CN113667767B - 一种快速、高通量检测幽门螺旋杆菌耐药性的方法 - Google Patents
一种快速、高通量检测幽门螺旋杆菌耐药性的方法 Download PDFInfo
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Abstract
本发明公开了快速、高通量检测幽门螺旋杆菌耐药性的方法。分离获取幽门螺旋杆菌样品，对幽门螺旋杆菌进行如下检测：检测克拉霉素耐药基因23S rRNA的突变位点：109G→A、2056A→G、2095T→C、2834C→T和蛋白infB的突变位点794Glu→Asp，如发生任一突变则幽门螺旋杆菌具有克拉霉素耐药性。本发明经研究发现现有的检测方案不能准确地预测HP中国流行株对抗生素的耐药情况，因此发明人建立了首个基于HP中国流行株耐药基因的分子表征库信息的基因‑表征关联模型，提出基于该模型的新型快速、高通量HP耐药检测方案。与现有技术相比，本发明能更精准地协助临床医生快速预测我国HP的耐药表型。
Description
技术领域：
本发明属于微生物技术领域以及医药技术领域，具体涉及一种快速、高通量检测幽门螺旋杆菌耐药性的方法。
背景技术：
幽门螺杆菌(Helicobacter pylori，HP)是一种革兰氏阴性、微需氧螺旋状细菌，与人类胃肠道疾病有关。HP感染可引起慢性胃炎，并导致多种并发症，如消化不良、消化性溃疡和胃癌。据统计，全球约90％的胃癌发病与HP感染密切相关。因此，HP感染严重威胁人类的健康、造成庞大的经济与社会损失，预防和治疗幽门螺旋杆菌感染具有巨大的应用研究价值。
抗生素治疗是目前清除HP的主要方案，但由于胃内存在大量胃酸及消化酶，可灭活绝大部分抗生素，因此现有的多数抗生素不能用于HP的治疗。迄今为止，仅5种抗生素在全球范围内获批用于HP的治疗：阿莫西林、克拉霉素、左氧氟沙星、甲硝唑及四环素。然而，大量研究数据指出，我国流行的HP对上述五种抗生素的耐药率在逐年爬升。至2017年，我国HP流行株对不同抗生素的耐药情况为：克拉霉素28.9％、甲硝唑63.8％、左氧氟沙星28.0％、阿莫西林3.1％、四环素3.9％。
HP耐药性检测的金标准为美国临床和实验室标准协会(Clinical andLaboratory Standards Institute，CLSI)提出的琼脂稀释法。该方法对实验人员专业水平、实验设备、实验耗材均有较高要求，难以在临床应用中开展。因此，寻求简便、准确率高的HP耐药性评判新标准有重要的应用意义。
目前认为HP耐受抗生素主要由该菌基因组中特定基因发生随机突变导致。HP基因发生突变后，抗生素难以结合到其作用靶点或药物难以在菌体内由前体药物转化为作用药物，均导致HP对抗生素耐药。因此，研究者通过大数据分析手段统计分析HP基因突变与耐药表型的规律，继而建立了基于HP特定基因突变预测其耐药表型的模型，为临床医生提供了一套简便、快速的HP药敏预测方案。
近年来，随着新一代测序技术的发展，微生物学家们逐渐发现HP是一个在种内具有高度变异性的物种，不同大洲人群感染的HP在基因组水平上差异极大。
发明内容：
本发明的第一个目的是提供一种准确地预测HP中国流行株对抗生素的耐药情况的快速、高通量检测幽门螺旋杆菌耐药性的方法。
本发明人团队在前期研究中发现，既往文献报道的其他国家发生的HP耐药基因突变并不具有预测中国流行HP株药敏表型的意义(图2)。
因此，本研究结合我国HP流行株的基因组特征，建立起HP中国流行株耐药基因的分子表征库，并设计出HP中国流行株的耐药基因组分子表征关联模型，针对我国流行HP药敏情况，对于三种易发生耐受的抗生素，设计出适用于我国HP分离株耐药性的快速检测方案。本方案对比现有方法，有利于协助临床医生对我国HP感染的治疗做出更为精准的判断。
本发明的快速、高通量检测幽门螺旋杆菌耐药性的方法，包括以下步骤：
