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       【摘要】 
       目的利用PCR技术鉴定干燥蛇粗毒的来源。方法从4种蛇粗毒中提取总DNA--其中包括1份已保存了7年的干燥眼镜蛇粗毒，并分别以从蛇粗毒和肌肉组织中提取的总DNA作为模板用1对线粒体16S rRNA基因引物进行扩增。结果测序结果提交NCBI，并与GenBank中的同源序列进行比对。结论序列比对和系统发育分析的结果证实，该扩增的即为正确的蛇粗毒来源物种的线粒体16S rRNA基因序列，该技术有可能推广用于对动物粗毒的来源进行准确和直接地鉴定。
       【关键词】  蛇粗毒 分子鉴定 16S rRNA基因 DNA测序
       Forensic Identification of Snake Crude Venom by mtDNA analysis
       CHEN Nian, ZHAO Shujin
       (School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, P.R. China;  Department of Pharmacology, General Hospital of Guangzhou Military Command, Guangzhou 510010, P. R. China)
       Abstract:ObjectiveSnake venom is a highly valuable industrial raw material. Forensic issues raised by identification of snake products include enforcement of related animal protection regulations, threats to endangered species, and compromise of the integrity of commercial products. MethodsIn this work, DNA was successfully extracted from four dried snake venoms, among which one sample had been preserved in a pharmaceutical factory for seven years. The mitochondrial 16S gene was amplified and sequenced from the four samples and all sequences were submitted to the National Center for Biotechnology Information (NCBI) and compared against their homologous sequences in the GenBank database. ResultsSequence alignment and phylogenetic analysis proved that these samples indeed contained the correct 16S gene from the respective original animals. ConclusionThis approach provides a straightforward and accurate method for determining the identity of snake crude venoms.
       Key words:Snake crude venom;   Molecular identification;   16S rRNA gene;   DNA sequencing
       
        Snake venom provides a rich source of biologically active substances such as neurotoxins, cardiotoxins, diverse enzymes, enzyme inhibitors, and some other factors ［1］. However, because of increasing human demands on food and medicinal products, many snakes are killed illegally in China. In order to curb the illegal processing and trading of venoms from endangered snake species, it is therefore essential to develop reliable methods to authenticate the snake products.
        Most analytical methods for snake venom identification are based on the analysis of proteins, such as ELISA ［2］, OIA ［3］, and Mass spectroscopy ［4］, etc. However, proteins suffer from denaturation in long-term storage, which may cause changes in anti-genicity and electrophoretic mobility of molecules and therefore, make these methods less sensitive. The discovery of DNA in animal crude venom is a breakthrough in this field ［5］. It enables the use of DNA markers to differentiate animal venoms. Mitochondrial DNA has been used widely as a marker in snake identification ［6］. It has been extracted successfully from a variety of snake tissues, such as shed skin, feces, and squama, etc. The mitochondrial encoded gene for 16S rRNA was selected in this work for snake specie identification because it has an adequate length and grade of mutation, exhibiting a typical mosaic structure of phylogenetically conserved and variable regions.
        In this study, we extracted the whole DNA directly from lyophilized snake venom, and subsequently sequences and analysis of its partial mitochondrial 16S gene. Considering the fact that the crude venom resolved in ultrapure water can be reused after lyophilization, our method has obvious advantages in non-destructive identification for very expensive specimens.
       1  Materials and methods
       1.1  Sample collectionOne Naja  atra crude venom sample had been kept for about seven years in a pharmaceutical factory affiliated with the General Hospital of Guangzhou Military Command; other snakes and venoms were collected during 2005 and 2006 at the JiaYu Snake Breeding Farm, Jiangxi province, China. All the specimens were identified by the Snake Venom Research Institute, Guangzhou, Guangdong province, China. Fresh crude venoms were lyophilized within 24 h, and long-term sample storage was at 4℃in a sealed container. Detailed information about the samples is listed in Table 1.Table1  Details of the venom samples from which genomic DNA were extracted and used in the amplification of the partial mitochondrial 16S rRNA gene.（略）aDNA template was also extracted and sequenced from muscle as a control
       1.2  Extraction of DNA from venomApproximately 100 mg of lyophilized venom sample was powdered in a 5ml centrifuge tube with a glass stick. Sterilized ultrapure water was then added to give a venom concentration of 25 mg/ml. The tube was vortexed until the venom was completely dissolved. It was then placed at room temperature for a few minutes and the precipitated cells were recovered by centrifugation at 10 000×g for 5 min. After carefully removing the supertant, the pellet was suspended in 1 ml ultrapure water and centrifuged at the same speed for 1 min. DNA was extracted from the pellet with the Wizard SV Genomic DNA Purification System (Promega Inc., USA), following the manufacturer"s protocol with some modifications. Positive controls extracted from the muscle tissues were prepared with the same kit.
       1.3  PCR amplification and sequencing of the partial 16S-rRNA geneA ca. 400-base pair segment of the mitochondrial 16S ribosomal RNA gene was amplified by PCR under optimized conditions in a Perkin Elmers PE-2400 thermal cycler, with the oligonucleotide primers［7］ 16H1 (5′-CTC CGG TCT GAA CTC AGA TCA CGT AGG -3′) and 16L1 (5′- CTG ACC GTG CAA AGG TAG CGT AAT CAC T-3′) synthesized by SBS Genetech Co., Ltd. Beijing. The PCR mixture contained ca. 5 ng template DNA, 0.8 mmol/L total dNTPs, 0.6 μmol/L of each primer, 10×PCR buffer (10 mmol/L Tris-HCl, pH9.0, 50 mM KCl, 1.5 mmol/L MgCl2) and 2 units of PrimeSTAR HS DNA polymerase (Takara, Japan) in 25 μl. For venom samples, 5 μl elution was added directly as template. A solution of genomic DNA from snake-muscle tissue and an equal volume of ultrapure water were added as positive and negative controls, respectively.
        The PCR program was run for 3 min at 94 °C, followed by 30 cycles of 30 s at 94 ℃, 1 min at 55 ℃, 1 min at 72 ℃, with a final extension step of 5 min at 72℃. Amplified products were analyzed by electrophoresis in an 1% agarose gel and visualized by ethidium bromide staining. DNA sequences were determined by use of a 3730XL genetic analyzer (ABI Inc.), following the manufacturer"s protocol for the sequence reactions.
       1.4  Sequence submission and analysisNew sequences were deposited in the GenBank under the accession numbers EF413641-EF413645. The identities of all the sequences were validated by a BLAST search in the GenBank and alignment (ca. 380 bp) against four 16S rRNA gene sequences from the genera Naja and Deinagkistrodon, viz. N. naja (Cobra or Indian cobra; GenBank accession no. Z46482), N. naja naja (L10674), Deinagkistrodon acutus (AF057235) and D. acutus isolate A223 (AY352716). The multiple alignments of sequences were performed by Clustal W incorporated in the BioEdit v7.0.5.3 ［8］with default gap opening and gap-extension values. Phylogenetic analyses were performed by DAMBE version 4.2.13 ［9］. Neighbor-joining trees based on P-distance were computed, the maximum parsimony analysis was performed by means of an heuristic search, and the minimum evolution analysis was also carried out using DAMBE with 1 000 bootstrap replicates in order to assess the statistical confidence of each node, respectively.
       2  Results
       2.1  Electrophoresis of PCR productsDNA was successfully extracted from the small amount of remaining intact cells in crude venom samples, the DNA concentration recovered in a 60 μl extraction volume being about 1 ng/μl (data not shown). Amplification of correctly sized fragments (approximately 400 bp; see Fig. 1) was successful for six samples (GenBank accession nos. EF413641-EF413645). Unknown milk-white components in A. halys crude venom preparations could not be dissolved in water or be digested with Proteinase K during the extraction; we suspect that this is the main reason that they resulted in no anticipate band in electrophoresis (cf Fig. 1, lane 5).
       
