CN111500708A - 一种与麻醉药物过敏相关分子标记及其应用和检测试剂盒 - Google Patents
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Abstract
本发明公开了一种与麻醉药物过敏相关分子标记及其应用和检测试剂盒。本发明首先提供了与麻醉药物过敏相关的基因突变位点rs10833049，可以提供检测人基因组中rs10833049的多态性的试剂在制备预测麻醉药物过敏制剂中的应用。将包含SNP位点区域的DNA模板通过PCR技术扩增，再使用sanger测序进行，获得准确的结果。本发明对于指导麻醉药物使用以及增加围术期的安全性具有积极的意义。
Description
技术领域
本发明属于分子生物学检测技术领域。具体为检测麻醉药物过敏相关的突变位点及其应用和检测试剂盒。
背景技术
过敏反应是“与引起过敏的物质接触后突然发生的可能致命的全身性反应”，随着社会发展及人类生存环境变化，过敏反应的发生率有逐年增高的趋势。过敏反应是围术期与手术、麻醉管理、既往合并症无关的最常见并发症。来自不同国家的多个系列研究估计麻醉期间临床过敏反应的发生率在1/353到1/18600之间，严重过敏比例在1/10000左右，麻醉药物中肌松药罗库溴铵是最常见的过敏原。
遗传因素在过敏性疾病中的作用得到了广泛关注及研究。研究发现HLA基因，Toll样受体基因，细胞因子包括IL-4、IL-10、IL-13等基因多态性参与食物过敏的发生。哮喘的发生与HLA基因多态性相关。Yang等发现HLA-DR9多态性可能参与青霉素类过敏反应等。基于申请人最近的研究，明确了一个与麻醉药物过敏相关的基因多态性位点：rs10833049。该位点可用于麻醉药物过敏的预测，提高患者围术期安全性。
发明内容
本发明的首要目的在于提供一种与麻醉药物过敏相关的分子标记，即人基因的rs10833049多态性位点A向G的突变。本发明是首次发现麻醉药物过敏相关的SNP分子标记。
该位点位于人MRGPRX2基因(序列见SEQ ID NO.1)全长的第185位，其碱基为A或G。该位点携带185G的患者对麻醉药物过敏后有保护作用，与野生型185A患者相比发生麻醉药物过敏的几率更低。
本发明所述麻醉药物过敏反应特指全身麻醉诱导后出现的过敏反应，临床表现及分级为I级：皮肤粘膜表现，包括皮肤潮红，皮疹，血管性水肿(局部或全身)，粘膜水肿；II级：存在可测量但不危及生命的症状，包括皮肤效应，血压下降≥30％及不明原因的心动过速，咳嗽或支气管痉挛但可实现机械通气；III级：存在危及生命的反应：心血管衰竭，心动过速或心动过缓，心律失常或严重支气管痉挛；IV级：心脏和/或呼吸停止。只要在全身麻醉诱导后出现上述任一种级别的临床表现就被确认为出现麻醉过敏反应。
本发明所述的麻醉药物包括全身麻醉诱导时所使用镇静催眠药、肌松药和阿片类镇痛药；镇静催眠药为咪达唑仑和依托咪酯；肌松药为罗库溴铵；镇痛药为舒芬太尼。
本发明所述的分子标记rs10833049位点基因型为AA、AG、GG；携带G的患者与携带A的患者相比发生麻醉药物过敏的几率更低；即AG和GG基因型比AA基因型发生麻醉药物过敏的几率低。
本发明所述的分子标记，rs10833049位点的扩增引物由rs10833049-F和rs10833049-R组成；引物序列如SEQ ID No.2和SEQ ID No.3。
本发明的第二个目的是提供上述的检测分子标记突变位点基因型的试剂在制备预测麻醉药物过敏制剂中的应用。
本发明的第三个目的是提供一种预测麻醉药物过敏的试剂盒。该试剂盒包含检测人基因的rs10833049位点基因型的试剂。本发明为首个预测麻醉药物过敏的SNP位点检测制剂。
本发明所述的试剂盒，包括DNA提取试剂、PCR扩增试剂、测序试剂。
本发明所述的试剂盒，PCR扩增试剂包括扩增rs10833049位点的引物，由rs10833049-F(正向引物)和rs10833049-R(反向引物)组成；引物序列如SEQ ID No.2和SEQID No.3。测序试剂里包含测序引物，引物序列如SEQ ID No.2。
该试剂盒检测的是人基因的rs10833049位点是否存在A向G的突变。
该试剂盒检测时，rs10833049位点基因型为AA、AG、GG；携带G的患者与携带A的患者相比发生麻醉药物过敏的几率更低；即AG和GG基因型比AA基因型发生麻醉药物过敏的几率低。
rs10833049是人染色体上的MRGPRX2基因的一个单核苷酸多态性位点，MRGPRX2基因的序列如SEQ ID No.1，该变异是A突变为G。所述rs10833049基因型为AA、AG、GG。所述AA是rs10833049位点为A的纯合型，所述AG是rs10833049位点A和G的杂合型，所述GG为rs10833049位点为G的纯合型。
本发明在一个来自中国南方汉族人群的样本中发现rs10833049是与麻醉药物过敏相关的单核苷酸多态性。本发明首先提供了与麻醉药物过敏相关的基因突变位点rs10833049，可以提供检测人基因组中rs10833049的多态性或基因型的试剂在制备筛查麻醉药物过敏患者产品、检测麻醉药物过敏易感性产品、检测与麻醉药物过敏相关的单核苷酸多态性产品、鉴定或辅助鉴定与麻醉药物过敏相关的单核苷酸动态性的产品中的应用。
