3本標(biāo)準(zhǔn)根據(jù)國標(biāo)委公布的2024年第一批國家標(biāo)準(zhǔn)計(jì)劃項(xiàng)目（國標(biāo)委發(fā)[2024]16本標(biāo)準(zhǔn)由全國生化檢測標(biāo)準(zhǔn)化技術(shù)委員會（SAC/TC387）提出并歸口?！癕ALDI-TOF”）是一種專門用于分析生物大分子的質(zhì)譜技術(shù)。其離子源（MALDI）因使質(zhì)譜技術(shù)能夠分析生物大分子而榮獲2002年諾貝爾化學(xué)獎。此外，MALDI-TOF還可作為多重PCR的質(zhì)譜檢測器，用于單核苷酸多態(tài)性位點(diǎn)的分析。酸分析。因此，它在基因分析科學(xué)研究、農(nóng)業(yè)、畜牧業(yè)疾病控制領(lǐng)域中的病蟲害遺傳分析、進(jìn)出口貨物鑒定、MALDI-TOF技術(shù)在單核苷酸多態(tài)性位點(diǎn)分析領(lǐng)域的應(yīng)用已經(jīng)歷了近二十年的發(fā)展。由于我國在核心技術(shù)（包括質(zhì)譜儀、分析軟件、試劑等）方面與發(fā)近年來，我國在MALDI-TOF質(zhì)譜儀的研發(fā)和制造方面取得了顯著進(jìn)步。目前，國內(nèi)已有超過十家企業(yè)涉足MALDI-TOF質(zhì)譜儀的研發(fā)、生產(chǎn)和銷售。其中，一些企業(yè)已經(jīng)開發(fā)出目前，我國已有一項(xiàng)行業(yè)標(biāo)準(zhǔn)和兩項(xiàng)團(tuán)體標(biāo)準(zhǔn)涉及MALDI-TOF單核苷酸多態(tài)性位點(diǎn)分析的相關(guān)內(nèi)容。然而，這些標(biāo)準(zhǔn)反映出當(dāng)前MALDI-TOF單核苷酸多態(tài)性位點(diǎn)分析技術(shù)存在的某些技術(shù)局限性，以及國外主導(dǎo)企業(yè)的專利技術(shù)4作為生物遺傳物質(zhì)分析領(lǐng)域的核心技術(shù)，MALDI-TOF單核苷酸多態(tài)性位點(diǎn)分析技術(shù)是我國企業(yè)通過自主研發(fā)所掌握的少數(shù)核心技術(shù)之智生物科技（青島）有限公司、山東英盛生物技術(shù)公司等在內(nèi)的多家企業(yè)，正積極致力于MALDI-TOF單核苷酸多態(tài)性位點(diǎn)分析的標(biāo)準(zhǔn)化工的標(biāo)準(zhǔn)立項(xiàng)建議。這一舉措旨在制定一個更具包容企業(yè)的專利技術(shù)制約，從而構(gòu)建一個由我國企業(yè)和科研機(jī)構(gòu)主導(dǎo)的MALDI-TOF單核苷酸點(diǎn)分析基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜法》標(biāo)準(zhǔn)項(xiàng)目進(jìn)行了預(yù)研，課題組成員廣泛收集了國內(nèi)外近百余篇標(biāo)準(zhǔn)、文獻(xiàn)，了解了國內(nèi)外相關(guān)技（3）2024年4月收到全國生化檢測標(biāo)準(zhǔn)化技術(shù)委員會生化標(biāo)[2024]5文件《關(guān)于下達(dá)2024年第一批推薦性國家標(biāo)準(zhǔn)計(jì)劃的通知文件》以及該標(biāo)委會轉(zhuǎn)發(fā)的國標(biāo)委發(fā)[2024]16號《國家標(biāo)準(zhǔn)委關(guān)于下達(dá)2024年第一批國家標(biāo)準(zhǔn)制計(jì)劃及相核苷酸多態(tài)性位點(diǎn)分析基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜法論，提出了寶貴的意見和建議。標(biāo)準(zhǔn)起草小組根據(jù)專家吸電離飛行時間質(zhì)譜法》標(biāo)準(zhǔn)的起草研制工作。