       2.2  Sequence analysisComparison with published sequences provided unambiguous identification of the snake DNA in the crude venom samples as originating from the genera Naja and Deinagkistrodon, respectively. A significant percentage of sequence divergence could be found between these two genera (12.03%-20.86%; Table 2), but the range of variance within these genera was only 0.79%-12.34% and 1.60%-2.41%, respectively. The 16S rRNA gene sequence (EF413645) obtained from the venom DNA template of Naja  atra (Jiangxi) differed from EF413644 (from muscle tissue) in only four nucleotides at position 34, 123, 128 and 163 (Fig 2), and the divergence between these two sequences is only 1.05%. These small differences between the Naja spp. and Deinagkistrodon spp. do not allow unambiguous determination of the species at this point; however, sequence information from additional DNA regions will provide a better differentiation capacity.Table 2    Percentage sequence divergence and number of transition/transversion for partial 16S rRNA gene sequences（略）
       
       2.3  Phylogenetic analysisIn order to test the reliability of the above conclusions, a phylogenetic analysis of the aligned homologous sequences (ca. 380 bp) was performed. From those trees we can see that these species are mainly divided into two groups. The first group includes six species of Naja and the second group includes three species of Deinagkistrodon. NJ and ME trees share the same topology (Fig 3; left) and the sequences of the genera Naja and Deinagkistrodon form two distinct clusters, but differ in bootstrap confidence level (BCL%; BCL of NJ and ME methods was indicated before and after diagonal). Compared with NJ and ME trees, the difference observed in the MP tree may be caused by the poor sampling in genus Deinagkistrodon (Fig 3; right). It has been reported that the result should be treated with caution if the BCL value is below 50% and that a result could be considered as reliable if the BCL is above 70%. In this analysis, six Naja spp. sequences congregate in the same single cluster with BCL values of 100% in all these trees.
       3  Discussion and conclusions
        This is the first report of the successful isolation and sequence analysis of mitochondrial DNA from dehydrated crude venoms from Naja spp. and Deinagkistrodon spp. The results presented here confirm that the correct mitochondrial DNA fragments have been amplified from the venom samples and that this technique is applicable in identifying the origin of dried snake venom.
        Our method provides a quick and simple solution to the problems in snake venom authentication. Small quantities of whole DNA can be recovered and purified from lyophilized venoms - even preserved for seven years at 4 ℃- under mild conditions, avoiding filtration, extraction with organic solutions or the use of any other compound that may inhibit the PCR reaction. The resulting DNA sequences can be reconciled against existing mtDNA haplotypes and the identity of the venom can be verified. However, it should be pointed out that in a strict sense, the generation of additional DNA sequence datasets can only contribute to a correct specie identification or raise relevant, case-specific questions. It is evident that evidence from morphology, ecology, and behavior should not be ignored in taxonomic appraisals.
       
        Because of the complex constitution of snake venom ［10,11］, which may have potential inhibitory effects on the PCR, and the fact that many cost-effective biological kits are available, we did not extract the total DNA by the classical phenol-chloroform method. Improvement of the efficiency of various commercial kits and optimization of their detailed protocols are still ongoing.
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