CN116121409B - 一种多重qPCR检测细菌的探针引物组、试剂盒以及检测方法 - Google Patents
一种多重qPCR检测细菌的探针引物组、试剂盒以及检测方法 Download PDFInfo
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Abstract
本发明提供了一种多重qPCR技术检测样本中四种细菌的探针引物组及检测方法。本发明针对鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌四种临床高频检出病原菌的基因序列,设计特异性引物和探针,利用多重qPCR同时鉴定样本中是否存在四种细菌的感染,优化了反应条件和检测流程,缩短检测时间,对临床感染患者而言,快速准确的鉴别诊断病原体对辅助治疗、用药和改善预后具有重要意义。同时,该多重引物探针组和检测方法的特异性好,准确性高、重复性好,还具有快速、实用、低成本和操作简便的特点,满足样本需求量大的临床检验实验室对四种细菌的检测需求。
Description
技术领域
本发明涉及生物检测领域,特别是涉及一种利用多重qPCR技术检测样本中多种细菌的方法。
背景技术
鲍曼不动杆菌(Acinetobacter baumannii)属于革兰氏阴性菌,是一种严格需氧、非乳糖发酵的条件致病菌,可广泛存在大自然中。该菌已经成为医院感染的主要来源,尤其是重症监护室,该菌通常会引起菌血症,肺炎,脑膜炎,腹膜炎,心内膜炎,以及泌尿道和皮肤感染等。医院感染性败血症和脓毒血症是不动杆菌属引起的最严重感染性疾病,病死率为32.0%。
肺炎克雷伯菌(Klebsiella pneumoniae)是为肠杆菌科中一类有荚膜的革兰氏阴性杆菌,其所致疾病占克雷伯氏菌属感染的95%以上,其对人致病性较强,是重要的条件致病菌和医源性感染菌之一。肺炎克雷伯氏菌为呼吸道感染的重要病原体,常引起重症肺炎,还可引起泌尿道感染、胆道感染、败血症和化脓性脑膜炎等严重疾病,尤其以败血症最为严重。其还经常通过污染的人工呼吸器、雾化器或各种导管侵入人体导致严重的医院内感染。
嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)是一种严格需氧革兰阴性杆菌,广泛存在于自然界,也可寄居于人的呼吸道和肠道中,为条件致病菌,是一种主要的医源感染的病原菌。该菌主要引起呼吸道感染,还可引起医院获得性肺炎、血流感染、腹腔感染、中枢神经系统感染、泌尿系统感染、皮肤软组织感染等。其医院感染有逐年上升的趋势,且其分离率在非发酵菌中,仅次于铜绿假单胞菌和鲍曼不动杆菌。
铜绿假单胞菌(Pseudomonas aeruginosa)原称绿脓杆菌,是广泛存在于自然界中的专性需氧革兰氏阴性杆菌。该菌为常见的条件致病菌,是医院内感染的主要病原菌之一。该菌经常引起术后伤口感染,也可引起褥疮、脓肿、化脓性中耳炎等,还是尿路感染的常见病原菌。其引起的很多感染发生在衰弱或免疫受损的住院病人,它是重症监护室感染的第二位最常见的病原菌,是呼吸机相关性肺炎的常见原因。除医院内获得感染外,HIV感染者很容易在社区获得该菌的感染。
长期以来,细菌感染的常规检测方法是培养法,其一直被视为病原体检测的“金标准”,但是该方法培养条件要求严格、耗时、易污染,且检出率低,无法满足快速精准检测的要求。实时荧光定量PCR(QuantitativeReal-timePCR)是指在PCR扩增反应体系中加入荧光基团,通过对扩增反应中每一个循环产物荧光信号的实时检测,最后通过标准曲线对未知模板进行定量分析的方法。其不仅实现了对模板的定量,且具有灵敏度高、特异性和可靠性更好、自动化程度高和无污染的特点。由此基础上发展而来的多重荧光定量PCR在病原的检测及鉴别诊断中变得越来越重要。因此迫切需要单管PCR同时定量检测以上4种病原菌的检测方法,使其在快速检测的同时可以节约成本,及时有效的为急危重症患者的治疗方案的制定提供参考依据。
发明内容
本发明的目的是将临床高频检出的四种病原菌通过多重荧光定量PCR技术来实现同时检测,弥补单重荧光定量PCR对多样本量检测时的高成本、费时耗力的缺点,降低劳动强度,缩短检测周期,减轻临床实验压力,简化操作流程,具有检测快速、灵敏度高、特异性强、操作方便等优点。