分离获取幽门螺旋杆菌样品，对幽门螺旋杆菌进行如下检测：
检测克拉霉素耐药基因23S rRNA的突变位点：109G→A、2056A→G、2095T→C、2834C→T和蛋白infB的突变位点794Glu→Asp，如发生任一突变则幽门螺旋杆菌具有克拉霉素耐药性；
或检测左氧氟沙星耐药基因的蛋白gyrA的突变位点：87Asn→Lys、91Asp→Asn/Gly/Tyr和蛋白gyrB的突变位点：684Glu→Asp，如发生任一突变则幽门螺旋杆菌具有左氧氟沙星耐药性；
或检测甲硝唑耐药基因的蛋白rdxA的突变位点：16Arg→Cys/His和蛋白rpsU的突变位点：56Val→Ile和蛋白sodB的突变位点：54Asp→Ala，如发生任一突变则幽门螺旋杆菌具有甲硝唑耐药性。
本发明的第二个目的是提供一种快速、高通量检测幽门螺旋杆菌耐药性的试剂盒，其包括：
检测克拉霉素耐药基因23S rRNA的突变位点：109G→A、2056A→G、2095T→C、2834C→T和蛋白infB的突变位点794Glu→Asp的试剂；
或检测左氧氟沙星耐药基因的蛋白gyrA的突变位点：87Asn→Lys、91Asp→Asn/Gly/Tyr和蛋白gyrB的突变位点：684Glu→Asp的试剂；
或检测甲硝唑耐药基因的蛋白rdxA的突变位点：16Arg→Cys/His和蛋白rpsU的突变位点：56Val→Ile和蛋白sodB的突变位点：54Asp→Ala的试剂。
本发明与现有技术相比，其有益效果为：
本发明经研究发现现有的检测方案不能准确地预测HP中国流行株对抗生素的耐药情况，因此发明人建立了首个基于HP中国流行株耐药基因的分子表征库信息的基因-表征关联模型，提出基于该模型的新型快速、高通量HP耐药检测方案。与现有技术相比，本发明能更精准地协助临床医生快速预测我国HP的耐药表型。
附图说明
图1是HP中国流行株耐药基因的分子表征库；HP中国流行株耐药基因的分子表征库部分内容：54株中国分离HP株对克拉霉素、左氧氟沙星、甲硝唑的药敏表型及耐药基因分子特征。54株中国分离HP株的进化关系如左侧进化树所示。长形方框代表菌株对相应抗生素的药敏表型，代表敏感、/>代表耐药；正方形代表菌株在特定基因位点的情况，/>代表与ATCC26695一致，无突变发生，/>代表存在突变。深灰色背景的突变位点为国外文献报道与耐药基因或蛋白突变位点，白色背景的突变位点为本发明公布的与中国流行株耐药相关的基因或蛋白突变位点。
图2是不同耐药基因、蛋白突变对HP中国流行株的三种抗生素MIC的影响；图A、B、C分别显示不同耐药基因或蛋白突变对HP中国流行株MIC的影响。白色为无突变HP分离株的MIC值，黑色为耐药基因突变HP分离株的MIC值。可见发生基因突变的HP株MIC值高于无突变株。图中*所标注的突变位点为本专利新发现的与HP中国流行株耐药相关的基因或蛋白突变位点。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
下述实施例中涉及的培养基及试剂如下：
培养基添加抗生素混悬液的制备：万古霉素10mg/mL、两性霉素B 5mg/mL、甲氧苄氨嘧啶20mg/mL、头孢磺啶10mg/mL，上述抗生素均采用灭菌水溶解。
HP选择性培养平板(g/L)：酵母浸粉3.0g/L、酪胨12.0g/L、动物组织消化物5.0g/L、牛肉浸粉3.0g/L、玉米淀粉1.0g/L、氯化钠5.0g/L、琼脂13.5g/L、无菌绵羊红细胞或胎牛血清70ml/L、抗生素混悬液10ml/L。HP增殖培养平板为不添加抗生素混悬液的HP选择性培养平板。
