CN117660642A - 外周血dna中辅助检测肺结节良恶性的甲基化生物标记物组合及其应用 - Google Patents
外周血dna中辅助检测肺结节良恶性的甲基化生物标记物组合及其应用 Download PDFInfo
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Abstract
本发明提供了外周血DNA中辅助检测肺结节良恶性的甲基化生物标记物的组合及其应用，所述甲基化生物标志物组合包括以下标记物：chr11:133938689‑133939345,chr19:10691814‑10692015,chr11:133938595‑133938877,chr5:175299506‑175299707,chr17:46655168‑46655474,chr7:142494474‑142495178,chr20:47444770‑47445043,chr7:87230068‑87230269,chr7:87230006‑87230207,chr8:72756691‑72756892,chr17:46655603‑46655876等。本发明发现最优的11‑marker、92‑marker组合以及作为备选的13‑marker、57‑marker、75‑marker组合等组合，其能有效地辅助检测肺结节良恶性，从而为肺癌患者制定合理诊疗方案等方面提供更为有效地辅助检测服务，还可以更全面分析肺癌发生、发展中的甲基化变化，应用于肺癌早期筛查、辅助诊断、疗效评估和复发监测等阶段。
Description
技术领域
本发明涉及生物技术领域，特别是涉及外周血DNA辅助检测肺结节良恶性的甲基化生物标记物组合及其应用。
背景技术
癌症是人类死亡主要原因之一，在2018年，全球癌症新发高达1810万人，而死亡高达960万人[Luo,H.,Wei,W.,Ye,Z.,Zheng,J.,&Xu,R.H..(2021).Liquid biopsy ofmethylation biomarkers in cell-free dna.Trends in Molecular Medicine.]。其中，肺癌是一种多步骤、多因素的疾病，有多种组织学亚型，是世界上最致命的癌症，据估计，2018年全球发生209万例肺癌新发病例，在所有癌症类型中排名第一。在中国，肺癌在男性和女性中的发病率近年来迅速上升，对人类健康构成了极大的威胁[Wanqing,Chen,Rongshou,Zheng,Hongmei,&Zeng,et al.(2015).Epidemiology of lung cancer inchina.Thoracic Cancer.]。
肿瘤防治中的早发现和早干预是至关重要的步骤。相比于欧美发达国家，我国大量病患的肿瘤发现即晚期，一个重要的因素是肿瘤早期检测普及力度不够，尤其对于肺癌，早发现和早干预的意义极为重要，来自《中国肺癌筛查与早诊早治指南(2021，北京)》的数据显示，肺癌患者生存时间与其临床诊断发现早晚密切相关，5年生存率随诊断分期升高而迅速降低，在Ⅰ期5年生存率为55.5％，而在Ⅳ期仅为5.3％，二者相差10.5倍[赫捷,李霓,陈万青,吴宁,沈洪兵,&江宇等.(2021).中国肺癌筛查与早诊早治指南(2021,北京).中华肿瘤杂志,43(3),26.]。
尽管早发现早治疗是改善癌症患者预后的最有效方法之一，但肿瘤早筛本身仍然面临诸多挑战。目前，临床上的肿瘤筛查方法(影像与肿标等)灵敏度有限且依从性较低，且大多数癌症缺乏针对性的非侵入性的癌症早筛工具。受此影响，不少科学家和临床医生注意到了液体活检的潜力，将其作为一种新的无创或微创筛查手段，用于不同癌症的早筛。液体活检是指通过微创或无创的方式收集血液或其他体液(包括尿液、腹水、胸腔积液等)，并对其中肿瘤相关的物质进行分析的一种检验方式，也是一种非常强大的癌症实时随访临床工具，它克服了组织活检的诸多局限性[Pantel,K.,&C Alix-Panabières.(2010).Circulating tumour cells in cancer patients:challenges andperspectives.Trends in Molecular Medicine,16(9),398-406.]。其中基于血浆的ctDNA甲基化检测具有很强的癌症早筛潜力，因为它可以在癌症发生的早期检测到。
DNA是遗传信息的主要载体，当细胞经历癌变过程时，DNA上会发生一系列的变异，既可以是染色体异常，又可以是基因变异，还可以是DNA上的一些化学修饰(如DNA甲基化)。因此肿瘤细胞的DNA与正常细胞的DNA存在诸多差异。细胞游离DNA(cell-free DNA，cfDNA)是在细胞凋亡、坏死等过程释放到血液中的核酸混合物，其组分中包含循环肿瘤DNA(circulating tumor DNA，ctDNA)，近年来被广泛应用于肿瘤诊疗、无创产前诊断、免疫系统疾病诊疗等领域[Newman,A.,Bratman,S.,To,J.et al.An ultrasensitive method forquantitating circulating tumor DNA with broad patient coverage.Nat Med 20,548–554(2014).]
