蛤蚧及其伪品COI条形码鉴别技术规程DB45／T 1856-2018.pdf

ICS 6 5.02 0.3 0 B 38 DB45 广西壮族自治区地方标准 DB 45/T 18562018 蛤蚧及其伪品 COI 条形码鉴别技术规程 Identification of Gekko gecko Linnaeus and Adult erants Using the COI Barcode 2018-10-20 发 布 2018-11-20 实 施广西壮族自治区质量技术监督局 发布 DB 45/T 1 85 6 20 18 I 目 次 前言.II 1 范围.1 2 术语和定 义.1 3 鉴别程序.2 附录 A（规范 性附录）蛤蚧伪品.3 附录 B（规范 性附录）蛤蚧及其伪品 CO I DN A 序列.4 DB 45/T 1 85 6 20 18 III 前 言 本标准按照G B/T 1.12 0 09给出的规 则起草。本标准由广西壮族自治区卫生和计划生育委员会提出。本标准由广西卫生标准化技术委员会归口。本标准起草单位：广西壮族自治区药用植物园。本标准主要起草人：徐永莉、李力、闫志刚、张月云、赵成坚、黄勇、谷颖乐。DB 45/T 1 85 6 20 18 1 蛤蚧及其伪品 COI 条形码鉴别技术规程 1 范围 本标准规定了蛤蚧（G ekk o ge ck o）及其伪品的C OI条形码鉴 别的术语和定义、鉴别程序。本标准适用于广西境内蛤蚧及其伪品CO I条形码 的鉴别。2 术语和定义 下列术语和定义适用于本文件。2.1 蛤蚧 G ek ko ge ck o 蛤蚧（Gek ko ge ck o）属爬行纲Re pt il i a、有鳞目Sq ua ma ta、壁 虎科Ge kk on i da e、壁虎属Ge kk o。蛤 蚧药材为除去内脏的干燥体。包括黑点蛤蚧和红点蛤蚧。2.2 蛤蚧伪品 Ge kk o ge ck o ad ul te ra nt s 市场上当蛤蚧销售和使用的动物。详见附录 A。2.3 D NA条形码 D NA b ar co ding 生物体内能够代表该物种的标准的、有足够变异的、易扩增且较短的D NA片段。2.4 C OI 细胞色素c氧 化酶亚基I(cy to ch ro me c o xi da se s u bu ni t I，CO I)，线粒体 基因组的蛋白质编码基 因。2.5 C OI条形码 C OI b ar co ding 集中存在于动物线粒体细胞色素C氧化酶亚单位I(C O I)特定区段的一段大约有6 5 0个碱基的D N A序列。2.6 近邻相接法 n e ig hb or-jo ining me tho d(NJ法)一种研究DN A 而建立亲缘关系的方法，由序列建立支序图或亲缘关系图。DB 45/T 1 85 6 20 18 2 2.7 bp 碱基对。3 鉴别程序 3.1 鉴别条件 PC R扩增仪、核酸电泳仪和水平电泳槽、凝胶成像系统、组织基因提取试 剂盒、序列 比 对 软 件。3.2 引物序列 L CO I4 90：5 G GT CA AC AAA TC AT AA AG ATA TT GG 3。H CO 21 9 8：5 T AA AC TT CAG GG TG AC CA AAA AA TC A 3。3.3 D NA 的提取 取药材 样 品，7 5 乙醇 表 面消毒，用 灭菌手 术 刀，取 样 品 皮 下 肌肉 约30 m g,剪碎，D N A提取研 磨 仪 研 磨 2 m in，用组织基因提取试 剂盒 提取 总 DN A，提取 步骤参 见 各产 品使用 说明书。3.4 PCR 扩增 反应 体 积 为2 5 L，所加 试 剂 包括5 U/L T aq DNA 聚合 酶(Ta K aRa)0.5 L，10 mm ol/L dNT P 1 L，10 pm ol/L 上下 游引 物 各 0.5 L，1 0 PC R b u ff er 2.5 L，Mg 2+1.5 L，D N A 模板 1 L。P C R 反应 在P C R扩增仪上 进 行，反应参数 为：95 3 m in，(9 4 4 5 s，55 4 5 s，7 2 45 s）39，7 2 5 m in，4 保 存。3.5 电泳 将 PC R 产 物5 L与 上 扬染料 3 L混合，加入 1.5 琼脂 糖 凝胶的点 样孔 中，同时设 10 0 bp 梯度 DN A标准 分子量作 为片段大 小 的标准。10 0 V 电 压 电泳约4 0 m in。3.6 成像及分析 将 电泳 后 的 琼脂糖 凝胶 置 于凝胶成像系统 观察窗 内，观察分析并 成像 保 存。3.7 D NA 样品测序 凝胶上出 现 一条60 0 bp 大 小 的特异性条 带 PC R扩增 产 物用试 剂盒 对PC R扩增 的 样 品 进 行 纯 化，纯 化的步骤参 见 各产 品使用 说明书，将检测纯 化 后 的 样 品 进 行 测 序。3.8 鉴别 将所得 序列 经 计 算机拼 接辅以 人 工校 对，采 用B ia st序列 相似 性 搜索比 对，蛤蚧COI 5 端 序列，即 蛤蚧DNA条形码序列，562 bp的 长度 用于种 间 水平的 分析。蛤蚧 同 其伪品 间 的序列 差 异 为 6.0 64.0，平 均 序列 差 异为35.0。通过 N J 法 并利 用 实验 序列和Ge nB an k 序列 所构 建系统 聚类树。基于CO I序列 构 建 的N J树将 蛤蚧 与 伪品区 分。