本发明实施例中，rs10833049应用sanger测序平台进行基因分型检测。将包含SNP位点区域的DNA模板通过PCR技术扩增，再使用sanger测序进行，获得准确的结果。
本发明的优点和积极效果
本发明首次提供了一个与麻醉药物过敏相关的基因多态性位点，并提供一种麻醉药物过敏易感位点检测分型试剂盒。对于指导麻醉药物使用以及增加围术期的安全性具有积极的意义。本发明采用基因检测领域的金标准-sanger测序进行基因分型，具有准确性高的优点。
附图说明
图1为rs10833049三种基因型分布测序峰图，绿色单峰为AA基因型，绿色和黑色双峰为AG基因型，黑色单峰为GG基因型。
具体实施方式
以下通过特定的具体实例进一步说明本发明，而不会形成对本发明保护范围的限制。
实施例：
1)与麻醉药物过敏相关的MRGPRX2基因多态性位点(rs10833049)的发现
本申请发明人通过病例对照研究分析证明MRGPRX2基因多态性位点(rs10833049)与麻醉药物过敏相关，具体的为根据Quanto软件计算研究所需样本量，经我院伦理委员会批准，患者知情同意后收集我院2017-2019年首次行全身麻醉手术且在麻醉诱导后发生过敏患者并排除使用抗生素、血浆、胶体等过敏的湖南汉族人群患者75人作为研究组，筛选性别年龄匹配接受全麻诱导后无过敏且既往无过敏史的75例湖南汉族患者作为对照组。过敏反应指全身麻醉诱导后出现的过敏反应，临床表现及分级为I级：皮肤粘膜表现，包括皮肤潮红，皮疹，血管性水肿(局部或全身)，粘膜水肿；II级：存在可测量但不危及生命的症状，包括皮肤效应，血压下降≥30％及不明原因的心动过速，咳嗽或支气管痉挛但可实现机械通气；III级：存在危及生命的反应：心血管衰竭，心动过速或心动过缓，心律失常或严重支气管痉挛；IV级：心脏和/或呼吸停止。只要出现上述任一种级别的临床表现就被确认为出现麻醉过敏反应。
麻醉药物涉及全身麻醉诱导时所使用镇静催眠药、肌松药和阿片类镇痛药。镇静催眠药为咪达唑仑和依托咪酯；肌松药为罗库溴铵；镇痛药为舒芬太尼。具体的为同时使用咪达唑仑0.05mg/kg体重，舒芬太尼0.5ug/kg体重，依托咪酯0.2mg/kg体重，罗库溴铵0.6mg/kg体重。
采集纳入研究患者的外周血2ml，使用天根DNA提取试剂盒提取血液中DNA，DNA经PCR扩增后行Sanger测序，比对筛选突变位点。
2)位点扩增引物及测序方法：引物序列为SEQ ID No.2-3，先行PCR扩增，扩增体系如表1.扩增条件为96℃ 1min预变性，之后96℃ 10s，50℃ 5s，60℃ 4min，共25个循环，最后4℃保温。
表1.PCR扩增体系
扩增后上机测序，测序引物为SEQ ID No.2。测序结果在软件中比对，在软件中查看峰图。三种基因型测序峰图如图1。统计计算：卡方检验及logistics回归分析比较两组具有明显差异的突变位点。
结果：三种基因型测序峰图如图1。等位基因分布如表2所示，等位基因G在过敏组的频率(20.7％)显著低于对照组(31.3％)。对过敏的发生具有保护作用(OR＝0.571，95％CI＝0.338-0.965)。计算所得显著性差异为P＝0.035。
本发明也对使用相应引物对其他位点进行研究，未发现明显差异，结果如表2所示。
表2.各位点等位基因分布及卡方检验结果
进行基因模型分析后发现，rs10833049位点在显性模型上两组具有差异(p＝0.047)，AG/GG基因型在过敏组的频率(33.3％)显著低于对照组(49.3％)，AG/GG基因型携带者接受麻醉药物后过敏发生率降低(OR＝0.514，95％CI＝0.265-0.993)。进行调整性别年龄后的回归分析发现，AG/GG基因型携带者接触麻醉药物后发生药物过敏的风险低于AA基因型携带者(OR＝0.503，95％CI＝0.255-0.991)，结果如表3所示。
表3.两组显性模型下基因型分布差异比较
3)一种麻醉药物过敏易感位点(rs10833049)检测分型试剂盒的设计
所述DNA提取试剂盒采用天根公司产品(DP332)，所述PCR扩增试剂主要包括扩增引物、BigDye、ddH2O。扩增引物为SEQ ID No.2-3。所述测序试剂使用Applied BiosystemsTM公司
Terminator v1.1循环测序试剂盒，测序引物为SEQ ID No.2。
该试剂盒使用的具体步骤为：1)首先将采集标本根据DNA提取试剂盒步骤提取样本基因组DNA；2)对DNA进行PCR扩增，使用试剂BigDye 4μl，上下游引物共1μl，DNA 1μl，ddH2O 1μl加入PCR扩增仪中。扩增条件为96℃ 1min预变性，之后96℃ 10s，50℃ 5s，60℃4min，共25个循环，最后4℃保温。扩增产物进行电泳：PCR产物取5μl 1％琼脂糖凝胶电泳，电泳参数：150V，100mA，10～20min电泳观察及纯化；3)产物加入进行Sanger测序，测序使用Applied BiosystemsTM公司
