CN115184516A - 一种快速检测肠杆菌β-内酰胺酶的方法 - Google Patents
一种快速检测肠杆菌β-内酰胺酶的方法 Download PDFInfo
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- CN115184516A CN115184516A CN202110358240.9A CN202110358240A CN115184516A CN 115184516 A CN115184516 A CN 115184516A CN 202110358240 A CN202110358240 A CN 202110358240A CN 115184516 A CN115184516 A CN 115184516A
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Abstract
本发明提供了一种快速检测肠杆菌β‑内酰胺酶的方法,以检测肠杆菌科细菌产生的碳青霉烯酶(IMP、VIM、OXA)、超广谱β‑内酰胺酶(TEM和CTX‑M)和AmpC(CMY‑2)。所述方法包括挑取单个菌落分散在缓冲液中,超声处理后经超滤离心,然后用胰蛋白酶进行膜上酶切,得到肽段;基于特异性肽段及同位素标记的特异性肽段的信息建立PRM(parallelreaction monitoring)靶向蛋白组方法,采用纳升液相及高分辨质谱联用检测肽段;用Skyline软件处理数据,根据所得参数判断β‑内酰胺酶是否阳性。
Description
技术领域
本发明涉及病原微生物及耐药微生物检测领域,具体地说,涉及一种基于LC-MS/MS的快速检测肠杆菌耐药酶的方法及应用。
背景技术
随着碳青霉烯类及β-内酰胺类抗菌药物在临床的广泛应用,细菌耐药形势日趋严峻。多重耐药和泛耐药菌株日益增多以及耐药酶的快速传播给临床抗感染治疗带来了极大的挑战,已成为严重的全球公共卫生问题。在2017年世界卫生组织(World HealthOrganization,WHO)发布的新型抗生素研发重点病原体清单中,碳青霉烯类药物耐药(CRE)、产超广谱β-内酰胺酶(ESBL)肠杆菌科处于极为重要(1类重点)的位置。
β-内酰胺酶是一类使β-内酰胺类抗生素失活,导致抗菌活性无效的耐药酶。根据β-内酰胺酶的一级结构,Ambler将其分为4类:А、B、C和D。在所有的β-内酰胺酶中,碳青霉烯酶最受关注;А和D类碳青霉烯酶是丝氨酸型水解酶,如KPC和OXA。B类碳青霉烯酶是金属水解酶,如NDM、IMP、VIM等。超广谱β-内酰胺酶(ESBL)如TEM和CTX-M组属于A类β-内酰胺酶。AmpCβ-内酰胺酶如CMY-2属于C类酶。肠杆菌科包括大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌等,携带多种β-内酰胺酶,给临床诊断和治疗带来巨大压力。
早期诊断和有效的药物治疗是解决抗生素耐药问题的关键手段。临床上迫切需要快速、准确的检测方法。较短的检测时间以及准确的诊断,有助于患者及时获得恰当的抗生素治疗。在过去的几十年中,传统的检测β-内酰胺酶的方法,如标准纸片扩散法、肉汤微量稀释法和琼脂稀释法等,耗时较长,只能确定其对哪类药物的耐药性,不能确定β-内酰胺酶的类型。以酶活性为基础的方法,如Carba-NP试验,对单一类型的碳青霉烯具有快速的特性但特异性较低。基于基因序列扩增的PCR技术已广泛应用于抗菌药物耐药基因型的检测。但由于耐药酶是基因表达的产物,基因检测的结果并不一定代表耐药酶的成功表达。此外,非特异性扩增引起的假阳性也会干扰正确诊断。