DNA甲基化是一种重要的表观遗传修饰，研究发现其对基因表达、基因组稳定性和肿瘤发生发展具有重要的作用。其中异常的DNA甲基化被认为是癌症发展的标志，借助基因转录的抑制或失活引起染色质的不稳定[Cheng Y,He C,Wang M,et al.Targetingepigenetic regulators for cancer therapy:mechanisms and advances in clinicaltrials.Signal Transduct Target Ther.2019；4:62.Published 2019Dec 17.doi:10.1038/s41392-019-0095-0]。启动子区域的高甲基化和整体DNA的低甲基化在癌症中相当普遍，且早期癌症中也存在不少甲基化变化，因此和甲基化相关的信号通常会被当作早期癌症追踪的首选。[Ma Y,Bai Y,Mao H,et al.A panel of promoter methylationmarkers for invasive and noninvasive early detection of NSCLC using a quantumdots-based FRET approach.Biosens Bioelectron.2016；85:641-648.doi:10.1016/j.bios.2016.05.067；Chen HF,Lei L,Wu LX,et al.Effect of icotinib on advancedlung adenocarcinoma patients with sensitive EGFR mutation detected in ctDNAby ddPCR.Transl Cancer Res.2019；8(8):2858-2863.doi:10.21037/tcr.2019.10.48；Liang W,Zhao Y,Huang W,et al.Non-invasive diagnosis of early-stage lungcancer using high-throughput targeted DNA methylation sequencing ofcirculating tumor DNA(ctDNA).Theranostics.2019；9(7):2056-2070.Published2019Apr 6.doi:10.7150/thno.28119；Sun K.Clonal hematopoiesis:background player in plasma cell-free DNA variants.Ann Transl Med.2019；7(Suppl 8):S384.doi:10.21037/atm.2019.12.97]。
若只看单个位点的甲基化信号变化，甲基化与碱基突变相比并没有优势，然而甲基化位点的数量远远超过点突变，且基因组上距离较近的CpG位点会同时被甲基化或是去甲基化，这就意味着可以将一簇CpG位点作为整体进行分析，通过横向模式和纵向丰度的多种组合，得到信号更强的甲基化小单元。在此基础上，还可将多个CpG位点簇相结合形成肿瘤特异的甲基化单倍体模式，这种联合判定方法能够进一步提升甲基化指纹的准确性[Guo,S.,Diep,D.,Plongthongkum,N.et al.Identification of methylation haplotypeblocks aids in deconvolution of heterogeneous tissue samples and tumortissue-of-origin mapping from plasma DNA.Nat Genet 49,635–642(2017).]。
综上所述，ctDNA甲基化可在肿瘤发生的早期被检测到，具备较好的稳定性，且具有简单、经济和组织特异性的特征，可用于肿瘤早期诊断、预后评估和疗效监测等，具有很高的临床应用价值，是肿瘤液体活检中一种极具价值的标志物。本发明旨在筛选通过外周血DNA辅助判定肺结节良恶性的甲基化生物标记物。
发明内容
基于此，本发明的目的之一在于提出一种用于通过外周血DNA辅助检测肺结节良恶性的甲基化生物标记物组合。
实现上述目的的技术方案包括如下。
本发明的第一方面，是提供一种用于通过外周血DNA辅助检测肺结节良恶性的甲基化生物标记物组合，所述甲基化生物标志物组合包括以下标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876；优选地，所述甲基化生物标志物组合还包括选自以下至少一种标记物：chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
在其中一些实施例中，所述甲基化生物标志物组合包括上述11种标记物，还包括表1中其他任意至少一种标记物。
在其中一些实施例中，所述甲基化生物标志物组合包括以下92种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
在其中一些实施例中，所述甲基化生物标志物组合包括上述92种标记物，还包括表1中其他任意至少一种标记物。
在其中一些实施例中，所述甲基化生物标志物组合包括以下13种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000。
在其中一些实施例中，所述甲基化生物标志物组合包括上述13种标记物，还包括表1中其他任意至少一种标记物；所述甲基化生物标志物组合还包括选自以下至少一种标记物chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
在其中一些实施例中，所述甲基化生物标志物组合包括以下57种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347。
在其中一些实施例中，所述甲基化生物标志物组合包括上述57种标记物，还包括表1中其他任意至少一种标记物；优选地，还包括选自以下至少一种标记物chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
在其中一些实施例中，所述甲基化生物标志物组合包括以下75种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901。
在其中一些实施例中，所述甲基化生物标志物组合包括上述75种标记物，还包括表1中其他任意至少一种标记物；优选地，所述甲基化生物标志物组合还包括选自以下至少一种标记物：chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
本发明的第二方面，还在于提出检测上述甲基化生物标志物组合或其检测试剂在制备预测、检测、分类、治疗监测、预后或其它评价肺结节良恶性的试剂盒中的应用。
本发明的第三方面，还在于还在于提出一种肺结节良恶性辅助检测试剂盒。
实现上述目的的技术方案如下：