DB 45/T 1 85 6 20 18 3 A A 附 录 A（规范性附录）蛤蚧伪品 无蹼壁虎G ekk o sw in ho nis、中国瘰螈 P ar am es ot rit on c hi ne nsi s、多疣壁虎 Ge kk o ja p on ic us、睑 虎 Gon iu ro sa ur us li ch te nf el de ri、西 藏沙蜥Ph ry no ce ph al u s t he ob al di、红瘰疣螈Ty lo to tr it o n ve rr uc os us、贵州疣螈T ylo to tr it on kwe ic ho we ns is、喜山岩蜥La ud ak ia h im ala ya na、变色树蜥Ca lo t es ve rs ic ol or、青海沙蜥Ph ry no ce ph alu s vl an ga li i、山溪鲵B atr ac hu pe ru s pi nc ho ni i、东方蝾螈C yno ps or ie nt al is 等。DB 45/T 1 85 6 20 18 4 B B 附 录 B（规范性附录）蛤蚧及其伪品 C OI D NA 序列 B.1 蛤蚧 G ek ko ch in en si s 562 bp g aa tt aa g tc aa cc a gg a g c ac tc ct t gg a aa c g ac ca ac ta ta ca at g t aa tc g ta ac a g ca ca c g c at tt g ta ataat ct tc tt ta ta g t g at g cc t g t ta ta at c gg gg gg tt t gg c aa tt g at ta g t cc cc ct aa t a at t gg c g ca cc gg a cata g ct tt tc ca c g a at aa ac aa c a ta a g ct tc t g ac t g ct cc c c cc a g c at ta tt cc tc ct ac ta g cc tc c g c g a g t g t gg a a g ct gg gg c gg gg ac a gg at g a ac t g t at ac cc cc ca ct a g c a g ct aa tc t a g ca ca t g ca gg c g ca tc t g t a g ac ct gg c ca tc tt tt cc tt ac ac ct a g c t gg a g tc tc tt cc at ct ta gg gg cc at ca at tt ta tc ac ca cc t g c at t a at at aaaac cc cc aa g c at aa cc ca at ac ac g ac ac cc c t g tt t g ta t g a t c gg t tc ta at ta ct g cc g t ac ta ct tc t c ct a g cacta cc a g t g ct t g c c g cc gg c at ca ct at ac t g c t g ac c g a cc g t aa cc tt aa ca ca tc at tc tt t g ac cc t g c c gg a gg c gg gg B.2 无蹼壁虎 G ekk o sw in ho nis 8 25 bp at g tt ta cc c g tt g at tt ct ct ca ac aa ac ca ta aa g a ca tt gg ca cc ct at at tt ac t g t t t gg t g t at g a g cc gg cat a g t gg g ca ca g ca ct g a g ct t g tt aa tt c g c g c gg a g t ta a g cc a g cc t g gg g ca ct ac t a gg gg at g at ca g at ctata at g tt at c g t aa ca g cc ca t g ca tt c g t aa ta at tt tt tt ta ta g tc at g c c c g tt at aa tt gg c gg c tt c gg ca att g a ct t g ta cc ac tt at aa tt gg c g ca cc c g at at gg ct tt tc ca c g aa t g aa ta at at aa g t tt tt g ac t g c t cc ca ccatc ac t g ct ac ta ct tc tc g c tt ct tc c gg a g t t g aa g ca gg t g ct gg aa cc g g tt g aa ct g t ct at cc cc ca tt a g ca g cta at ct t g ca ca t g c gg g c g ca tc a g tt g a cc ta g t ta tt tt ct ct ct ac at tt gg ca gg t g t at ct tc aa t c tt a g ggg ct at ca at tt ta tc ac ca cc t g ca tt aa ta ta aa at ct ac t g c c a t g tc g ca g ta ca at ac g c cc ct at tt a t tt g at c gg t at ta at ta cc g cc g ta ct ac tc tt ac tt g c cc ta cc gg tt ct gg ct g cc g g a at ta ct at ac tt ct aa cc g ac c g caacc tt aa ca ct ac at tc tt t g ac cc t g c gg gg g g c gg a g at cc t g ta ct at at ca ac ac ct at tc t g at tt tt t gg g c accca g aa g ta ta ta tt tt aa tt ct