Terminator v1.1循环测序试剂盒，据试剂盒要求加样后在ABI3730XL仪器中测序。
序列表
<110> 中南大学湘雅医院
<120> 一种与麻醉药物过敏相关分子标记及其应用和检测试剂盒
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6426
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
ctctgtacag gaaatcacag ggttgttgag catcttgata agcttcactc agtcatcagg 60
tttcactcca gatcctgcag gcatctcccc atcctcagct gtttgccagt cccaggaaag 120
cacttctcaa ctcaccaact ccagtagaaa gaagggtgtt aaggtaagag tttgttcaag 180
aaccatcttc tttcaaaggc agttttggtt tttaccttag tccacatcct gatgaattca 240
ttaggaaaag aagacaggaa gctcctttga ggaagctttg gatgctggca gcttagatgc 300
tgggagatcc gaatggggtc aagcaggttc gtcccacttc tcttctatct accaatattc 360
tccccttctc ttcgaccatt caaatctgaa actatggaga gaaagatttt gctgatttta 420
atttaggggc aggctagaat gaagtcatta cttccctaag ctaaattgtt atttaaaacc 480
tcaggttgca ctgattttct tgttattcaa aaggtttttc cttcatatct gtcattgtcc 540
cagcagaaaa cacacatcac acactctaaa ggggtagttt agaggagttt agtgaagaga 600
ctgtttacaa cggcgtgaac agggattaag gaatgatgaa gccctcaaag gcaagtgata 660
gtggagggct gttaccgttg ctaggtctga agtgataagg agaaagagaa gttctgagaa 720
ctcagaatgt gctgtagcgg taactgtaac tgtaactata gctgtggcta caggaaaggg 780
ccaccaggca gggctatgtc cttaggtaga aaaacactgc cactgccaac tcacagccct 840
tcagggcgca gggagagagc caggaaattt ttaaaaaatc atcccccaat ctactgtcaa 900
tgtgtccctt tggctgaaaa aaaaagtcac cctccaatct cctgtcaatg tgtacccttt 960
ggagcctgag tgaaagacag cccattgacg aggcacagac atgtctcctc ccaggatgca 1020
aaggcaggta gagaaggatg gacatgagat cctaatagca aataggaaag tccatttctt 1080
cccatagcct cttctcagtt tgtctttcct gagactttct ctattaatgt gattgaatca 1140
atttctcatt ctatcacctc cctttttttt aattgtttga ccttcccatc tagtgtcact 1200
tctttggact agtctctcac tatcatcata aatgccttga gaatggaatg tggttgggaa 1260
aaaaagggat tgggagtaca taggtactcc cagctataag tacacaggta tgtgtaatgt 1320
aagtttatga ttttggcttc tctaaaaaga aacctattat agtgataatc aaaaagaatg 1380
tttactagac ttgggctgat aactctaaga catcaaacat caaggctcct tccaatttct 1440
atttccccat ccctaaagcg gtacccttac cctcatggtc ctacatggct cccaaacaca 1500
tcagcattcc agccagagag aagagaaaaa aagaaagagt acaggagtag ctggttatat 1560
cactttctct cacctcccct tgaccatcac atatggttac atggtgccat tagcataaag 1620
ggaagctgag aaatgggcct ttttctgatc taagactcag aattattatg tgtgagaaaa 1680