一种肺结节良恶性辅助检测试剂盒，其包括检测上述甲基化生物标志物或其组合的甲基化差异程度的试剂。
在其中一些实施例中，上述试剂盒是采用聚合酶链式反应技术、原位杂交技术、酶学突变检测技术、化学剪切错配技术、质谱分析技术、基因芯片技术或基因测序技术或它们的组合制备而成。
在其中一些实施例中，上述试剂盒采用的检测技术中聚合酶链式反应技术(PCR)包括但不限于RT-PCR、免疫PCR、巢式PCR、荧光定量PCR、原位PCR、膜结合PCR、锚定PCR、固着PCR、原位PCR、不对称PCR、长距离PCR、降落伞PCR、梯度PCR、数字PCR等；高通量检测技术包括但不限于简化基因组甲基化测序，全基因组甲基化测序，DNA富集测序，焦磷酸盐测序，亚硫酸盐转化测序；基于质谱的GC-MS、LC-MS、MALDI-TOFMS、FT-MS、ICP-MS、SIMS的检测技术；基于芯片检测平台，如450K和850K甲基化检测技术。
本发明的目的之一还在于提出上述试剂盒在预测、检测、分类、治疗监测、预后或其它评价肺结节良恶性中的应用。
本发明的目的之一还在于提出一种辅助检测肺结节良恶性的方法。
实现上述目的的技术方案如下：
一种辅助检测肺结节良恶性的方法，其包括以下步骤，
提取待测的生物样品基因组DNA；
对所述DNA进行亚硫酸氢盐转化；
对上述甲基化生物标志物或其组合的甲基化差异程度的检测。
在其中一些实施例中，上述方法包括但不限于以下技术：甲基化特异性PCR，亚硫酸盐PCR测序，实时定量甲基化特异性PCR等；高通量检测技术包括简化基因组甲基化测序，全基因组甲基化测序，DNA富集测序，焦磷酸盐测序，亚硫酸盐转化测序等；基于质谱等检测平台的检测技术；基于芯片检测平台，如450K和850K甲基化检测技术。
在其中一些实施例中，上述生物样品为外周血(血浆)、组织切片、唾液、胸腔积液、腹水、羊水、骨髓或培养的动物细胞。
本发明的在研究中发现包括最优的11-marker、92-marker组合以及作为备选的13-marker、57-marker、75-marker组合等，并采用随机森林等方式在肿瘤组织和血浆样本中建立预测模型，发现其能有效地辅助检测肺结节良恶性，同时还能克服单个DNA甲基化信号低的问题，并提高检测的灵敏度和特异性，从而为肺癌患者制定合理诊疗方案等方面提供更为有效地辅助检测服务。并且，基于对样本中这些DNA甲基化标记物的甲基化状态进行检测，还可以更全面分析肺癌发生、发展中的甲基化变化，应用于肺癌早期筛查、辅助诊断、疗效评估和复发监测等阶段，为临床提供更准确灵敏的检测服务和相关学术研究。
附图说明
图1为实施例中挑选组织模型重要性排序位于前1-前2000的特征，再次在组织样本上建立随机森林模型时，测试集AUC均值的变化趋势。
图2为实施例中在组织样本上使用未筛选的全部7282个甲基化生物标志物绘制的热图，每一列代表一个样本，每一行代表一个甲基化生物标志物。
图3为实施例中在所有血浆样本(其被分为训练、测试、验证集)上使用未筛选的全部7282个甲基化生物标志物绘制的热图，每一列代表一个样本，每一行代表一个甲基化生物标志物。
图4为实施例中挑选组织模型重要性排序位于前1-前500的特征，在血浆样本的训练集上建立随机森林模型时，血浆样本测试集AUC的变化趋势。
图5为实施例中挑选组织模型重要性排序位于前11的特征，再次在血浆样本的训练集上建立随机森林模型时，血浆样本测试集和血浆样本验证集结果对应的ROC图。
图6为实施例中挑选组织模型重要性排序位于前13的特征，再次在血浆样本的训练集上建立随机森林模型时，血浆样本测试集和血浆样本验证集结果对应的ROC图。
图7为实施例中挑选组织模型重要性排序位于前57的特征，再次在血浆样本的训练集上建立随机森林模型时，血浆样本测试集和血浆样本验证集结果对应的ROC图。
图8为实施例中挑选组织模型重要性排序位于前75的特征，再次在血浆样本的训练集上建立随机森林模型时，血浆样本测试集和血浆样本验证集结果对应的ROC图。
图9为实施例中挑选组织模型重要性排序位于前92的特征，再次在血浆样本的训练集上建立随机森林模型时，血浆样本测试集和血浆样本验证集结果对应的ROC图。
具体实施方式
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
在整个说明书和权利要求书中，以下术语具有与本文明确相关的含义，除非上下文另有明确规定。在本发明中使用的短语“在一个实施方案中”不一定指代相同的实施方案，尽管其可能是。此外，在本发明中使用的短语“在另一实施方案中”不一定指代不同的实施方案，尽管其可能是。因此，如下所述，可以容易地组合本发明的各个实施方案，而不脱离本发明的范围或精神。
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
以下结合具体实施例对本发明作进一步详细的说明。
实施例1
本实施例公开了本发明中的11-marker组合(包含区域chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876)、13-marker组合(包含区域chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000)、57-marker组合(包含区域chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,
chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,
chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,
chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,
chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,
chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,
chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,
chr1:166135146-166135347)、75-marker组合(包含区域chr11:133938689-133939345,
chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,
chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,
chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,
chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,
chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,
chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,
chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,
chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,
chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,
chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,
chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,
chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,
chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,
chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,
chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,
chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,
chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,
chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,
chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,
chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,
chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,
chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,
chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,
chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,
chr1:181454885-181455261,chr14:60952518-60952901)、92-marker组合(包含区域
chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,
chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,
chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,
chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,
chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,
chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,
chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,
chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,
chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,
chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,
chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,
chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,
chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,
chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,
chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,
chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,
chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,
chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,
chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,
chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,
chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,
chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,
chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,
chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,
chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,
chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,
chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,
chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,
chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,
chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026)等，均可用于辅助检测肺结节良恶性的检测方法，具体包括如下步骤：
一、样本信息
1、426例包含肺结节的组织样本队列被用于筛选和鉴定辅助判定肺结节良恶性的甲基化生物标记物。上述样本包含132例恶性样本，294例良性样本，其中包括占主导的恶性亚型为78例IA、24例MIA，占主导的良性亚型为50例TB、37例INF、35例FUN、32例GRAN、26例HAM。
2、655例包含肺结节的血浆样本队列被用于筛选和鉴定辅助判定肺结节良恶性的甲基化生物标记物。该血浆样本队列被大致平均分为三份，分别对应后分析中的训练集(train)、测试集(test)、验证集(validation)等，并且使每一份数据集中不同病理信息特征的样本数目尽量保持平衡。上述样本总共包含441例恶性样本(训练集147，测试集147，验证集147)，214例良性样本(训练集71，测试集72，验证集71)，其中包括占主导的恶性亚型为315例IA(训练集105，测试集110，验证集100)、49例MIA(训练集17，测试集13，验证集19)、44例AIS(训练集18，测试集15，验证集11)、20例SC(训练集5，测试集5，验证集10)，占主导的良性亚型为72例GRAN(训练集14，测试集31，验证集27)、66例INF(训练集20，测试集24，验证集22)、14例HAM(训练集7，测试集4，验证集3)、10例TB(训练集5，测试集1，验证集4)；总共包含335例女性(训练集108，测试集122，验证集105)，320例男性(训练集110，测试集97，验证集113)；年龄小于70岁的604例(训练集202，测试集201，验证集201)，大于或等于70岁的51例(训练集16，测试集18，验证集17)；恶性样本主要以癌症早期中的I期占主导，该分期总共有341例样本(训练集109，测试集116，验证集116)；吸烟情况为183例一直/曾经吸烟(训练集64，测试集59，验证集60)，472例不吸烟(训练集154，测试集160，验证集158)。
3、借助公司内部过往项目积累的甲基化数据库，参考TCGA公开数据库、公共文献报道等，再经由一系列实验和生物信息质控等步骤进行筛选，最终得到7282个甲基化生物标记物，作为本专利中各项测试的初始筛选对象。
4、将上述有恶性肺结节的病人的血浆和组织样本进行高通量靶向甲基化测序，包括但不限于分离组织基因组DNA或血浆游离DNA(cfDNA)，亚硫酸氢盐转化，以及AnchorIRISTM靶向甲基化测序等步骤。
二、建库流程和方法
根据现有的方法，进行建库，具体包括以下步骤：
1、样本DNA提取及甲基化建库
1.1、样本DNA的提取
对于肺癌外周血样本，DNA的提取步骤按照QIAGEN公司的DNeasy Blood&TissueKit操作说明进行。对于肺癌组织样本，DNA的提取步骤按照QIAGEN公司的DNeasy Blood&Tissue Kit操作说明进行。
1.2、转化
将提取的样本DNA(50ng)进行亚硫酸氢盐转化，使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶，而甲基化的胞嘧啶保持不变，得到亚硫酸氢盐转化后的DNA，转化具体操作按照Zymo Research的EZ DNA Methylation-Lightning Kit说明书进行。
1.3、末端修复
以下步骤1.3至步骤1.9使用AnchorDx IRIS甲基化建库试剂盒。
将上述转化后的17ul样本加入以下试剂进行反应：
| 组分 | 体积(μl) |
| 转化后样本 | 17 |
| MEB1缓冲液 | 2 |
| MEE2酶 | 1 |
| 总体积 | 20 |
置于PCR仪中按照以下程序进行反应：
| 37℃ | 30min |
| 95℃ | 5min |
| 热盖 | 105℃ |
当PCR反应第二步(95℃)达到5min时，立即将样本从PCR仪中取出，直接插入冰中，放置2min以上再进行下一步操作。
1.4、连接I
配制如下反应液：
| 组分 | 单个用量(μl) |
| 上一步反应产物 | 20 |
| H2O | 4 |
| MLB1缓冲液 | 8 |
| MLR1试剂 | 2 |
| MLR5试剂 | 2 |
| MLE1酶 | 2 |
| MLE5酶 | 2 |
| 反应混合体积 | 40 |
置于PCR仪中按照以下程序进行反应：
1.5、扩增I
配制如下反应液
| 组分 | 单个用量(μl) |
| 上一步反应产物 | 40 |
| H2O | 35 |
| MAB2缓冲液 | 20 |
| MAR1试剂 | 2 |
| MAR2试剂 | 2 |
| MAE3酶 | 1 |
| 反应混合体积 | 40 |
置于PCR仪中按照以下程序进行反应：
1.6、纯化I：
加入166ul 1:6倍稀释的Agencourt AMPure Beads(需提前于室温平衡半小时)对扩增I反应后的产物进行纯化，用21μl EB进行洗脱，纯化具体步骤如下：
取上一步反应产物并进行离心，每个样本加入166μl 1:6倍稀释的Agencourt AMPure Beads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μl 80％ EtOH，静置30s，吸走乙醇，重复一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇，开盖干燥磁珠2-3min，注意不要过干。加入21μl EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取20μl上清于新的PCR管中。
1.7、连接II
配制如下反应液：
置于PCR仪中按照以下程序进行反应
| 温度 | 时间 | 循环数 |
| 37℃ | 30min | 1 |
| 95℃ | 5min | 1 |
| 10℃ | Hold | 1 |
1.8、Indexing PCR(扩增产物文库构建)
配制如下反应液：
| 组分 | 体积(μl) |
| 上一步反应体积 | 40 |
| H2O | 6 |
| 2X KAPA HiFi Hot Start Ready Mix | 8 |
| I5接头引物 | 2 |
| I7接头引物 | 2 |
| 总体积 | 100 |
置于PCR仪中按照以下程序进行反应
1.9、纯化II
加入Agencourt AM Pure Beads(需提前于室温平衡半小时)对Indexing PCR反应后的产物进行纯化，用41μl EB进行洗脱，纯化具体步骤如下：
取上一步反应产物并进行离心，每个样本加入71μl未稀释的Agencourt AM PureBeads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μl80％ EtOH，静置30s，吸走乙醇，重复步骤一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇。开盖干燥磁珠2-3min，注意不要过干。加入41μl EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取20μl上清于新的PCR管中。Qubit定量：取1μl用Qubit dsDNAHS Assay Kit对文库进行定量。