随着液质联用技术的日趋完善,LC-MS/MS逐渐成为最热门的分析手段之一。作为已经比较成熟的技术,其目前己在蛋白质分析、生化分析、天然产物分析、药物和食品分析以及环境污染物分析等许多领域得到了广泛的应用。近几年,以SRM(selected reactionmonitoring)和MRM(multiple reaction monitoring)为代表的靶向蛋白质组学逐渐应用于耐药酶检测领域。基于Orbitrap高分辨、高精度质谱的PRM(parallel reactionmonitoring,平行反应监测)技术,能够对目标肽段进行选择性检测,从而实现对目标肽段进行相对及绝对定量。随着技术的不断进步,目前PRM技术在肽段检测数目方面有大幅度提升,展现出巨大的优势。
发明内容
鉴于目前临床产耐药酶肠杆菌的诊断和治疗困难,本发明旨在应用高灵敏度、高分辨率、高精确度的蛋白质谱检测技术(Nano LC-Orbitrap Fusion Lumos)和靶向蛋白组学分析方法平行反应监测(Parallel Reaction Monitoring,PRM)建立CRE、ESBL及AmpC超级细菌感染的早期、快速、准确的诊断方法,为临床产耐药酶肠杆菌的诊断和治疗提供重要依据和技术支撑。
本发明主要提供了一种快速检测肠杆菌β-内酰胺酶的方法,包括:
样品处理:挑取单个菌落,悬浮在缓冲液中,超声处理,超滤离心后去除缓冲液,然后进行膜上酶切,得到肽段;
反相色谱分离:用反相色谱柱分离肽段,流动相A为包含甲酸的水溶液,流动相B为流动相A与乙腈的混合物;
质谱法检测特异性肽段;和
分析质谱数据,根据参数值判断β-内酰胺酶是否阳性,其中,所述特异性肽段为选自以下的一种或多种:
LVVPSHSEVGDASLLK;
VQATNSFSGVNYWLVK;
NSFGGVNYWLVK;
LDEGVYVHTSFEEVNGWGVVPK;
LAEAEGNEIPTHSLEGLSSSGDAVR;
DGDELLLIDTAWGAK;
NNGLTEAWLESSLK;
IINHNLPVK;
ADIANNHPVTQQTLFELGSVSK;
TLQQGIALAQSR;
QLTLGHALGETQR;
TEPTLNTAIPGDPR;
LIAQLGGPGGVTAFAR;
APLILVTYFTQPQPK;
SDLVNYNPIAEK;和
VGYIELDLNSGK。
在一些优选实施方案中,本发明的方法包括质谱法检测特异性肽段和同位素标记的特异性肽段;计算特异性肽段和同位素标记的特异性肽段的峰面积之比,对特异性肽段进行相对定量,所述特异性肽段为选自以下的一种或多种:
LVVPSHSEVGDASLLK;
VQATNSFSGVNYWLVK;
NSFGGVNYWLVK;
LDEGVYVHTSFEEVNGWGVVPK;
LAEAEGNEIPTHSLEGLSSSGDAVR;
DGDELLLIDTAWGAK;
NNGLTEAWLESSLK;
IINHNLPVK;
ADIANNHPVTQQTLFELGSVSK;
TLQQGIALAQSR;
QLTLGHALGETQR;
TEPTLNTAIPGDPR;
LIAQLGGPGGVTAFAR;
APLILVTYFTQPQPK;
SDLVNYNPIAEK;和
VGYIELDLNSGK,
所述同位素标记的特异性肽段的标记位点为下划线标记氨基酸。
在一些优选实施方案中,所述单个菌落的直径>2mm。
在一些优选实施方案中,用蛋白酶进行酶切,优选采用胰蛋白酶进行酶切,更优选地,用测序级胰蛋白酶进行酶切。
在一些优选实施方案中,使用C18柱分离肽段。
在一些优选实施方案中,流动相A为包含0.1%甲酸的水溶液(体积百分比,v/v),流动相B为含0.1%甲酸的80%乙腈(体积百分比,v/v)。