tc cc gg at tc gg ca ta g t at c g ca ta tc at ta c g ta ct at g ca gg a aa aa aa g aa cc g tt c gg ct ac at g gg g at a g tc t gg g ct B.3 中国瘰螈 P ara me so tr it on ch in en si s 2 69 0 bp g tt aa gg t gg c a g a g cc c gg t aa tt g ca aa a g ac ct aa g c cc tt tc ca cc a g a gg t tc aa tt cc tc tt ct ta ac t g t g ta ta ct at at cc g c cc tc tt aa ac cc at ta ct at at at ta tc cc a g ta tt at ta g cc g t gg c ct tt tt aa ct ct t g tt g aac g aa aa g ta ct a gg g ta ta ta ca ac t g c gg a aa gg ac ca aa ca ta g t gg gg c c a g ta gg cc tt ct tc aa cc aa tc g ca g at gg ac ta aa ac ta tt ta tt aa a g aa cc c g ta c g cc ca tc aa ca tc g t ca ca aa ct ct g t tt ct ca tt at ac cc at aa t a g c g ct aa ct ct g g ca ct aa tt a t tt g aa tc cc ac ta cc ca t g c cc tt ta ca c t g tc ta at ct aa at ct gg gg g tt ct tttcc tt ct at cc ct at ct a g cc t g g cc g ta ta ct ca at cc ta gg g tc c gg ct gg t c g t ca aa ct ca aa at at g c cc ta g tt gg a g ca ct tc g c g c a g ta g ca ca g ac ta tc tc a t at g aa g t ta ca ct a gg a ct aa tc ct ac ta t g cc tt at tt ta ct aa c a gg gg g c tt ca ta tt aa ca aa ct tt aa ta tc tc g ca a g a at ca ac ct g a tt ta tt at tc ca g c at g ac ct at a g ca ac aat g t g a tt ta tc tc ac cc ct a g ca g aa ac aa ac c g aa cc cc at t t g at ct aa ca g a gg gg g a at ca g aa ct a g t ct cc gg attt aa c g ta g a at at g ca gg a gg g cc at tc g ca ct at tt tt tc t gg ca g a at a t tc ca ac at t t ta ct ta ta a a ta cc ctatc a g ct at cc ta tt cc tt gg cc aa ac aa ca a g c ca ca tt ca a c ca g aa at at ca ac aa tt aa cc ta at ta tt aa a g c atc g a DB 45/T 1 85 6 20 18 5 ca ct at ca at c g ta tt tc tc t g aa tt c g ag c at cc ta cc c a c gat tt c gg t a t g at ca ac ta at ac ac tt aa tc t g aaaa aa ct tc tt ac cc ct aa ca at ta ca at aa ct ct ca ta ca ca tc tc ac ta cc aa tt ac aa ta gct gg ta tc c c cc ct ttatc ct agg ac g tg t g cc cg aa ag a t a gg gg c ca ct tt gat a ga gt gg cc ga ta ga gg t tc aa a c cc tc tc ac a t cc ca ctaaa aa aa ca gg a c t c gaa cc ta t c cc ta g agg a t ca aa ac cc c t c gt gca cc c a ct ac ac ta t c tt tt aa gg t a aa at aa g ct aa at ca a g ct tt t gg g cc ca ta cc cc aa ac at gtt gg c ga aa ac cc tt ct tt ta ct aa t g a g cc cc ta ta ca ct gtccat at ta at at ca a g tc tt gcc g t a gg aa ca at ta cc ac ac ta tc aa gc ta tc ac t gg t tt ct a g ca tg aa ta gg ac ta g a g a tc aa ca ca tt gg ct at ta tt cc ac t g at aa ct aa aa t g ca cc ac cc cc gg gcc ac a ga at ct gca ac aa a gta ct ttcta ac ac aa gc ca ta gcc tc