ggagaatggc ttttggggca acaagcagtc tctatagccc gattctcttt tagcatctgt 1740
gctcacctgg agatttttct caacactgcc tcacaaatag tgaatgatgt agacatggaa 1800
cagaaataaa cctaggacag ggccccagga gactggagct ggtattggac ctgcttttca 1860
ccatgtgatt caggaaactc ttttgtacac gttaagcctg tctttctttg ttaaatgagc 1920
gtattacaat agatggcctc caagggatat gtgttggagg ctggggatat aatagtgaac 1980
aaccacaaca aaaatgttga gcttaatgag ggaagtaaaa gaaaaaacag acatacactt 2040
aaaaatacag gtgcagggcc gggcacggtg gctcacgcct gtaatcctgg cactttggga 2100
ggccgaggcg ggcagatcac aaggtcagaa gttcgagacc atcctggcta acacagtgaa 2160
accccgtctc tactgaaaaa aaaaatacaa aaaattagcc agtcgtggtg gtgggcacct 2220
gtagtcccag ctactcggga ggctgaggca ggagaatggc gtgagcccag gaggcggagc 2280
ttgcagtgag ccgagatcga gccactgcac tccagcctgg gtgacagagc gagactccat 2340
ctcaaaaaaa aaaaaaaata caggtgcaaa ctgtgttcag tgccacgaat aaaaaggcca 2400
acctgccctg agggaggaca acagaagtgg ttttcttcca tatcctcaca aggccaacca 2460
gggctacagg aactaagaca gcatgtgatg gatgcattca gtccttgatg ccaattctgc 2520
acccccatgg aggacacatt cactttcccc ttggaggcct ccaacccatg agagttttct 2580
gcagtgtgga gtgattgacg gctgtttttc tgaagcaaga aagagctctt gcattcagtt 2640
gcttattctc agttggcttc tattcatatc tcttctcctt atttgattaa tctctcttcc 2700
ttactgtttc ttaaagttgc ttaatctagg cttaaaaaac tgcaatttcg acattccatt 2760
ttggatttga tatgagcatt acttgttgct tttctccttt cttcatgtgt agttggtatt 2820
gaacccttgc aatggggctg caaggctggt taagaacaag ccctctgaca cagacctgtg 2880
ggttcaaatg caggctctac cacttgtttg tgagacctga agcatattac tcaatacatt 2940
tgtgctttaa tctccttctc tgtaaaatgg gactaataat agtactgacc tcaccaggct 3000
ctgaagaaga tttaaagagg ctatacctga agagcattca gccagctgct attattcacc 3060
ccattatgag taagggctcc ttcccaagtg taatttgcat aagcatctgc cttttcccct 3120
caaagccctg cacagtgtag acatggttct tttttctaat ttcccactcc tttccccaaa 3180
taatcctgtt cacaaatgct ttaatttgca aagaaagctc aaaatcatct gggggaagta 3240
atgggtccac cttagatgta ttcataccac aacaccctct agttacctct agacactaga 3300
cactctcaat tgctgcctct ctctcctttt cccaaattgc ccccttttcc tccaagatgc 3360
agagaaattc agataaaccc tgaatcctct ccataaactg gaagtttaaa ctcttttggg 3420
tgctgagcaa aagccatata ctagcccttc ctggattgaa gatatgacag tggccattat 3480
tcactatctg tgtaggcaga agcaatccct tcaaatacac cagagctttc ccagaaggaa 3540