在一些实施方案中,采用梯度洗脱法进行洗脱。在一些优选实施方案中,洗脱从11%的流动相B开始,然后从11%的流动相B上升到13%的流动相B,持续2min,16min后梯度上升到32%的流动相B,7min后梯度上升到42%的流动相B,1min后梯度上升到95%的流动相B,保持4min。
在一些优选实施方案中,质谱检测的参数为:
MS1光谱:Orbitrap分析;分辨率,60000;质量范围,350~2000m/z;RF lens,30%;AGC靶,2.0×105;最大注射时间,50ms;和
MS2分析,HCD伴随以下条件:碰撞能量,30%;AGC,5.0×104;最大注射时间,特异性肽段,54ms;同位素标记的特异性肽段,22ms;Orbitrap分辨率,特异性肽段,30000;同位素标记的特异性肽段,7500;隔离窗口,1.4Da。
在一些优选实施方案中,利用Skyline软件分析处理质谱数据。
附图说明
附图用来提供对本申请技术方案的理解,并且构成说明书的一部分,与本申请的实施例一起用于解释本申请的技术方案,并不构成对本申请技术方案的限制。
图1示出了β-内酰胺酶的特异性肽段以及同位素标记的特异性肽段的信息。
图2示出了不同酶切条件对特异性肽段的丰度的影响。
图3示出了特异性肽段经冻融三次后的稳定性。
图4示出了特异性肽段在样品槽(10℃)0、1、3或4天的稳定性。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚明白,下文中将结合附图对本申请的实施例进行详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互任意组合。
仪器和试剂
实施例中使用到的仪器:高分辨液质联用仪(Thermo Fisher Scientific,NanoLC1200-Orbitrap Fusion Lumos)、摇床培养箱(上海智城分析仪器制造公司,ZWY-100H)、金属浴(Thermo Fisher Scientific,Thermal Mixer)。
试剂:质谱级碳酸氢铵(Merck,MS grade)、超滤膜(Pall,OD030C35)、胰蛋白酶(Promega,V5280);肽段浓度测定试剂盒(Thermo Fisher Scientific,23275);培养基:Luria-Bertani(LB)Agar培养基。
实施例1:单菌落靶向蛋白组快速检测方法的建立。
从NCBI(https://www.ncbi.nlm.nih.gov/protein/)下载酶的氨基酸序列,并用PeptideCutter(https://web.expasy.org/peptide\u/)对潜在肽段进行评价。鉴于肽在质谱中的不同响应能力,使用表1示出的4株菌株通过数据依赖采集模式的液质联用评估肽的电离能力,并通过BLASTp搜索评估肽的特异性。选择具有较好信号稳定性、保留时间稳定和相对稳定氨基酸残基的特异性肽段作为IMP、VIM、OXA、CMY、CTX-M和TEM的肽标记。Val(13C5,15N)、Gly(15N)和Ala(13C3,15N)用于标记肽段(图1)。
表1:方法评价所用菌株含β-内酰胺酶信息。
a Kwon T,Jung Y H,Lee S,et al.Comparative genomic analysis ofKlebsiella pneumoniae subsp.pneumoniae KP617 and PittNDM01,NUHL24835,and ATCCBAA-2146reveals unique evolutionary history of this strain[J].Gut Pathogens,2016,8(1):1-16.
