淀粉酶检测讲解学习

淀粉酶检测FeatureofsalivaandsalivarystainSalivaryglandsSalivasecretion唾液的分泌Salivacomposition

唾液成分正常纯净唾液：无色无味接近中性液体,低渗通常情况为泡沫状、稍混浊粘稠液体正常分泌量：成人每日1.0~1.5L分泌量及性质受精神因素、刺激强度等影响

Physical&ChemicalProperties唾液的理化性质①唾液清水漱口自然流出试管/唾液斑/灭活酶②口腔拭子棉签/纱布浸湿反复擦拭口腔粘膜无创，简便，对照样本Collectofsaliva新鲜唾液收集什么情况会留下唾液斑？唾液斑提取:整件提取/二步转移性侵犯案皮肤咬痕、舔舐痕提取Objective&requirement检验目的与要求Isitsalivarystain?

Isithumansalivarystainoranimalsalivarystain?

IndividualizeofsalivarystainConfirmationofSalivaryStain

唾液斑的证明检出淀粉酶口腔脱落上皮细胞确证唾液斑Detectionofamylase淀粉酶检测Highlevelsofamylaseinthesaliva.Canbefoundinotherbodyfluids

saliva:263000-376000U/LUrine:263-940U/LBlood:110U/LSemen:35U/LNasalsecretion:undetectablelevelsSweat:undetectablelevels

Amylaseenzymecanbreakdownstarch(淀粉)Twokindsoftest:Starch-iodineassay淀粉碘实验PhadebasReagentPhadebas试剂Detectionofamylase淀粉酶检测Starch-IodineTest

淀粉碘实验principleIodine+starch→turnblueAmylasebreaksdownstarch淀粉酶检测starch37℃Iodine阴性蓝色无淀粉酶/失活阳性：无色/淡黄色有淀粉酶设置已知阳性和阴性对照！假阳性假阴性生成糖的证明淀粉碘阳性反应液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-IodineTest

淀粉碘实验Geldiffuse凝胶法:GelcontainingstarchisstainedbluewithiodineSampleisaddedtowellingel.Ifamylase(saliva)ispresent,thebluecolorbeginstovanishORbeforenegativepositive含碘淀粉凝胶无淀粉酶有淀粉酶淀粉酶检测PhadebasReagent

菲德倍斯试剂法StarchlinkedtoacoloreddyePresenceofamylasereleasesthedye

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。TwomethodsforusingPhadebasReagent:PressTest按压法TubeTest试管法淀粉酶检测ThePhadebas®ForensicPresstestStep1:Placeitemtobetestedonaflatandcleansurface.DampentheitemwithwaterStep2:CovertestingareawithPhadebas®paper,reagentsidedown.DampenPhadebas®paperwithwaterStep3:TraceanoutlineofthetestingareaonthePhadebaspaperStep4:CoverthePhadebas®paperwithacleanglassboard.WeighdowntheArrangementStep5:Startatimerandobservethetestfrequentlyduring40minutes.Recordthetimeofpositivereaction(distinctareaofdiffusebluecolor).ThePhadebas®ForensicTubeTestSmallpiececontainingstainisplacedintubeWaterandPhadebasreagentadded,incubatedin37℃CentrifugedAmountofcolorintopliquidismeasuredbyaspectrophotometer.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）PhadebasReagent

菲德倍斯试剂法Oralepithelialcells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Immunochromatographicstrip(ICS)test

免疫层析试剂条原理：免疫胶体金技术检测人唾液α-淀粉酶（α-amylase）

灵敏（1微升唾液），具有高度种属特异性，不受尿液、汗液等干扰。

Individualizeofsalivarystain

唾液斑个人识别ABO血型蛋白多态性酶型DNA分析Absorption-inhibition

test

凝集抑制试验ABO血型测定无A抗原抗A抗体不结合效价不变A红细胞凝集Absorption-inhibition

test

凝集抑制试验含A抗原抗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按照最大抗血清稀释倍数稀释抗血清原液DNAAnalysisWithsomebiologicalstains,DNAtestingismoreimportantthanidentifyingthestain.Ifatestdestroyscells,itmaynotbedonebeforeDNAtesting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DNA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：