Asiatic black bears, Ursus thibetanus, are hampered by difficulties associated with identifying such items. We extracted DNA from bile crystals of unknown species origin and generated partial cytochrome b (cyt b) sequences using either universal primers (positioned in conserved regions of cyt b), or primers designed on existing U. thibetanus sequences (UT). Species origin was determined by aligning resolved sequences to reference sequence data. The universal primers were unsuitable for U. thibetanus identification when multiple species templates were present in the samples. The UT primers amplified U. thibetanus DNA from all sample extracts, including those containing mixed species templates. The amplified fragment can distinguish U. thibetanus from the most closely related species, U. americanus, a distinct advantage of DNA sequencing over the methods currently used to analyze suspected U. thibetanus bile.     

Validation of cytochrome b sequence analysis as a method of species identification

One of the stages of dealing with biological material submitted to forensic laboratories is species identification. The aim of the present work was to validate and assess the possibility of applying sequence analysis of the region coding cytochrome b as a method of species identification in the field of forensic science. DNA originating from individuals from major phyla of vertebrates was isolated by the organic method from various specimens. Extracted DNA was subjected to PCR and direct cycle sequencing using a universal pair of primers. The validation process, performed according to TWGDAM recommendations, revealed that the technique is a very sensitive and reliable method of species identification allowing analysis of tiny amounts of material and also degraded material, and can be useful in the field of forensic genetics. The case example presented here, concerning the determination of species origin of biological evidence collected from fatal road accident, confirms that analysis can be carried out even when there is no reference sample, and the sequences obtained can be assessed through analysis of their similarity to sequences for cytochrome b present in DNA databases.     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

Species identification using sequences of the trnL intron and the trnL-trnF IGS of chloroplast genome among popular plants in Taiwan

Forensic botanical comparison can be hampered by the lack of appropriate DNA databases. While DNA sequence databases for many mitochondrial loci have been established for the identification of animal species, less is known regarding the genomes of plants. We report on the use of the trnL intron and the trnL-trnF intergenic spacer (IGS) in the chloroplast genome and establish a DNA sequence database for plant species identification. The DNA sequences at these two loci from commonly encountered plants, including monocots and dicots, were aligned to establish a DNA database of local plants. The database comprises 373 individual sequences representing 80 families, 206 genera and 269 species. These plant species can be grouped to species level using both sequence and length polymorphisms at these loci. To validate the database for future forensic purposes, we sequenced 20 blind samples and searched the local database and the databases of GenBank and EMBL. Fifteen of these 20 samples used in blind trial testing matched their respective species from our local DNA database but only 6 matched species registered in the GenBank and EMBL databases. The sequences of two species used in the blind trial did not match any sequence registered in any of these databases. Cluster analysis was performed to demonstrate the family and genus distribution of samples. Neighbor-joining trees of the two DNA regions from 70 samples of the local database and 10 of the species used in the blind trials were constructed and clustered to both family and genus. The bootstrap values of the trnL intron were higher than most of those of the trnL-trnF IGS. The sequence database described in this study can be used to identify plant species using DNA sequences of the trnL intron and trnL-trnF IGS of chloroplast genome and illustrates its value in plant species identification.     

人与动物mtDNA细胞色素b基因的序列差异

目的 探讨人与动物之间mtDNA细胞色素b(Cyt-b)基因序列差异及其种属鉴定。方法 采用1对Cyt-b基因通用引物对人和19种动物共171例样本的mtDNA进行PCR扩增,琼脂糖凝胶检测扩增产物,ABI 377测序仪及荧光测序技术分析扩增产物的DNA序列。结果 所有样本均检测到1条358bp的扩增片段;任何两种动物扩增片段的序列都不相同,人与19种动物的序列差异在18.9％-30.0％,19种动物之间的序列差异在5.9％-32.9％。同种动物不同个体间只有人、驴及小白鼠存在变异,最多有4个碱基变异位点(1.3％),其它动物未发现种内变异。结论 人与不同种动物的Cyt-b基因序列存在差异,以此可区分不同种属的动物。     

Cytochrome b gene for species identification of the conservation animals

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.     

Establishing the rDNA IGS structure of Cannabis sativa

The rDNA intergenic spacer (IGS) structure of Cannabis sativa was established and can be used for classification and identification of this species. In this study, DNA fragments of rDNA IGS were amplified by PCR from Cannabis sativa plant extracts and a 1387 bp fragment was obtained. DNA sequence analysis revealed six different repeat motifs. In the middle of the IGS sequence, there were three sequence motifs, and the same three sections of DNA were then repeated with minor variation in sequence. The terminal region of the IGS was composed of another three different repeat units; multiple copies of these terminal repeat motifs were present in no discernible order. Within six repeat motifs, point variations were observed in five. The DNA sequence of the locus was compared with all the plant sequences registered in GenBank by the Fasta program of GCG software with the result that this DNA fragment was significantly different from any other DNA sequence recorded to date. The most similar sequence was that of Hops (Humulus lupulus), but with a similarity of only 88.9% over 579 bp. These specific and complex variations of IGS may be related to the species and geographic distributions.     