b PCR及测序验证。
样品处理:通过枪尖或10μL环提取直径>2mm的单个菌落,悬浮在200μL 50mM碳酸氢铵缓冲液(50mM,PH 8.0)中,超声处理1min(3s工作,6s休息),然后95℃处理5min,并用带有Nanosep超滤离心管的超滤离心装置去除缓冲液。加入50mM碳酸氢铵缓冲液和测序级胰蛋白酶,水浴微波加热,55℃酶切消化。脱盐后用肽段浓度测定试剂盒(Thermo FisherScientific,23275)测定肽段浓度后上机。在EASY-nlc1200-Orbitrap Fusion Lumos平台上采用数据依赖采集方法建立谱图库。
液相分离方法如下:将肽段溶液以600nL·min-1的流速装入预柱((Reposil Pur120C18-AQ(3μm,Dr.Maisch GmbH,德国)20×0.05mm)上,然后装入分析柱(Reposil Pur120C18(1.9μm,Dr.Maisch GmbH,德国)120×0.15mm)。流动相A由含0.1%(体积百分比,v/v)甲酸的水组成,流动相B为含0.1%甲酸的80%乙腈(体积百分比,v/v)。进样后,梯度从11%的流动相B开始,然后从11%的流动相B上升到13%的流动相B,持续2min,16min后梯度上升到32%的流动相B,7min后梯度上升到42%的流动相B;1min后梯度上升到95%的流动相B,保持4min。
MS参数:MS1(Orbitrap分析;质量范围:350~1550m/z;分辨率,120000;AGCtarget,5×105;RF lens,50%;最大注入时间,50ms),MS2(HCD,碰撞能量,32%;最大注入时间,22ms;AGC,5×104;隔离窗口,1.6Da;Orbitrap分辨率,15000)。原始数据以从NCBI下载的细菌β-内酰胺酶FASTA文件作为背景库,通过Thermo ScientificTMProteomeDiscovererTMversion 2.2(PD2.2)软件搜库。
通过Skyline软件对采集的数据进行分析。以从NCBI下载的β-内酰胺酶的氨基酸序列作为背景库并导入数据依赖采集模式原始数据建立数据库。选择特异性肽段并导出特异性肽段的相关参数,建立PRM方法。靶向蛋白质组学在Thermo Scientific OrbitrapFusion Lumos和EASY nLC 1200平台上进行。我们建立了30min Nano LC-MS方法,特异性肽段和同位素标记的特异性肽段的参数如图1所示。为了提高鉴定质量,对特异性肽段和同位素标记的特异性肽段采用了不同的Orbitrap分辨率。在每个样品后加入30min的清洗程序,以避免携带效应引起的假阳性。所用柱和洗脱梯度与上述相同。
PRM参数:MS1光谱(Orbitrap分析;分辨率,60000;质量范围,350~2000m/z;RFlens,30%;AGC靶,2.0×105;最大注射时间,50ms)和MS2分析,HCD伴随以下条件:碰撞能量,30%;AGC,5.0×104;最大注射时间,特异性肽段,54ms;同位素标记的特异性肽段,22ms;Orbitrap分辨率,特异性肽段,30000;同位素标记的特异性肽段,7500;隔离窗口,1.4Da。将多肽信息(RT、m/z和电荷)导入质谱方法。质谱采集数据后,将特异性肽段和同位素标记的特异性肽段的信息导入Skyline,导入PRM数据,针对每个靶肽,计算检测到的特异性肽段和同位素标记的特异性肽段的峰面积之比,用同位素标记的特异性肽段计算靶肽的相对浓度。CRE/ESBL酶是阳性还是阴性取决于肽:保留时间(RT)与同位素标记的特异性肽段相似,rdotp>0.95,Library dotp>0.8。
图1示出的特异性肽段均可以成为某β-内酰胺酶或某β-内酰胺酶亚型的标志物,从而对β-内酰胺酶或β-内酰胺酶亚型进行检测或定量。
实施例2:单菌落蛋白组快速提取酶切方法建立。