acatgaagaa tgatgttttc ttcatttctt tacatgtgtg ttttttagtg actaatttca 3600
ttttaaaatg ttggtgttcc acttagccca aattttcaac tactcttggc tccccatgca 3660
tgcatgcatg cattcattca ttcatttata tgtggtacaa acttcttgac cctccactat 3720
gccctcacat tgagctaggc actgaggaca cagaaatgga taagagaaaa actctttctg 3780
ttttcataaa gctgacccac tgagcaattt ctagtctgac aggcctggct gtcctcagac 3840
ctgtcctagc aagaagtcac ctctccgtta tgaccctaaa tcacaatctc cattctgact 3900
gttgatggac tcctctcagt cattaagggt ggccatgggt catggcctgg gtcatttctt 3960
ctggcccctg agaggaaatc tgtatgccag gataaaaaga atcctgaact cagagtactg 4020
tctcttgctg ccccacatat tctgcctaga cttttctctt cctgcctcca ggagacatga 4080
cacagtgaca gtgagtgggg gtgtttgggc cttagaatat tcccatacca gcagaggatc 4140
taccttgtaa tagaaagagc ccaggattta gagtcagcaa gaaatgagtt tgaatccagg 4200
tgctaggact ccctggccct taataaatga cttaatctct tcaagcctct gatttcctct 4260
cctgtaaaac aggggcggta attaccacat aacaggctgg tcatgaaaat cagtgaacat 4320
gcagcaggtg ctcaagtctt gtttttgttt ccaggggcac cagtggaggt tttctgagca 4380
tggatccaac caccccggcc tggggaacag aaagtacaac agtgaatgga aatgaccaag 4440
cccttcttct gctttgtggc aaggagaccc tgatcccggt cttcctgatc cttttcattg 4500
ccctggtcgg gctggtagga aacgggtttg tgctctggct cctgggcttc cgcatgcgca 4560
ggaacgcctt ctctgtctac gtcctcagcc tggccggggc cgacttcctc ttcctctgct 4620
tccagattat aaattgcctg gtgtacctca gtaacttctt ctgttccatc tccatcaatt 4680
tccctagctt cttcaccact gtgatgacct gtgcctacct tgcaggcctg agcatgctga 4740
gcaccgtcag caccgagcgc tgcctgtccg tcctgtggcc catctggtat cgctgccgcc 4800
gccccagaca cctgtcagcg gtcgtgtgtg tcctgctctg ggccctgtcc ctactgctga 4860
gcatcttgga agggaagttc tgtggcttct tatttagtga tggtgactct ggttggtgtc 4920
agacatttga tttcatcact gcagcgtggc tgattttttt attcatggtt ctctgtgggt 4980
ccagtctggc cctgctggtc aggatcctct gtggctccag gggtctgcca ctgaccaggc 5040
tgtacctgac catcctgctc acagtgctgg tgttcctcct ctgcggcctg ccctttggca 5100
ttcagtggtt cctaatatta tggatctgga aggattctga tgtcttattt tgtcatattc 5160
atccagtttc agttgtcctg tcatctctta acagcagtgc caaccccatc atttacttct 5220
tcgtgggctc ttttaggaag cagtggcggc tgcagcagcc gatcctcaag ctggctctcc 5280
agagggctct gcaggacatt gctgaggtgg atcacagtga aggatgcttc cgtcagggca 5340
ccccggagat gtcgagaagc agtctggtgt agagatggac agcctctact tccatcagat 5400