完成了《單核苷酸輔助激光解吸電離飛行時間質(zhì)譜法》標(biāo)準(zhǔn)的編制說明，會匯報(bào)了《單核苷酸多態(tài)性位點(diǎn)分析基質(zhì)輔助激光解吸電離飛行案）進(jìn)一步完善和修改情況，向全國生化檢測標(biāo)準(zhǔn)化技位點(diǎn)分析基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜法》標(biāo)準(zhǔn)（征求并參照GB/T6379.1-2004《測量方法與結(jié)果的準(zhǔn)確度（正確度與精密度）第1部分總則與定義》和GB/T6379.2-2004《測量方法與結(jié)果的準(zhǔn)確度（正確度與精密度）第2部分確定2、確定國家標(biāo)準(zhǔn)主要內(nèi)容（如技術(shù)指標(biāo)、參數(shù)、長度8~25）bp；熔解溫度（Tm55~65）℃;表1PCR反應(yīng)體系組成成分濃度/體積說明DNA模板（1~100）ng來源：全血、組織、唾液等PCR預(yù)混液含Taq酶、MgCl?、dNTP混合液引物（正/反）每種引物在反應(yīng)液中的終濃度為（0.1~0.2）μM無RNA酶水補(bǔ)足至5μL/每對引物擴(kuò)增的單重?cái)U(kuò)增產(chǎn)物電泳檢測為單一條帶；瓊脂糖凝膠電泳（2%）。產(chǎn)物濃度≥5ng/μL；使用UV分光光度計(jì)或熒光儀（如Qubit）檢測。表2SAP反應(yīng)體系組成成分濃度/體積說明PCR產(chǎn)物來自前述PCR反應(yīng)體系SAP緩沖液（0.17~1）μL廠商提供（含Mg2?)SAP酶（0.5~1）U依據(jù)廠商推薦調(diào)整用量無RNA酶水補(bǔ)足至7μL/）；表3MPE延伸反應(yīng)體系組成成分濃度/體積說明SAP處理后的PCR產(chǎn)物上一步產(chǎn)物延伸緩沖液配合延伸酶使用，根據(jù)廠商提供的參考用量MPE引物特異性延伸引物延伸酶（如ThermoSequenase）0.6μL熱穩(wěn)定聚合酶，根據(jù)廠商提供的參考用量混合ddNTP含不同分子質(zhì)量標(biāo)記，保證峰分辨）：表4純化體系成分濃度/體積說明MPE反應(yīng)產(chǎn)物來自前一步延伸反應(yīng)純化樹脂（3~10）mg（固體）每次實(shí)驗(yàn)根據(jù)廠商建議稱取去離子水用于稀釋反應(yīng)體系及助于均勻分散樹脂樣品與樹脂混勻，震蕩（或顛倒混勻10~30）min；）；表5基質(zhì)配制與添加方法步驟操作說明1配制3-HPA溶液，過濾除雜（0.22μm）2將延伸產(chǎn)物以1:1體積比與基質(zhì)混合3點(diǎn)樣：每孔加1μL基質(zhì)，干燥后加1μL基質(zhì)產(chǎn)物樣品混合液4室溫自然干燥結(jié)晶（優(yōu)于PAGE純化級別）、SAP酶、SAP緩沖液、單堿基延伸酶（MPE酶）、單堿基延陰性質(zhì)控：應(yīng)有標(biāo)準(zhǔn)物質(zhì)溯源信息或經(jīng)測序確認(rèn)；陽性質(zhì)控：應(yīng)有標(biāo)準(zhǔn)物質(zhì)溯源信息MALDI-TOFMS：質(zhì)譜儀的質(zhì)量范圍須滿足最小值≤1500，最大值≥10000；質(zhì)量分所有樣本均視為有潛在的感染性，操作及運(yùn)輸提取的模板DNA純度要求紫外分光光度計(jì)讀數(shù)（A260/A280）在1.7~將PCR試劑取出，在冰上融解，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)目，按照表6的比例在適合的離心管中配制PCR擴(kuò)增反應(yīng)混合液（DNA模板除外），然后將試配制好的PCR反應(yīng)體系渦旋振蕩混勻，瞬時離心，按3μL/孔的量加入PPCR反應(yīng)管底部。每次反應(yīng)選擇兩個孔分別加入2μL陰表6PCR反應(yīng)體系試劑名稱反應(yīng)孔/μL成分及反應(yīng)液濃度PCR預(yù)混液2.0dNTP混合液（0.1μM）無酶水補(bǔ)充體系/PCR引物DNA模板2.0總體積5.0/表7PCR反應(yīng)程序擴(kuò)增溫度95℃95℃59℃72℃60℃4℃反應(yīng)時間15min30s30s15minHold備注45cycles將SAP酶和SAP緩沖液平衡至室溫，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)目，按照表8的比例在適合的離心管中配制PCR擴(kuò)增反應(yīng)混合液（DNA模板除外），上述表8SAP反應(yīng)體系試劑名稱反應(yīng)孔/μL無酶水SAP緩沖液（10×)0.17SAP酶（1.7U/μL）0.3總體積2.0將SAP反應(yīng)管板放置于PCR儀中，運(yùn)行表9表9SAP反應(yīng)程序擴(kuò)增溫度反應(yīng)時間37℃40min5min4℃Hold將MPE酶和緩沖液、ddNTP混合液和MPE引物平例在適合的離心管中配制MPE反應(yīng)混合液，混勻后按4μL每SAP反應(yīng)孔分裝。