c gc ct ta at tt ta tt ct ca ac aa tt at aa ac gc at gaa t ga ca gg ag aa tg a a c aa tc ataa g ca ta aa tc ac ca ta ta tc aa ca ac aa tc tt aa ca at t g ct tt a g ca at ta aa ct a gg a at c g ca cc at tt ca cc ta t g at ta cc a g ac gt tc ta ca a gg a ct aa at at aa ta ac tt gc ct aa tc ct gt ca ac at ga ca aa aa tt a g cc cc ca ta gcctta ct ca tt tt ga ca a g cc ac ca ac ta aa cc ca aa cc ta ct aa tt at aa ta gc ac ta ac at cc at gct t gta g gg gg at g a gg gg gc ct aa at ca aa c g ca aa ca c g aa ag at ta ta g c at ac tc at cc at c g c ac ac tt a gg c t ga at aa ct at aa tt g tat cc tt tt cc cc ca at ct a g cg ct ac ta aa cc ta tt a g ta ta ca tt at cc t g a cc tc at ct a t g t tt at aa c a ct aa taacc tt aa at tc aa ca aa ca ta aa ca aa ct ct ca ac ct c g tg ac ta aa aa ca cc aa ca ct a g ca gc ct tt ac aa ta at t g ttct aa tgg cc ct t gg agg g ct cc cc cc ca ca tc a gg a tt tt ta cc aa aa t g ac ta at tt ta ca a g a aa ta ac c a aa ca acacc tc aa t g ca ct ct cc gc ca ca at a g cc at at ta gcc tt ac t c a gc ct at tt tt tt at ct ac gtt ta tc at a t ac aa tctca at aa cc tc ac cc cc tc ac at ct ca aa ct ca a g cc ta at tt gac gg aa aa a a ac aa ac ct a c at at aa tc c c aa tt ataat ta ta tt gt ca gc aa ta tt aa ta cc aa ta ac tc ca ac ac t g ct aa ta at aa ac ta a gg a ct ta gg at aa ca a g ac ca a a g a cc tt ca aa gc c tt aa g ca ga a g tt aa aa tc tt ct a g tc ct t g ac aa aa cc t g ca gg a ta c t ac cc ca ca t c tt ct g aa tg ca ac cc a g ac ac tt ta at ta a g ct aa a g cc tt ct a g at ta gca gg ct tt g ac cc ta ca ac ct tt ta gt ta ac a g c taaat ac tc aa tc aa c g ag ct tt aa tc ta ct tc tc cc gct t gtt g a g aa aa aa a g c gg g ag aa gc cc c gg ca aa a gct ac ttt g c tc tt ta aa at tt gc aa tt tt at at gct g aa ca tc ac a g ag c t t gg t aa aa aa a gg a ct at aa cc tt ta t g a c a a gg g cta ca ac ct t g c a c ct g ct tc gg c ca tt tt ac c a g tg at aa tc ac tc gat g a c t at tt tc ta c B.4 多疣壁虎 G ekk o ja po ni cus 8 25 bp at at tc ac cc gtt g at tt ct ct ca ac aa ac ca ta a gg a ta tc gg ca cc ct ct at tt ac t g t t t gg t gt ct gg gcc ggcat gg tc gg ca cc gca tt aa g tc tt ct ta tt c g c g ct gaa ct g a g tc ag cc cg gt gcc ct cc t c gg c gac g at ca gat ttaca ac gtc at t gt ca cc gcc ca c gca tt c gt aa ta at tt tt tt ta ta gt aa ta cc gg tc at ga tc gg cgg c tt c gg ta att g a ct cg ta cc cc tt at aa tc gg c g ca cc t g ac at gg cc tt cc ca c g aa ta aa ca ac at aa gc tt ct ga ct tc t g c cc ccctc tc ta ct ac tt ct ac tc gc ct cc tc t gg a gt gg agg cc gg c gct gg ca cc g gtt gg ac t gt at ac cc cc ca ct c g ca g caa ac ct c g ca ca c g cc gg cg cc tc c g tc ga cc tc gt ta tc tt tt ca tt gc at tt gg cc gg gg t c t c gt ca at tc tt gg t g ca at ca ac tt ca tt ac ca cc t g ta tt aa ta ta aa at cc ac ac ct at aa ca ca at ac aa ca c g c cc ct g tt ta tt t g a tca g t tc ta at ca ct gcc g ta ct ac tc tt gct c gc tt t g cc ag ta tt a g cgg cc g ga at ta cc at ac tt ct aa cc gat c gtaatc tc aa ca cc ac tt tt tt t g at cc a g ct gg gg gc gg cg ac cc c g ta ct gt at ca ac at ct at tt t g at tc tt c gg tc accca ga gg ta ta ta tt tt aa tt ct cc cc gg tt tc gg ca ta gt at ct ca ta tt at ca ca ta tt at gca gg g aa aa a gg a gccctt cgg ct ac at a gg aa tgg tc t ga gcc B.5 睑虎G on iu ros au ru s li cht en fe ld er i 5 62 bp ga gct ta ac c a g cc tgg gg c gct cc tc gg c gac g ac ca aa tt ta ca at gt aa tc gta ac c gcc ca t gc ct tt gtc ataat tt tt tt ca ta gta at ac ct g t t a t gat t gg gg gct tc gg a aa ct gg ct ag t ac cc tt aa t a at t gg gg cc cc t g a catag ct tt cc cc c g a at aa ac aa t a ta a gct tc t ga ct tt ta cc cc ca tc tt ta ct tc ta ct cc ta gct tc tt c a gg t gtag aa DB 45/T 1 85 6 20 18 6 g ct gg t g ct g g aa ca gg g t g g ac c g t at ac cc cc ca ct a g c c g ca aa cc t c g ca ca c g ca gg a g ca tc c g t a g ac cttac aa tc tt tt cc ct ac at ct c g c c gg t g tt tc ct ca at tc ta gg gg ca at ta ac tt ca tt ac ta cc t g t at t a ac at aaaac cc cc t g ca at at ca ca at at aa ca cc cc g t t g tt t g t g t g a t c a g t tc ta at ca ca g ca g t ac ta ct ac t t ct at cacta cc t g ta ct t g c c g ct gg ca t t ac aa ta ct a c ta ac a g at c g aa ac ct aa a c ac gg ct tt t t tt g at cc t g c a gg gg g a gg gg B.6 西藏沙蜥 P hry no ce ph al us th eo ba ld i 6 61 bp ac ac tt ta ct tc ct at tc gg c ac ca ta g cc gg ct tc ac t g g ct ca ac a g t ca g cc tt at g a t t c g ac ta c a ac ta attca ac cc gg cc aa ca ca ac gg a g ac ac aa ta ta ca ac ac tt tc at ta ca ct tc at g c ct tc at ta ta at ct tc tt ta ta g taa ta cc aa tc at aa tc gg a gg a tt c gg ca ac t g at ta g ta cc ac tc at ac ta gg c g cc cc a g a ca ta g ca tt tc ca c g aata aa ta ac at aa g ct tc t gg ct cc ta cc ac ca tc g t tc tt tt ta ct tc ta at g tc at ca aa a t tc gg c g ac g g a g ta gg aac a gg at g aa ca at tt at cc tc ca tt a g ca gg a aa ta tt g ca ca ct ca a g cc ca tc aa ta g a ca ta ac aa tt tt c g c actac at ct a g cc gg a g ca tc ct ca at ct t gg gg g cc at ta ac tt ta tt ac aa cc t g ca tt aa ta ta g cc cc ac a c tc aa caacc cc at ac aa ct g ac ca ct g tt c g tc t g at cc g ta tt cc tt a c c g c aa tc tt ac tc ct ac t g g cc at cc ca g t at ta g ca g c a g cc at ca cc at g ct tc ta a c a g ac c g a aa cc ta aa ca ca ac at tc tt c g a cc cc g cc gg a gg a gg a g a c c ca at tctct t cc aa ca cc t a B.7 红瘰疣螈 T ylo to tr it on ve rr uc os us 28 89 bp c g ct at ta a g g tt c g tt t g t tc aa c g at ta aa g tc ct ac g t g at ct g a g t tc a g ac c gg a g ta at cc a gg t c a g t ttcta tc ta ta aa aa at tt tt tc ta g ta c g aa a gg a c c g aa aa aa ta g a g c cc at ta ct aa ac aa t g ct ct ta ca ca g tt g aaaa ca aa ta aa ac ac g c ac ac aa aa ca ca cc cc ca a g aa ca gg g ta aa g tt aa g at gg ca g a g c c c gg ta at t g ca aa a g acc ta a g cc ct tt ca cc ca g a gg tt ca ac tc ct ct tc tt aa ct at g ca ta tc c t ac aa ac ac t c at ca ac cc a t ta ct atata tt at tc cc at ct ta tt a g ct g ta g ca tt cc tc ac ac ta g t c g aa c g aa aa g ta tt a gg at at at ac a g ct cc g ca aa gg c cc aa ac at c g t a gg ac ca at a gg a ct at ta ca ac ca at c g c c g ac gg a g t aa aa ct at tt at ta a gg a ac cc at cc g acc at cc ac at cc tc ac a g ac ct ta tt ta tc ac aa t g cc ta ta at a g ca ct aa ca ct a g ca ct aa t g at ct g a a tt cc ccttc ca at g c ca tt ta ta ct at ca a g cc tt aa cc ta gg a g ta ct g tt ca ta tt a g cc at at ca a g ct ta g ct g t a ta ct caatc tt a gg ct ca g g at g at ca tc ca ac tc aa aa ta t g ca ct aa tc gg gg c g ct ac g c g ca g ta g ca ca g ac ca tc tc at a t g a a g ta ac ac ta gg ac tt at tc tc ct ct g t ct ta tt tt aa t g a c a gg aa g ct tt ac at ta at aa g ct tc aa c a ca ct acaa g aa cc aa ca t g a ct ta tc at a c ca g ca t g ac ca at a g ca ac aa ta t g at tt at ct ca ac at ta g ca g aa ac aa at c g a g cc cc at tt g a tt ta ac gg aa gg a g aa tc a g aa ct a g ta tc a g g ct tt aa t g t a g aa ta c g cc gg a gg a cc at tc g ca ctatt tt tt ct a g ca g a at ac tc aa at at ct ta ct aa ta aa t g cc ct ct ca ac ta tc tt at tt ct a gg aa ca ac a a tt aa tcacc ta ca ac ca g a aa ta ct ta ca at ta ac ct aa ta at aa aa g c at cc g c cc ta tc aa tt at at tt ct at g a g t ac g a g catca ta tc ca c g at tc c g ct at g a cc aa tt aa ta ca cc tt at ct g a aa aa ac tt cc tc cc aa tt ac aa tt gg a c t ta ca ctaat ac at at ct ca ct ac ca at tt ta ac ct cc gg a at tc cc tc t g ca ct at a gg a c a t g t g c cc g aa a g at a gg g ct ta cttt g at a g a g ta ac ac at a g a gg t tc aa ac cc tc tc at ct cc t g t at aa aa aa a t a gg at tt g a ac ct ac cc ca a gg a g atcaa aa cc cc tc g t g c tt cc ac ta ca