药敏检测用抗生素悬液的制备：(1)克拉霉素：称取100mg克拉霉素，溶解于0.2mL甲醇或冰醋酸中配成贮存液，使用时采用pH 6.5、浓度为0.1mol/L的磷酸盐缓冲液稀释至所需浓度；(2)左氧氟沙星：称取100mg左氧氟沙星，加0.2mL水后缓慢滴加浓度为0.1mol/L氢氧化钠直至溶解配成贮存液，使用时使用无菌水稀释至所需浓度；(3)甲硝唑：称取100mg甲硝唑，溶解于0.2mL二甲基亚砜中配成贮存液，使用时采用无菌水稀释至所需浓度；(4)阿莫西林：称取10mg阿莫西林，溶解于1mL pH 6.0、浓度为0.1mol/L的磷酸盐缓冲液中配成贮存液，使用时采用pH 6.0、浓度为0.1mol/L的磷酸盐缓冲液稀释至所需浓度；(5)四环素：称取抗生素粉末直接加无菌水配成所需浓度。
实施例1幽门螺杆菌的分离、培养与鉴定
(1)HP的分离及培养
①HP的分离：在无菌环境下，取慢性胃炎患者胃粘膜组织，涂布于HP选择性培养平板上，后放置于37℃微需养环境中(5％O2、10％CO2、85％N2)培养5-7天。挑取平板上形态典型的菌落至新的HP选择性培养平板上进行划线纯化，纯化2次后获得纯化的HP分离株。
②HP的培养：从含HP的选择性培养平板上挑取单菌落，接种于HP增殖培养平板上，37℃微需养环境中培养72-96小时，加入生理盐水洗脱获得HP分离株菌液。
(2)HP的鉴定
采用基于16S rRNA基因序列的方法对分离菌株进行分子鉴定：采用细菌DNA提取试剂盒(Mabio，CHINA)进行提取HP的基因组DNA，后采用2×PCR mix(Dongshengbio，CHINA)进行PCR扩增。PCR扩增引物采用16S rRNA基因通用引物，上游引物序列为27F：5’-AGA GTTTGA TCC TGG CTC AG-3’；下游引物序列为1492R：5’-CTAC GGC TAC CTT GTT ACG A-3’。PCR反应条件为：预变性95℃5min；95℃30s，56℃30s，72℃1min 30s共30个循环，72℃退火延伸10min。对PCR产物进行切胶回收，后进行Sanger测序。获得的16S rRNA基因序列后比对NCBI数据库(https://blast.ncbi.nlm.nih.gov)，结果提示其与HP同源性最高，且Identity和Coverage均大于99％，可确定分离菌株为HP。本研究采用的54株中国HP分离株16S rRNA基因比对结果显示与HP标准株ATCC26695高度同源，Identity和Coverage均大于99％。
实施例2幽门螺旋杆菌的药敏检测
采用琼脂稀释法对HP进行抗生素药敏检测，采用HP ATCC 43504作为质控菌株。根据美国临床和实验室标准协会(The Clinical&Laboratory Standards Institute，CLSI)制定的方法和标准进行测定。
琼脂稀释法检测HP对不同抗生素的敏感性：将阿莫西林、克拉霉素、左氧氟沙星、甲硝唑及四环素五种药物的粉末根据CLSI推荐的方式溶解于相应溶质中，配置成抗生素贮存液，后根据所需检测的药敏浓度，分别加入至HP选择性培养平板中，制成含相应浓度抗生素的HP药敏检测平板。将HP菌悬液浓度调整为1×107cfu/mL，并接种至含相应浓度抗生素的检测平板上，在37℃微需养环境中培养72小时，观察含相应浓度抗生素的检测平板上有无HP菌落形成。以不能形成菌落的最低浓度药物平板中的药物浓度为该种抗生素对所检HP的最小抑菌浓度(The minimum inhibitory concentrations，MIC)。每个药物浓度进行平行操作3次，得出最小抑菌浓度的平均值。HP对抗生素耐受(Resistant，R)的评判标准为：阿莫西林≥0.125mg/L、克拉霉素≥0.25mg/L、左氧氟沙星≥1mg/L、甲硝唑≥8mg/L、四环素≥1mg/L。
实施例3基于全基因组测序的幽门螺旋杆菌中国流行株耐药表征及分子特征库构建
3.1.HP的全基因组测序及基因注释