2、通过对已建库后的样本进行寡核苷酸探针捕获富集，得到特定区域的上机终文库。杂交捕获试剂盒为IDT公司的xGen Lockdown Reagents，具体按照说明书进行操作。
3、采用Illumina公司的测序仪对杂交捕获后的样本进行测序，得到测序结果。
4、下机数据的分析
对测序仪的下机原始数据，进行常规的生物信息学分析处理，先通过fastp过滤低质量(QC低，长度短、太多N等)的读长(reads)，然后去除reads双端的adapter、共有序列、PolyA/T，得到理想的插入片段序列(target区间)，使用bismark将这些reads比对hg19对应的位置后，根据UMI对reads进行去重，得到每份样本被探针捕获得到的真实reads数据(bamfile)，对bam文件进行统计和分析，得到甲基化数据，用于后续的数据再分析。
5、对原始测序数据进行了相关清理及处理分析[Liang,W.,et al.,Non-invasivediagnosis of early-stage lung cancer using high-throughput targeted DNAmethylation sequencing of circulating tumor DNA(ctDNA).2019.9(7):p.2056.]，并基于对reads读数来确定每一个区域甲基化胞嘧啶的百分比(β值)。
6、统计分析方法
R统计软件(v3.5.1，贝尔实验室，美国新泽西州默里山)用于所有相关统计检验分析。威尔科克森秩和统计检验(Wilcoxon rank-sum test)被用于检测差异甲基化的生物标记物。在Random Forest R模块中部署实现了随机森林预测模型，并借助pROC模块绘制了ROC曲线。
7、针对这426例包含肺结节的组织样本(含132例恶性样本，294例良性样本)的甲基化检测信息，通过采用随机森林(Random Forest，RF)方法建立多个预测模型尝试对样本良恶性进行分类。统计模型输出重复33次的3折交叉验证的结果，并绘制受试者工作特征曲线(Receiver Operator Characteristic Curve，ROC)和计算曲线下面积(Area UnderCurve，AUC)，以此来评估这些模型的性能，结果如图1和表1所示。其中图1展示了挑选组织模型重要性排序位于前1的特征，并按照该次序逐个增加特征数目直到包含前2000的特征，将上述挑选到的特征子集再次在组织样本上建立随机森林模型时，测试集AUC均值的变化趋势。可以发现这条AUC对挑选特征数目的曲线，在前500特征附近存在一个明显的拐点，这说明当取到前500个特征时，随机森林模型在组织样本中能够获得最优的良恶性分类效果(前1-前7282特征的相关结果均有被计算，此处仅选取前1-前2000进行必要展示，其足以体现相关趋势，前2001-前7282的AUC均值持续单调降低)。表1显示了组织样本随机森林模型重要性在前1-500的特征以及它们在基因组上的相关注释信息(注：Synonyms:hg19)。
表1组织样本随机森林模型重要性在前1-500的特征及其注释信息
在通常情况下，组织中的甲基化信号远高于血浆且更为可靠，借助图2和图3的全体特征分布的热图，也可看出组织样本和血浆样本按照良恶性类别聚类能力的差距。因此，血浆中甲基化生物标志物的筛选通常亦需参考组织样本的数据分析结果，而并不建议仅仅在血浆样本中直接进行差异分析，一方面为了让备选的甲基化生物标志物具备更可靠的生物学意义，以此提升信号的强度，另一方面也减少不必要的干扰噪声，从而减少了假阳性的可能性，最终从正反两方面入手，提高了整体的信噪比。
因此，根据以上思路，利用上述基于组织数据的分析结果及模型特征重要性排序，采用随机森林(Random Forest，RF)方法，在血浆数据的给定训练集(218例包含肺结节的血浆样本(含147例恶性样本，71例良性样本)队列)中建模，并将所建立的模型在血浆数据的给定测试集(219例包含肺结节的血浆样本(含147例恶性样本，72例良性样本)队列)中进行初步评估。图4展示了挑选组织模型重要性排序位于前1的特征，并按照该次序逐个增加特征数目直到包含前500的特征，将上述挑选到的特征子集在血浆数据的训练集上建立随机森林模型后，所得模型在血浆数据的测试集上AUC的变化趋势。可以发现这条血浆数据的测试集AUC对挑选特征数目的曲线，在前11、前13特征附近存在一个明显的拐点，它们在测试集上对应的AUC值分别为(0.682、0.682)；在前57、前75、前92特征附近取到测试集中的性能峰值，它们在测试集上对应的AUC值分别为(0.706、0.705、0.707)。上述评估结果说明，当取到前57、前75、前92个特征时，随机森林模型在血浆样本中能够获得最优的良恶性分类效果(可称为“优化性能的marker”)。然而实际应用场景中可能受到成本、时间等原因限制，则取到前11、前13个特征时，随机森林模型在血浆样本中的良恶性分类效果性价比较高(可称为“优化性价比的marker”)。
实施例2
为进一步评估所挑选得到的特征子集的性能，本实施例纳入实施例1中未经使用的验证集样本进行更为详细地评估。具体方式为，使用实施例1中挑选得到的处于AUC对挑选特征数目的曲线拐点处的前11、前13特征，以及处于AUC对挑选特征数目的曲线峰值点处的前57、前75、前92特征，将它们在血浆数据给定训练集先进行模型建立(218例包含肺结节的血浆样本(含147例恶性样本，71例良性样本)队列)，然后在血浆数据的给定测试集(219例包含肺结节的血浆样本(含147例恶性样本，72例良性样本)队列)和血浆数据的给定验证集(218例包含肺结节的血浆样本(含147例恶性样本，71例良性样本)队列)中进行综合性能评估。检测方法参照实施例1所述。
图5、图6、图7、图8、图9分别对应了选取前11、前13、前57、前75、前92特征所建立的模型，它们各自在测试集和验证集中代表模型性能的ROC曲线。对比同样处于拐点的前11、前13特征的模型性能可知，二者性能接近(前者略优)，但前11的特征数目更少，从简化模型角度出发，选择前11优于前13特征。对比同样处于峰值点的前57、前75、前92特征的模型性能可知，三者在测试集中的性能接近，但前92特征的验证集性能与测试集相差最小，从模型泛化能力角度出发，选择前92优于前57、前75特征。
表2展示了拐点处最优选的前11特征，以及峰值点处最优选的前92特征，它们的综合性能评估情况。表中，前11、前92等，表示的是前面11个、前面92个这些标记物的组合。固定测试集特异性/敏感性，表示尝试选取特定阈值，使得测试集样本中判定的特异性/敏感性在所期望的值附近，此时将该阈值同样运用至验证集中，从而得到验证集在该设定条件下的相关性能。
表2在血浆样本中使用上述组织模型特征重要性排序在前11与前92进行建模的统计结果