上述操作須表10MPE反應(yīng)體系試劑名稱反應(yīng)孔/μLddNTP混合液MPE酶緩沖液MPE酶（1.5U/μL）0.6MPE引物（2~5μM）總體積4.0將MPE反應(yīng)管板放置于PCR儀中，運(yùn)行表1表11MPE反應(yīng)程序擴(kuò)增溫度95℃95℃48℃72℃4℃反應(yīng)時間30sHold備注40cycles5cycles將PCR試劑取出，在冰上融解，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)目，），配制好的PCR反應(yīng)體系渦旋振蕩混勻，瞬時離心，按3μL/孔的量加入PPCR反應(yīng)管底部。每次反應(yīng)選擇兩個孔分別加入2μL陰表12PCR反應(yīng)體系試劑名稱反應(yīng)孔/μL成分及反應(yīng)液濃度PCR預(yù)混液2.0混合液（0.1μM）無酶水補(bǔ)充體系/PCR引物DNA模板1.0~2.0總體積5.0/將PCR反應(yīng)管放置于PCR儀中，運(yùn)行表表13PCR反應(yīng)程序擴(kuò)增溫度95℃95℃60℃72℃60℃4℃反應(yīng)時間15min30s30s15minHold備注45cycles將SAP酶和SAP緩沖液平衡至室溫，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)表14SAP反應(yīng)體系試劑名稱反應(yīng)孔/μL無酶水SAP緩沖液（10×)0.17SAP酶（1.7U/μL）0.3總體積2.0將SAP反應(yīng)管板放置于PCR儀中，運(yùn)行表15表15SAP反應(yīng)程序擴(kuò)增溫度反應(yīng)時間37℃40min5min4℃Hold將MPE酶和緩沖液、ddNTP混合液和MPE引物平例在適合的離心管中配制MPE反應(yīng)混合液，混勻后按4μL每SAP反應(yīng)孔分裝。上述操作須表16MPE反應(yīng)體系試劑名稱反應(yīng)孔/μLddNTP混合液MPE酶緩沖液MPE酶（1.5U/μL）0.6MPE引物（2~5μM）總體積4.0將MPE反應(yīng)管板放置于PCR儀中，運(yùn)行表1表17MPE反應(yīng)程序擴(kuò)增溫度94℃94℃58℃72℃4℃反應(yīng)時間30sHold備注40cycles5cycles將PCR試劑取出，在冰上融解，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)目，），配制好的PCR反應(yīng)體系渦旋振蕩混勻，瞬時離心，按3μL/孔的量加入PPCR反應(yīng)管底部。每次反應(yīng)選擇兩個孔分別加入2μL陰表18PCR反應(yīng)體系試劑名稱反應(yīng)孔/μL成分及反應(yīng)液濃度PCR預(yù)混液2.0混合液（0.1μM）無酶水補(bǔ)充體系/PCR引物DNA模板2總體積5.0/將PCR反應(yīng)管放置于PCR儀中，運(yùn)行表擴(kuò)增溫度95℃95℃65℃72℃60℃4℃表19PCR反應(yīng)程序反應(yīng)時間15min30s30s15minHold備注45cycles將SAP酶和SAP緩沖液平衡至室溫，振蕩混勻（3~5）s，瞬時離心。根據(jù)檢測樣本數(shù)），表20SAP反應(yīng)體系試劑名稱反應(yīng)孔/μL無酶水SAP緩沖液（10×)0.17SAP酶（1.7U/μL）0.3總體積2.0將SAP反應(yīng)管板放置于PCR儀中，運(yùn)行表21表21SAP反應(yīng)程序擴(kuò)增溫度反應(yīng)時間37℃40min5min4℃Hold將MPE酶和緩沖液、ddNTP混合液和MPE引物平例在適合的離心管中配制MPE反應(yīng)混合液，混勻后按4μL每SAP反應(yīng)孔分裝。