通过枪尖或10μL环提取直径>2mm的单个菌落,悬浮在200μL 50mM碳酸氢铵中,超声处理1min(3s工作,6s休息),然后95℃处理5min,并用带有Nanosep超滤离心装置去除缓冲液。加入50mM碳酸氢铵缓冲液和测序级胰蛋白酶,分别用微波炉水浴5min、10min或15min或者微波炉水浴2min后在55℃金属浴保持15min、30min或45min后离心,收集肽段。采用纳米液相色谱-质谱联用技术对多肽进行检测,总处理时间不超过1h。
如图2所示,所有的酶解条件,包括微波炉水浴5min,均可快速进行蛋白组的酶解并且得到有效肽段。对于大多数肽,消化时间越长,丰度越高,而不同的消化条件对LAEAEGNEIPTHSLEGLSSSGDAVR、VQATNSFSGVNYWLVK、IINHNLPVK和NSFGGVNYWLVK没有影响。实施例3:靶向蛋白组检测细菌β-内酰胺酶特异性肽段检测变异系数评价。
以肺炎克雷伯杆菌(Klebsiella pneumoniae)ATCC BAA-2146、ATCC BAA-1705、17-R66和阴沟肠杆菌17-R42为模式菌株,采用上述蛋白组快速提取及检测方法对其β-内酰胺酶肽段检测变异系数进行评价,每株菌各有6个生物学重复。结果如表2所示,所有肽段的变异系数均小于8%。
表2:特异性肽段变异系数及丰度比值。
实施例4:靶向蛋白组检测细菌β-内酰胺酶特异性肽段检测稳定性评价。
稳定性是方法开发中需要评价的一个重要性质。在不含β-内酰胺酶的大肠杆菌DH5α肽溶液中加入一定浓度的特异性肽段,以检测肽段在生物样品环境中的稳定性。检测特异性肽段经过冻融三次的稳定性,每个样品六个生物学重复,结果如图3所示。
如图所示,DGDELLLIDTAWGAK和VGYIELDLNSGK在冻融三次后含量降低较为明显(冻融三次后含量<80%);LIAQLGGPGGVTAFAR和APLILVTYFTQPQPK有轻微的含量降低(冻融三次后含量80%~90%)。表明该四条肽段在使用时应避免反复冻融。
实施例5:靶向蛋白组检测细菌β-内酰胺酶特异性肽段检测稳定性评价。
将一定浓度的特异性肽段加入在不含β-内酰胺酶的大肠杆菌DH5α肽溶液中,检测特异性肽段在样品槽内(10℃)0、1、3或4天的稳定性,每个样品六个生物学重复。
结果如图4所示。DGDELLLIDTAWGAK含量在第1天显著下降(70%),第4天下降至6%。VGYIELDLNSGK的含量在第3天显著下降(64%),而APLILVTYFTQPQPK的含量在第4天下降到82%左右。说明该三条肽段应尽量减小肽段在样品槽中的停留时间,现配现用。
实施例6:液质联用检测临床菌株β-内酰胺酶。
采用上述单菌落β-内酰胺酶快速提取与检测方法对51株临床菌(25株大肠埃希菌,25株肺炎克雷伯菌,1株阴沟肠杆菌)进行检测;同时用PCR从基因水平进行检测,PCR引物见表4,用GoTaq Green Master Mix进行PCR扩增,检测结果如表5所示。IMP、VIM、OXA(NNGLTEAWLESSLK)和CTX-M(APLILVTYFTQPQPK)的特异性肽段灵敏度为100%,其他肽段灵敏度略低。所有肽段特异性均为100%。
表4 PCR引物
如表5所示,因密码子的简并性,氨基酸序列和基因分型并不能完全匹配,但对比PCR结果,所列肽段均可成功鉴定出相应耐药酶是否存在,这为临床耐药酶的检测提供了新的技术依据。此外,同一耐药酶的多个特异性肽段组合可以极大的提高检测的灵敏度和特异性,因此,以PRM为基础手段的靶向蛋白组学可以在肽段组合的基础上保证检测的灵敏度和特异性。
上述实施例显示,用以上所述方法可以有效检测特异性检测样品中耐药酶的存在。
本申请描述了多个实施例,但是该描述是示例性的,而不是限制性的,并且对于本领域的普通技术人员来说明显的是,在本申请所描述的实施例包含的范围内可以有更多的实施例和实现方案。尽管在附图中示出了许多可能的特征组合,并在具体实施方式中进行了讨论,但是所公开的特征的许多其它组合方式也是可能的。除非特意加以限制的情况以外,任何实施例的任何特征或要素可以与任何其它实施例中的任何其他特征或要素结合使用,或可以替代任何其它实施例中的任何其他特征或要素。