采用Illumina Nextseq 550测序平台对HP进行全基因组测序。细菌基因组DNA提取方法同前。二代测序采用AMT Rapid DNA-Seq Kit for Illumina(CISTRO,CHINA)建库，采用High Output v2.5 kit(Illumina,USA)试剂盒测序。下机数据采用Trimmomatic(v0.39)软件进行质控，后采用SPAdes(v3.13.1)软件进行组装。组装后HP基因组采用Quast(v5.0.2)软件进行组装质控评估，对于质控合格的基因组，采用Prokka(v1.13)软件进行注释。
3.2.HP耐药基因的点突变检测
从HP注释后的核苷酸序列文件及氨基酸序列文件中提取待分析的耐药基因的核苷酸序列及耐药蛋白的氨基酸序列。选择待分析的目标基因或蛋白序列导入CLCworkbench(Qiagen，Germany)软件，采用Classical Sequence Analysis中的Alignmentand Tree分析模块，比对已知的HP抗生素敏感标准菌株ATCC 26695的参考序列，进行基因/蛋白序列特征图谱的分析比对。与ATCC 26695相应的基因核苷酸序列相比，若出现基因突变，则在数据库中录入相应基因位点及其核苷酸的突变类型。与ATCC 26695的相应蛋白氨基酸序列相比，若出现氨基酸突变，则在数据库中录入相应突变的氨基酸位点及氨基酸的突变类型。
3.3.HP中国流行株耐药表型-分子特征关联模型的构建与应用
综合HP中国流行株的耐药表型谱、耐药基因突变谱及耐药蛋白突变谱信息，整理获得HP中国流行株耐药基因的分子表征库(图1，表1和表2)。采用Pearson相关分析检测耐药基因核苷酸或氨基酸序列突变与耐药表型的相关性，采用P<0.05认为两者存在相关性。由附图2分析可知，多数由其它地区报道的HP耐药分子特征与中国HP流行株的耐药表型并不存在相关性。而采用发明人所整理的HP中国流行株耐药基因的分子表征库，可建立HP中国流行株的耐药基因组分子表征关联模型，采用模型则可根据其它中国流行株的耐药基因突变预测其耐药表型，分析发现存在耐药突变的HP菌株对抗生素的MIC高于野生株(图2)。
实施例4高通量、快速检测幽门螺旋杆菌耐药性
在患者胃黏膜分离获得HP单菌落后，参照前述方法进行全基因组HP全基因组测序，并提取23S rRNA基因的核苷酸序列及infB、gyrB、rdxA、rpsU及sodB共5个蛋白的氨基酸序列。对比附表1、附表2检测相应的核苷酸位点及氨基酸位点，读取HP分离株对克拉霉素、左氧氟沙星及甲硝唑三种抗生素为耐药或是敏感。对比现有方案，采用本发明能检出采用旧方案不能检出的HP耐药情况，即本发明提供的检测方案对于耐药的HP中国流行株检出有更高的敏感性(附表3)。
表1 HP耐药基因核苷酸突变位点与对应抗生素药物参照表
表2 HP耐药蛋白基因氨基酸突变位点与对应抗生素药物参照表
表3现有方案与本发明方案对54株HP中国分离株耐药情况的判读准确率对比
从上可知，本发明是基于HP中国流行株的基因及耐药谱特征建立的预测模型，本发明主要针对中国株普遍耐受的抗生素药敏表型进行检测，更契合中国流行株的基因组特征情况，具有高通量、高效、准确预测中国HP流行株耐药情况的作用。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (1)
1.一种快速、高通量检测幽门螺旋杆菌耐药性的试剂盒，其特征在于，包括检测克拉霉素耐药基因23S rRNA的突变位点：109G→A、2056A→G的试剂；
使用试剂盒的操作步骤为：分离获取幽门螺旋杆菌样品，对幽门螺旋杆菌进行如下检测：
检测克拉霉素耐药基因23S rRNA的突变位点：109G→A、2056A→G，如发生任一突变则幽门螺旋杆菌具有克拉霉素耐药性。
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