上述操作須表22MPE反應(yīng)體系試劑名稱反應(yīng)孔/μLddNTP混合液MPE酶緩沖液MPE酶（1.5U/μL）0.6MPE引物（2~5μM）總體積4.0將MPE反應(yīng)管板放置于PCR儀中，運(yùn)行表2表23MPE反應(yīng)程序擴(kuò)增溫度95℃95℃52℃72℃4℃反應(yīng)時間30sHold備注40cycles5cycles將校準(zhǔn)品和無核酸酶水按照體積比1:13的比例，配制校準(zhǔn)品混合液。在每個反應(yīng)孔中樣品的每個孔對齊。然后輕敲樹脂管，使樹脂落入樣本樹脂的樣品板放置在翻轉(zhuǎn)混勻儀中，以20rpm的速率混勻30min。混勻結(jié)束后瞬時離心，將純化后的產(chǎn)物與基質(zhì)混合，取適量體積點(diǎn)在靶板上（通過質(zhì)譜軟件，分析基因位點(diǎn)的形態(tài)。應(yīng)能夠給出檢測樣本中SNP位點(diǎn)的定性分析結(jié)表24耳聾判定結(jié)果表突變所在基因突變所在位點(diǎn)描述野生(陰性）突變（陽性）雜合GJB2c.35delGGDELG/DELc.257C>GCGC/G突變所在基因突變所在位點(diǎn)描述野生(陰性）突變（陽性）雜合c.427C>TCTC/Tc.167delTTDELT/DELc.176_191del16ACACGTTCTTGCAGCCDELACACGTTCTTGCAGCC/DELc.235delCCDELC/DELc.299_300delATATDELAT/DELc.512insAACGDELAACGDEL/AACGSLC26A4c.281C>TCTC/Tc.589G>AGAG/Ac.1174A>TATA/Tc.1226G>AGAG/Ac.1229C>TCTC/Tc.1975G>CGCG/Cc.2027T>ATAT/Ac.2162C>TCTC/Tc.2168A>GAGA/GIVS7-2A>GAGA/GIVS15+5G>AGAG/A12srRNAm.1494C>TCTC/Tm.1555A>GAGA/GGJB3c.538C>TCTC/Tc.547G>AGAG/AUSH2A2314delGGDELG/DEL表25地中海貧血判定結(jié)果表突變所在基因突變所在位點(diǎn)描述野生(陰性）突變（陽性）雜合α-珠蛋白基因c.369C>GCGC/Gc.427T>CTCT/Cc.377T>CTCT/Cβ-珠蛋白基因c.45dupGGdupGG/dupGc.52A>TATA/Tc.85dupCCdupCC/dupCc.94delCCDELC/DELc.126_129delCTTTCTTTDELCTTT/DELc.130G>TGTG/Tc.217dupAAdupAA/dupAc.2T>GTGT/Gc.-82C>ACAC/Ac.-80T>CTCT/Cc.-79A>GAGA/G突變所在基因突變所在位點(diǎn)描述野生(陰性）突變（陽性）雜合c.-78A>GAGA/Gc.-50A>CACA/Cc.92+1G>TGTG/Tc.316-197C>TCTC/Tc.92+5G>CGCG/Cc.79G>AGAG/A表25心血管指導(dǎo)用藥判定結(jié)果表突變所在基因突變所在位點(diǎn)描述野生(陰性）突變（陽性）雜合ADRB1rs1801253,G>CGCG/CAGTR1rs5186,A>CACA/CALDH2rs671,G>AGAG/AAPOErs7412,C>TCTC/Trs429358,T>CTCT/CCYP2C19rs12248560,C>TCTC/Trs4244285,G>AGAG/Ars4986893,G>AGAG/ACYP2C9rs1799853,C>TCTC/Trs1057910,A>CACA/CCYP2D6rs1065852,G>AGAG/ASLCO1B1rs4149056,T>CTCT/Crs11045852,A>GAGA/GVKORC1rs9923231,C>TCTC/Trs9934438,G>AGAG/A實(shí)驗(yàn)方法：采用梯度稀釋法對檢測限樣本進(jìn)行驗(yàn)證，將陰陽質(zhì)控品、樣品稀釋至10判定標(biāo)準(zhǔn)：在10ng/μL、5ng/μL的濃度下，所有樣本的所有位點(diǎn)均應(yīng)被均應(yīng)被檢出，且檢出的分型結(jié)果與理論位點(diǎn)及分型表26耳聾陽性質(zhì)控品(10ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCCTTTTTTTTTT6CCCCCCCCCCc.299_300