福州晋安河水域浮游微藻种群18SrDNA分析

目的应用分子生物学方法分析浮游微藻18SrDNA序列,分析福州晋安河浮游微藻种群分布。方法在晋安河分段设立11个点采集和水样本,采用优化CTAB法提取水样总DNA,通过PCR技术扩增并对浮游微藻18SrDNA序列进行测序,应用NCBI数据库序列进行比对筛选,分析该河段浮游微藻分布情况。结果筛选出67条浮游微藻序列,11个采样点部分藻类分布存在差异,常见种群为硅藻门藻类占40%,绿藻门藻类占12%,金藻门藻类占43%,原生动物类占5%,其中硅藻和金藻为优势种群。结论晋安河福州市河段内浮游微藻种群分布有一定特征,浮游微藻18SrDNA序列分析可望成为法医学溺水死亡鉴定的新方法。     

NCBI数据库在常见嗜尸性蝇类种属鉴别中的应用

目的探讨NCBI数据库比对分析对常见嗜尸性蝇类种属鉴别中的应用价值。方法收集2009年1月至2012年12月重庆市常见嗜尸性蝇类不同发育历期2科5属7种样本52份,采用Chelexl00法提取mtDNA,利用2对引物扩增细胞色素C氧化酶辅酶I基因,分别截取498bp和841bp相同长度的序列,采用MEGA软件计算种内及种间进化分歧情况,并分别在NCBI数据库进行序列BLAST搜索种属同源性比对分析。结果所得序列种内进化分歧均数在0％～0．7％之间,种问进化分歧均数在7．5％～16．1％之间;7个种属的样本序列Ⅰ和序列Ⅱ分别有5个和6个种属完全比对正确,样本总体的正确率分别达到96．15％和98．08％,Maxident值均在97％以上。结论采用序列同源性比对分析,并借助NCBI数据库强大的检索分析功能,可准确进行常见嗜尸性蝇类的种属鉴定,为法医学死亡时间推断提供重要参考依据。     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

The application of mitochondrial DNA cytochrome oxidase II gene for the identification of forensically important blowflies in Western China

Blowflies found on human corpses are important for the estimation of the postmortem interval and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, and therefore a molecular method was elaborated for species identification. Here, we describe a molecular method for rapid identification of these insects. Specific insect DNA fragments were amplified using the polymerase chain reaction, followed by direct DNA sequencing of the amplification products. Analysis of the cytochrome oxidase II sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, ie, the species within the Lucilia sericata and Lucilia cuprina groups. The molecular data support the existing species group separation of the taxa within the Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach enables quick identification of species used for estimation of postmortem interval.     

提取CO Ⅰ基因片段鉴定嗜尸性蝇类

目的通过扩增蝇类COⅠ基因片段,结合形态学特征鉴定嗜尸性蝇类的种类。方法运用改良平衡酚—tris饱和酚法提取蝇类DNA,进行COⅠ序列扩增和测序,与数据库序列进行比对和分析。结果改良平衡酚—tris饱和酚提取DNA方法可获得有效的蝇类DNA,并可应用于COⅠ基因片段扩增,进而进行蝇类种类的鉴定。结论平衡酚—Tris饱和酚法提取的噬尸性蝇类虫体DNA作为模板,用于COⅠ序列扩增,测序后与数据库目的序列分析比对,可准确鉴定嗜尸性蝇类的种类;和传统的昆虫形态学特征鉴定方法比较,该体系更准确,应用范围更广。     

Substitution of human for horse urine disproves an accusation of doping*

In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproducible and clear PCR-RFLP patterns and DNA sequence match with those expected for horse, while the urine sample results were coincident with human. These results allowed us to exclude the urine sample from the questioned stallion and determine its human species origin, confirming the manipulation of urine sample.     

Species identification of Kachuga tecta using the cytochrome b gene

A DNA technique has been established for the identification to species level of tortoises. The test on the shell of the animal was used to identify samples from the species Kachuga tecta. A total of 100 tortoise shell specimens collected from the National Council of Agriculture (COA), Taiwan, were used in this study. Primer pairs were designed to amplify partial DNA fragments of cytochrome b within the mitochondrial genome. The DNA data showed that among the 100 samples, there were four distinct haplotype DNA sequences, within which there were a total of 90 variable sites. Between haplotypes I and II, there was only 1 nucleotide difference at position 228. Between haplotypes I and III, 65 nucleotide differences were observed; haplotypes I and IV, 62 nucleotide differences; and haplotypes III and IV, 56 nucleotide differences were observed. There were 66 and 63 nucleotide differences between haplotypes II and III and haplotypes II and IV respectively. All four haplotypes were compared with the DNA sequences held at the GenBank and EMBL databases. The most similar species were K. tecta (haplotype I and II), Morenia ocellata (haplotype III) and Geoclemys hamiltonii (haplotype IV), and their respective mtDNA similarities were 99.5%, 99.3%, 89.9% and 99.5%. However, as haplotype III was only 89.9% homologous with M. ocellata, it would seem that this haplotype shows only a limited relationship with a similar species registered currently in these databases. The method established by this study is an additional method for the identification of samples protected under Convention International Trade in Endangered Species (CITES) and will improve the work for the preservation of the endangered species.     

Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.