序列表
<110> 中国医学科学院医药生物技术研究所
<120> 一种快速检测肠杆菌β-内酰胺酶的方法
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Claims (10)
1.一种快速检测肠杆菌β-内酰胺酶的方法,包括:
样品处理:挑取单个菌落,悬浮在缓冲液中,超声处理,超滤离心去除缓冲液,然后进行酶切,得到肽段;
反相色谱分离:用反相色谱柱分离肽段,流动相A为包含甲酸的水溶液,流动相B为流动相A与乙腈的混合物;和
质谱法检测β-内酰胺酶的特异性肽段,
其中所述特异性肽段为选自以下的一种或几种:
LVVPSHSEVGDASLLK;
VQATNSFSGVNYWLVK;
NSFGGVNYWLVK;
LDEGVYVHTSFEEVNGWGVVPK;
LAEAEGNEIPTHSLEGLSSSGDAVR;
DGDELLLIDTAWGAK;
NNGLTEAWLESSLK;
IINHNLPVK;
ADIANNHPVTQQTLFELGSVSK;
TLQQGIALAQSR;
QLTLGHALGETQR;
TEPTLNTAIPGDPR;
LIAQLGGPGGVTAFAR;
APLILVTYFTQPQPK;
SDLVNYNPIAEK;和
VGYIELDLNSGK。
2.一种快速检测肠杆菌β-内酰胺酶的方法,包括:
样品处理:挑取单个菌落,悬浮在缓冲液中,超声处理,超滤离心去除缓冲液,然后进行酶切,得到肽段;
反相色谱分离:用反相色谱柱分离肽段,流动相A为包含甲酸的水溶液,流动相B为流动相A与乙腈的混合物;和
质谱法检测β-内酰胺酶的特异性肽段和同位素标记的特异性肽段,
其中所述特异性肽段为选自以下的一种或几种:
LVVPSHSEVGDASLLK;
VQATNSFSGVNYWLVK;
NSFGGVNYWLVK;
LDEGVYVHTSFEEVNGWGVVPK;
LAEAEGNEIPTHSLEGLSSSGDAVR;
DGDELLLIDTAWGAK;
NNGLTEAWLESSLK;
IINHNLPVK;
ADIANNHPVTQQTLFELGSVSK;
TLQQGIALAQSR;
QLTLGHALGETQR;
TEPTLNTAIPGDPR;
LIAQLGGPGGVTAFAR;
APLILVTYFTQPQPK;
SDLVNYNPIAEK;和
VGYIELDLNSGK,
上述特异性肽段下划线标记为同位素标记位点。
3.根据权利要求1或2所述的方法,其中所述单个菌落的直径>2mm。
4.根据权利要求1或2所述的方法,其中用蛋白酶进行酶切,优选采用胰蛋白酶,最优选采用测序级胰蛋白酶进行酶切。
5.根据权利要求4所述的方法,其中使用C18柱分离肽段。
6.根据权利要求5所述的方法,其中流动相A为包含0.1%甲酸的水溶液,流动相B为含0.1%甲酸的80%乙腈。
7.根据权利要求6所述的方法,其中洗脱为梯度洗脱。
8.根据权利要求7所述的方法,其中洗脱梯度为:从11%的流动相B开始,然后从11%的流动相B上升到13%的流动相B,持续2min,16min后梯度上升到32%的流动相B,7min后梯度上升到42%的流动相B,1min后梯度上升到95%的流动相B,保持4min。
9.根据权利要求8所述的方法,其中质谱检测的参数为:
MS1光谱:Orbitrap分析;分辨率,60000;质量范围,350~2000m/z;RFlens,30%;AGC靶,2.0×105;最大注射时间,50ms;和
MS2分析,HCD伴随以下条件:碰撞能量,30%;AGC,5.0×104;最大注射时间,特异性肽段,54ms;同位素标记的特异性肽段,22ms;Orbitrap分辨率,特异性肽段,30000;同位素标记的特异性肽段,7500;隔离窗口,1.4Da。
10.根据权利要求9所述的方法,其中利用Skyline软件分析处理质谱数据。
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