delAT4CCCCCCCCCCGGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGTTTTTTTTTTCCCCCCCCCCAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGUSH2AGGGGGGGGGG表27耳聾陽性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCCTTTTTTTTTT6123456789CCCCCCCCCCc.299_300delATc.512insAACG4CCCCCCCCCCGGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGTTTTTTTTTTCCCCCCCCCCAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGUSH2AGGGGGGGGGG表28耳聾陽性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCCTTTTTTTTTT6CCCCCCCCCCc.299_300delATc.512insAACG4CCCCCCCCCCGGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCC123456789GGGGGGGGGGTTTTTTTTTTCCCCCCCCCCAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGUSH2AGGGGGGGGGG表29耳聾陰性質(zhì)控品(10ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGTTTTTTTTTTc.176_191del16c.235delCc.299_300delATc.512insAACG4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2A表30耳聾陰性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2A表31耳聾陰性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGTTTTTTTTTT123456789c.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2A表32地中海貧血陽性質(zhì)控品(10ng/μL)SNP分析結(jié)果地中海貧血陽性質(zhì)控品(10ng/μL)123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCC地中海貧血陽性質(zhì)控品(10ng/μL)123456789GGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTCCCCCCCCCCAAAAAAAAAA表33地中海貧血陽性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTCCCCCCCCCCAAAAAAAAAA表34地中海貧血陽性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果地中海貧血陽性質(zhì)控品(2.5ng/μ123456789GGGGGGGGGGCCCCCCCCCCCCCCCCCCCC地中海貧血陽性質(zhì)控品(2.5ng/μ123456789TTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTCCCCCCCCCCAAAAAAAAAA表35地中海貧血陰性質(zhì)控品(10ng/μL)SNP分析結(jié)果地中海貧血陰性質(zhì)控品(10ng/μL)123456789CCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAA地中海貧血陰性質(zhì)控品(10ng/μL)123456789AAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGG表36地中海貧血陰性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789CCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGG表37地中海貧血陰性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果地中海貧血陰性質(zhì)控品(2.5ng/μ123456789CCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAA地中海貧血陰性質(zhì)控品(2.5ng/μ123456789CCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGG表38心血管指導(dǎo)用藥陽性質(zhì)控品(10ng/μL)SNP分析結(jié)果123456789CCCCCCCCCCCCCCCCCCCCAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGTTTTTTTTTTAAAAAAAAAA表39心血管指導(dǎo)用藥陽性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789123456789CCCCCCCCCCCCCCCCCCCCAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGTTTTTTTTTTAAAAAAAAAA表40心血管指導(dǎo)用藥陽性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果123456789CCCCCCCCCCCCCCCCCCCCAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAACCCCCCCCCCGGGGGGGGGGTTTTTTTTTTAAAAAAAAAA表41心血管指導(dǎo)用藥陰性質(zhì)控品(10ng/μL)SNP分析結(jié)果123456789123456789GGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCAAAAAAAAAAGGGGGGGGGGTTTTTTTTTTAAAAAAAAAACCCCCCCCCCGGGGGGGGGG表42心血管指導(dǎo)用藥陰性質(zhì)控品(5ng/μL)SNP分析結(jié)果123456789GGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCAAAAAAAAAAGGGGGGGGGGTTTTTTTTTTAAAAAAAAAACCCCCCCCCCGGGGGGGGGG表43心血管指導(dǎo)用藥陰性質(zhì)控品(2.5ng/μL)SNP分析結(jié)果123456789123456789GGGGGGGGGGAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCAAAAAAAAAAGGGGGGGGGGTTTTTTTTTTAAAAAAAAAACCCCCCCCCCGGGGGGGGGG判定標(biāo)準(zhǔn)：陰陽質(zhì)控品的所有樣本的所有位點(diǎn)均從表44~52可以看出，陰陽質(zhì)控品的所有樣本的所有位點(diǎn)均應(yīng)被檢出，且檢出的分型結(jié)果與理論位點(diǎn)及分型信息完全一致，樣品的7次測定結(jié)果一致，表明重復(fù)性與方法準(zhǔn)確表44耳聾陽性質(zhì)控品SNP分析結(jié)果述耳聾陽性質(zhì)控品SNP分析結(jié)果1234567GGGGGGGCCCCCCCCCCCCCCTTTTTTTCCCCCCC述耳聾陽性質(zhì)控品SNP分析結(jié)果1234567CCCCCCCGGGGGGGAAAAAAAGGGGGGGCCCCCCCGGGGGGGTTTTTTTCCCCCCCAAAAAAAAAAAAAAGGGGGGGCCCCCCCAAAAAAACCCCCCCGGGGGGGUSH2AGGGGGGG表45耳聾陰性質(zhì)控品SNP分析結(jié)果述耳聾陰性質(zhì)控品SNP分析結(jié)果1234567GGGGGGGTTTTTTTTTTTTTTAAAAAAATTTTTTTAAAAAAATTTTTTTCCCCCCCAAAAAAATTTTTTTGGGGGGGGGGGGGG述耳聾陰性質(zhì)控