atatgtggct ttgagaggca actttgcccc tgtctgtctg atttgctgaa ctttctcagt 5460
cctgatttta aaacagttaa gagagtcctt gtgaggatta agtgagacag tgcctatgaa 5520
acaaacacta agtgcagtgt ctctggaact gccttactca caggcttcca ccacagccct 5580
atgagagctt tgccaactct gcggtccatg actgttccca cttttaatga atcctacctt 5640
tcgcagaagg ctgaaagcag ggcagaaaag atctacattt ctttggacac tgcacttgat 5700
agggactcaa agaatgttat atttttaatt aatttctttt tctcttccgt acaatttctg 5760
tctcaacaaa attagaagaa ttaaatttaa aactagctcc aaaagagcag tcgtctttca 5820
ttttggcaga ccttagaata tccccctagc ttaataaatc tttgttgaat ggcttaatga 5880
atgaataaac tggttaatgt ttaagttaaa cctctgaaaa gtctccattt accagatttg 5940
agtcactaaa tttattgctt tcactacttt tgaattttgc aaacatgaaa ttaagtttta 6000
taattagata aatcaatgtc aacacatatt taaagtttga ggtacactgt cttcctgtgg 6060
tttcctttca catgccatcc cttaaaatcc cagctacacg ccttcccatt ccttcccctt 6120
tgcctttgtt ctaatcttcc ctctctgggg gctctctaat tcgtcctgga agtttccagt 6180
ggtcttatag actccatgtt cttggaggac aggctgtatg tcagatttac cttttattcc 6240
gaagaactcg gagcatttat tttgttaatt aaattgcaca tatttttaaa agttacgtgt 6300
tccacagaat aaaatactaa ttgtaaatgc tgcatctttt aataattttt tattattttt 6360
aattaaggta taattaagag aatgatatgc acagatcata aatgtacagc ttaatgagtt 6420
ttgtta 6426
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aagcctctga tttcctctcc tgtaa 25
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
acgctgcagt gatgaaatca aa 22
Claims (10)
1.一种与麻醉药物过敏相关的分子标记，人基因的rs10833049位点A向G的突变。
2.根据权利要求1所述的分子标记，其特征在于，所述rs10833049位点基因型为AA、AG、GG；AG和GG基因型比AA基因型发生麻醉药物过敏的几率低。
3.根据权利要求2所述的分子标记，其特征在于，所述的麻醉药物包括全身麻醉诱导时所使用镇静催眠药、肌松药和阿片类镇痛药；其中镇静催眠药为咪达唑仑和依托咪酯；肌松药为罗库溴铵；镇痛药为舒芬太尼。
4.根据权利要求2所述的分子标记，其特征在于，rs10833049位点的扩增引物由rs10833049-F和rs10833049-R组成；引物序列分别如SEQ ID No.2和SEQ ID No.3。
5.检测权利要求1-4任一项所述的分子标记突变位点基因型的试剂在制备预测麻醉药物过敏制剂中的应用。
6.一种预测麻醉药物过敏的试剂盒，其特征在于，包含检测人基因的rs10833049位点基因型的试剂。
7.根据权利要求6所述的试剂盒，其特征在于，包括DNA提取试剂、PCR扩增试剂、测序试剂。
8.根据权利要求7所述的试剂盒，其特征在于，PCR扩增试剂包括扩增rs10833049位点的引物，由rs10833049-F和rs10833049-R组成；引物序列如SEQ ID No.2和SEQ ID No.3。
9.根据权利要求6-8任一项所述的试剂盒，其特征在于，该试剂盒检测的是人基因的rs10833049位点是否存在A向G的突变。
10.根据权利要求9所述的试剂盒，其特征在于，rs10833049位点基因型为AA、AG、GG；AG和GG基因型比AA基因型发生麻醉药物过敏的几率低。
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