综合上述结果表明，以组织样本筛选得到的500-marker组合为基础，通过血浆样本进一步筛选，可得到一组“最优化性价比的marker组合”：11-marker组合(包含区域chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876)。若放宽外部限制条件(成本、时间等)，还可得到一组“最优化性能的marker”：92-marker组合(包含区域chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,
chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,
chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,
chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,
chr17:21355825-21356026)。此外，未被作为最优组合选中的13-marker组合(包含区域
chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,
chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,
chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,
chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,
chr7:87229799-87230000)；57-marker组合(包含区域chr11:133938689-133939345,
chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,
chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,
chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,
chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,
chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,
chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,
chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,
chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,
chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,
chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,
chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,
chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,
chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,
chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,
chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,
chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,
chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,
chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,
chr8:67874085-67874286,chr1:166135146-166135347)；75-marker组合(包含区域
chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,
chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,
chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901)，鉴于它们相对较高的性能，亦可作为备选。这些组合在血浆样本中具有有效的区分能力，可用于辅助检测肺结节良恶性。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (15)
1.外周血DNA中辅助检测肺结节良恶性的甲基化生物标记物的组合，其特征在于，所述甲基化生物标志物组合包括以下11种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876。
2.根据权利要求1所述的组合，其特征在于，所述甲基化生物标志物组合包括权利要求1中所述11种标记物，还包括表1中其他任意至少一种标记物；优选地，所述甲基化生物标志物组合包括权利要求1中所述11种标记物，还包括以下至少一种标记物chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
3.根据权利要求2所述的组合，其特征在于，所述甲基化生物标志物组合包括以下92种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
4.根据权利要求3所述的组合，其特征在于，所述甲基化生物标志物组合包括权利要求3中所述92种标记物，还包括表1中其他任意至少一种标记物。
5.根据权利要求2所述的组合，其特征在于，所述甲基化生物标志物组合包括以下13种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000。
6.根据权利要求5所述的组合，其特征在于，所述甲基化生物标志物组合包括权利要求5中所述13种标记物，还包括表1中其他任意至少一种标记物；优选地，所述甲基化生物标志物组合包括权利要求5中所述13种标记物，还包括以下至少一种标记物chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
7.根据权利要求2所述的组合，其特征在于，所述甲基化生物标志物组合包括以下57种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347。
8.根据权利要求7所述的组合，其特征在于，所述甲基化生物标志物组合包括权利要求7中所述57种标记物，还包括表1中其他任意至少一种标记物；优选地，所述甲基化生物标志物组合包括权利要求7中所述57种标记物，还包括以下至少一种标记物chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901,chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
9.根据权利要求2所述的组合，其特征在于，所述甲基化生物标志物组合包括以下75种标记物：chr11:133938689-133939345,chr19:10691814-10692015,chr11:133938595-133938877,chr5:175299506-175299707,chr17:46655168-46655474,chr7:142494474-142495178,chr20:47444770-47445043,chr7:87230068-87230269,chr7:87230006-87230207,chr8:72756691-72756892,chr17:46655603-46655876,chr8:72755937-72756138,chr7:87229799-87230000,chr2:182322618-182322819,chr6:100050649-100050850,chr19:38183129-38183330,chr10:22764801-22765002,chr8:72756260-72756461,chr1:101702398-101702769,chr17:46824646-46824847,chr19:38183239-38183440,chr2:182322187-182322399,chr2:145277709-145277972,chr5:158527736-158527937,chr2:73518581-73518782,chr7:87229471-87229672,chr17:46655737-46655938,chr17:46655480-46655681,chr12:104852358-104852566,chr8:72755703-72755904,chr14:57274596-57274859,chr14:21121443-21121644,chr19:40724227-40724508,chr1:197888773-197888974,chr4:55093736-55094091,chr14:57275265-57275466,chr2:105478535-105478736,chr21:27012192-27012393,chr1:101702701-101702902,chr2:145281541-145281742,chr12:54321532-54321733,chr14:70654558-70654759,chr6:28303954-28304241,chr19:10691733-10691941,chr7:27205260-27205461,chr1:12123433-12123634,chr19:38182869-38183070,chr1:166134265-166134530,chr10:8079270-8079471,chr10:101287631-101287832,chr20:2781235-2781603,chr13:28363998-28364199,chr6:28303851-28304336,chr7:27135356-27135557,chr12:103352173-103352534,chr8:67874085-67874286,chr1:166135146-166135347,chr20:21082151-21082352,chr1:155830143-155830344,chr6:5996999-5997200,chr20:21081670-21081871,chr7:155248041-155248242,chr8:9763952-9764153,chr5:170735900-170736101,chr1:65731311-65731512,chr12:133481439-133481640,chr1:166134161-166134362,chr7:128430888-128431089,chr7:27135602-27135860,chr1:91189329-91189530,chr1:228651627-228651828,chr1:166134618-166134819,chr1:91176462-91176807,chr1:181454885-181455261,chr14:60952518-60952901。
10.根据权利要求9所述的组合，其特征在于，所述甲基化生物标志物组合包括权利要求9中所述75种标记物，还包括表1中其他任意至少一种标记物；优选地，所述甲基化生物标志物组合包括权利要求9中所述75种标记物，还包括以下至少一种标记物：chr10:101290618-101290819,chr18:2847944-2848145,chr17:77019960-77020161,chr6:28304370-28304571,chr14:36004016-36004217,chr14:29229374-29229575,chr6:6004207-6004408,chr2:223169505-223169706,chr3:172166113-172166314,chr5:169064409-169064610,chr7:158936551-158936859,chr2:145277525-145277726,chr1:47698000-47698201,chr6:150285394-150285754,chr2:175191675-175191876,chr12:3602165-3602366,chr17:21355825-21356026。
11.权利要求1-10任一项所述甲基化生物标志物组合或其检测试剂在制备辅助预测、检测、分类、治疗监测、预后或其它评价肺结节良恶性的试剂盒中的应用。
12.一种辅助检测肺结节良恶性的试剂盒，其特征在于，其包括检测权利要求1-10任一项甲基化生物标志物或其组合的甲基化差异程度的试剂。
13.根据权利要求12所述的试剂盒，其特征在于，所述试剂盒是采用聚合酶链式反应技术、原位杂交技术、酶学突变检测技术、化学剪切错配技术、质谱分析技术、基因芯片技术或基因测序技术或它们的组合的试剂制备而成。
14.根据权利要求13所述的试剂盒，其特征在于，所述聚合酶链式反应技术选自RT-PCR、免疫PCR、巢式PCR、荧光定量PCR、原位PCR、膜结合PCR、锚定PCR、固着PCR、原位PCR、不对称PCR、长距离PCR、降落伞PCR、梯度PCR、数字PCR。
15.根据权利要求12所述的试剂盒，其特征在于，所述基因测序技术选自简化基因组甲基化测序，全基因组甲基化测序，DNA富集测序，焦磷酸盐测序，亚硫酸盐转化测序；
或所述质谱分析技术选自基于质谱的GC-MS、LC-MS、MALDI-TOFMS、FT-MS、ICP-MS、SIMS的检测技术。
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