品SNP分析結(jié)果1234567AAAAAAATTTTTTTGGGGGGGTTTTTTTAAAAAAAUSH2A表46樣品耳聾SNP分析結(jié)果述樣品SNP分析結(jié)果1234567GGGGGGGTTTTTTTTTTTTTTAAAAAAATTTTTTTAAAAAAATTTTTTTCCCCCCCAAAAAAATTTTTTTGGGGGGGGGGGGGGAAAAAAATTTTTTTGGGGGGGTTTTTTTAAAAAAAUSH2A表47地中海貧血陽性質(zhì)控品SNP分析結(jié)果12345671234567GGGGGGGCCCCCCCCCCCCCCTTTTTTTTTTTTTTGGGGGGGAAAAAAACCCCCCCGGGGGGGGGGGGGGCCCCCCCTTTTTTTTTTTTTTCCCCCCCAAAAAAA表48地中海貧血陰性質(zhì)控品SNP分析結(jié)果CCCCCCCTTTTTTTTTTTTTTGGGGGGGAAAAAAACCCCCCCCCCCCCCGGGGGGGAAAAAAATTTTTTTCCCCCCCTTTTTTTAAAAAAAAAAAAAACCCCCCCAAAAAAAGGGGGGGCCCCCCCGGGGGGGGGGGGGGCCCCCCC表49地中海貧血樣品SNP分析結(jié)果CCCCCCCTTTTTTTTTTTTTTGGGGGGGAAAAAAACCCCCCCCCCCCCCGGGGGGGAAAAAAATTTTTTTCCCCCCCTTTTTTTAAAAAAAAAAAAAAAAAAAAAGGGGGGGCCCCCCCGGGGGGGGGGGGGGCCCCCCC表50心血管指導(dǎo)用藥陽性質(zhì)控品SNP分析結(jié)果1234567CCCCCCCCCCCCCCAAAAAAATTTTTTT1234567CCCCCCCTTTTTTT9AAAAAAAAAAAAAATTTTTTTCCCCCCCAAAAAAACCCCCCC1GGGGGGGTTTTTTTAAAAAAA表51心血管指導(dǎo)用藥陰性質(zhì)控品SNP分析結(jié)果1234567CCCCCCCCCCCCCCAAAAAAATTTTTTTCCCCCCCTTTTTTTAAAAAAAAAAAAAATTTTTTTCCCCCCCAAAAAAACCCCCCCGGGGGGGCCCCCCCCCCCCCC表52心血管指導(dǎo)用藥樣品SNP分析結(jié)果1234567G,AG,AG,AG,AG,AG,AG,ACCCCCCCAAAAAAAGGGGGGG1234567CCCCCCCTTTTTTTCCCCCCCGGGGGGGGGGGGGGCCCCCCCAAAAAAAG,AG,AG,AG,AG,AG,AG,ATTTTTTTAAAAAAAC,TC,TC,TC,TC,TC,TC,T收集了50管心血管疾病病人的全血，采用天根生化科技（北京）有限公司（磁珠法通用型基因組DNA提取試劑盒,貨號：DP705）。按試劑盒中提供的操作說明書提取樣本表53耳聾50個樣本SNP分析結(jié)果123456789GGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2AGGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2AGGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2AGGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2AGGGGGGGGGGTTTTTTTTTTc.299_300delAT4TTTTTTTTTTAAAAAAAAAATTTTTTTTTTAAAAAAAAAATTTTTTTTTTCCCCCCCCCCAAAAAAAAAATTTTTTTTTTGGGGGGGGGGGGGGGGGGGGAAAAAAAAAATTTTTTTTTTGGGGGGGGGGTTTTTTTTTTAAAAAAAAAAUSH2A表54地中海貧血50個樣本SNP分析結(jié)果123456789CCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGAAAAAAAAAATTTTTTTTTTCCCCCCCCCCTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGAAAAAAAAAACCCCCCCCCCC