淀粉酶检测.ppt

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1、Identification of Salivary stainNie Shengjie,Assoc.Prof Section of Forensic Biology 2010.09.20OutlineFeature of saliva and salivary stainObjective&requirementConfirmatory testIndividual identificationFeature of saliva and salivary stainSalivary glandsSaliva secretion 唾液的分泌唾液的分泌Saliva composition 唾液成

2、分唾液成分正常纯净唾液：无色无味接近中性液体正常纯净唾液：无色无味接近中性液体,低渗低渗 通常情况为泡沫状、稍混浊粘稠液体通常情况为泡沫状、稍混浊粘稠液体 正常分泌量：成人每日正常分泌量：成人每日1.01.01.5L1.5L 分泌量及性质受精神因素、刺激强度等影响分泌量及性质受精神因素、刺激强度等影响 Physical&Chemical Properties唾液的理化性质唾液的理化性质唾液唾液清水漱口清水漱口自然流出自然流出试管试管/唾液斑唾液斑/灭活酶灭活酶口腔拭子口腔拭子棉签棉签/纱布浸湿纱布浸湿反复擦拭口腔粘膜反复擦拭口腔粘膜 无创，简便，对照样本无创，简便，对照样本Collect of

3、 saliva 新鲜唾液收集新鲜唾液收集什么情况会留下什么情况会留下唾液斑？唾液斑？唾液斑提取唾液斑提取:整件提取整件提取/二步转移二步转移性侵犯案皮肤咬痕、舔舐痕提取性侵犯案皮肤咬痕、舔舐痕提取Objective&requirement 检验目的与要求检验目的与要求Is it salivary stain?Is it human salivary stain or animal salivary stain?Individualize of salivary stainConfirmation of Salivary Stain 唾液斑的证明唾液斑的证明检出淀粉酶口腔脱落上皮细胞确证唾液斑D

4、etection of amylase 淀粉酶检测淀粉酶检测High levels of amylase in the saliva.Can be found in other body fluids saliva:263 000-376 000 U/L Urine:263-940 U/L Blood:110 U/L Semen:35 U/L Nasal secretion:undetectable levels Sweat:undetectable levelsl Amylase enzyme can break down starch(淀粉淀粉)l Two kinds of test:St

5、arch-iodine assay 淀粉碘实验淀粉碘实验 Phadebas Reagent Phadebas 试剂试剂Detection of amylase 淀粉酶检测淀粉酶检测Starch-Iodine Test 淀粉碘实验principleIodine+starch turn blueAmylase breaks down starch淀粉酶检测starch3737Iodine阴性阴性 蓝色蓝色无淀粉酶无淀粉酶/失活失活阳性：阳性：无色无色/淡黄色淡黄色有淀粉酶有淀粉酶设置已知阳性和阴性对照！设置已知阳性和阴性对照！假阳性假阳性 假阴性假阴性生成糖的证明淀粉碘淀粉碘 阳性阳性反应液反应液

6、碱性铜氯化三苯基四氮唑液短时加热阳性 红色沉淀阴性 无色/浅红色阴性 无沉淀阳性 红色沉淀Starch-Iodine Test 淀粉碘实验Gel diffuse 凝胶法:Gel containing starch is stained blue with iodine Sample is added to well in gel.If amylase(saliva)is present,the blue color begins to vanishORbeforenegativepositive含碘淀粉凝胶含碘淀粉凝胶无淀粉酶无淀粉酶有淀粉酶有淀粉酶淀粉酶检测Phadebas Reagent

7、菲德倍斯菲德倍斯试剂法试剂法nStarch linked to a colored dye nPresence of amylase releases the dye 淀粉包裹染料制成微球，淀粉酶能将淀粉分淀粉包裹染料制成微球，淀粉酶能将淀粉分解，染料释放使溶液呈蓝色反应。解，染料释放使溶液呈蓝色反应。Two methods for using Phadebas Reagent:Press Test 按压法Tube Test 试管法淀粉酶检测The Phadebas Forensic Press testStep 1:Place item to be tested on a flat and

8、clean surface.Dampen the item with waterStep 2:Cover testing area with Phadebas paper,reagent side down.Dampen Phadebas paper with waterStep 3:Trace an outline of the testing area on the Phadebas paperStep 4:Cover the Phadebas paper with a clean glass board.Weigh down the ArrangementStep 5:Start a t

9、imer and observe the test frequently during 40 minutes.Record the time of positive reaction(distinct area of diffuse blue color).The Phadebas Forensic Tube TestSmall piece containing stain is placed in tubeWater and Phadebas reagent added,incubated in 37CentrifugedAmount of color in top liquid is me

10、asured by a spectrophotometer.灵敏、特异灵敏、特异，羊、马、牛、猪、猫、狗等动物唾液（斑）检测结果为阴性，尿液、血液、精液、汗液、鼻腔分泌物等反应不敏感 干扰：粪便污染（淀粉酶含量与唾液接近）Phadebas Reagent 菲德倍斯菲德倍斯试剂法试剂法Oral epithelial cells口腔脱落上皮细胞口腔脱落上皮细胞HE染色：形态以多角形为主，胞质呈粉红色，胞核圆形，较小，蓝染不特异不特异Immunochromatographic strip(ICS)test免疫层析试剂条原理：原理：免疫胶体金技术检测人唾液-淀粉酶（-amylase）灵敏灵敏（1微升唾

11、液），具有高度种属特异性高度种属特异性，不受尿液、汗液等干扰。Individualize of salivary stain唾液斑个人识别ABO血型蛋白多态性酶型DNADNA分析分析Absorption-inhibition test凝集抑制试验凝集抑制试验-ABO-ABO血型测定血型测定无A抗原抗A A抗体不结合效价不变A红细胞凝集Absorption-inhibition test凝集抑制试验凝集抑制试验含A抗原抗A A抗原抗体结合，游离A抗体减少效价下降A红细胞凝集减弱或不凝集关键：抗血清的标化关键：抗血清的标化抗血清的标化抗血清的标化效价：产生明显凝集反应的最大抗血清稀释倍数标化抗血清

12、 按照最大抗血清稀释倍数稀释抗血清原液DNA AnalysisnWith some biological stains,DNA testing is more important than identifying the stain.nIf a test destroys cells,it may not be done before DNA testing样本预处理PCR 扩增毛细管电泳荧光检测有 机 法Chelex-100法DNA提取STR 分型DNADNA分析分析PAGE电泳银染检测 某市多家单位发生盗窃。犯罪嫌疑人作案时均戴手套作案，无法取到有价值的指掌纹，虽然作案手法相似，但无并案依据。现场勘验时发现在多个案发现场提取的烟头或嫌疑人在现场喝过水的纸杯。问题：如何正确提取烟蒂及水杯以尽量避免唾液斑的污染和损失？盗窃案中生物样本的意义？某年某月某日，某地旅馆发现一具女尸，提取死者阴道拭子及现场卫生纸。一房客未退房就离开，其住处卫生间遗留1把牙刷。问题：性侵犯案件中，应该如何发现提取生物样本？如何进行牙刷上脱落细胞检测？某年某月某日，某市某一居民小区发生入室抢劫案，勘验现场时发现卧室地上一口香糖胶，系嫌疑人所留。问题：如何进行口香糖类检材的提取？如何处理口香糖以获取尽可能多的脱落细胞？summary