一种多重qPCR检测细菌的探针引物组,用于单反应同时检测鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌的四重荧光PCR反应,包括:
用于对鲍曼不动杆菌进行检测的正反向引物对,核苷酸序列如SEQ ID NO.1-2所示;
用于对肺炎克雷伯菌进行检测的正反向引物对,核苷酸序列如SEQ ID NO.4-5所示;
用于对嗜麦芽窄食单胞菌进行检测的正反向引物对,核苷酸序列如SEQ IDNO.13-14所示;
用于对铜绿假单胞菌检测的正反向引物对,核苷酸序列如SEQ ID NO.10-11所示。
所述的探针引物组中还包括分别对扩增产物进行报告的探针组合;所述的探针组合包括:
用于对鲍曼不动杆菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.3所示;
用于对肺炎克雷伯菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.6所示;
用于对嗜麦芽窄食单胞菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.15所示;
用于对铜绿假单胞菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.12所示。
所述探针的5’端修饰有报告基团,所述报告基团为FAM、HEX、ROX和Cy5;四种探针采用不同的报告基团修饰。
所述探针的3’端修饰有淬灭基团,所述猝灭基团为BHQ1和BHQ2;SEQ ID NO.3所示核苷酸序列与SEQ ID NO.6所示核苷酸序列采用淬灭基团BHQ1修饰,SEQ ID NO.15所示核苷酸序列与SEQ ID NO.12所示核苷酸序列采用淬灭基团BHQ2修饰。
本发明提供了一种检测方法,含有上述的四重荧光PCR引物组合和荧光探针组合,可以采用荧光定量PCR单反应同时检测鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌。
所述的样本包括但不限于肺泡灌洗液、痰液、血液、脑脊液、心包积液、胸水、尿液、脓液、拭子和组织等。
本发明提供了一种基于多重荧光qPCR技术同时检测四种高频检出病原菌的检测方法,包括以下步骤:
1)从样品中提取核酸;
2)以提取的核酸为模板,用上述所述的引物组及上述所述探针组进行多重荧光定量PCR扩增反应并收集荧光信号;
3)根据荧光信号判定样品中是否含有鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌。
进一步的,上述步骤2)中的多重荧光定量PCR扩增反应体系为:
所述引物探针混合液中含有上述所述的引物组及上述所述探针组。
进一步的,上述步骤2)中多重荧光qPCR扩增反应程序为:95℃预变性5min;95℃变性15s,58.5℃退火30s,共45个循环;
优选的,可将探针和引物提前混合一周的量,而不影响检测结果。
结果判定:鲍曼不动杆菌和肺炎克雷伯菌:待测样本孔有Cq值和明显的S型扩增曲线,说明该孔对应菌种为阳性检出;待测样本孔无Cq值和明显的S型扩增曲线,说明该孔对应菌种为阴性检出。嗜麦芽窄食单胞菌和铜绿假单胞菌:当NTC无Cq值时,待测样本孔有Cq值和明显的S型扩增曲线,说明该孔对应菌种为阳性检出;当NTC有Cq值时,待测样本满足CqNTC-Cq样本>1,说明该孔对应菌种为阳性检出;当NTC无Cq值时,待测样本孔无Cq值和明显的S型扩增曲线,说明该孔对应菌种为阴性检出;当NTC有Cq值时,待测样本无Cq值或满足CqNTC-Cq样本≤1,说明该孔对应菌种为阴性检出。
一种试剂盒,其中包含上述的多重qPCR检测细菌的探针引物组。
有益效果
本发明突破了单荧光PCR检测的限制,可实现同时检测鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌,进一步减少了实验时间和成本,且为样本量需求大的实验室降低劳动强度,减轻临床实验压力,缩短检测周期。本发明还具有快速、扩增效率高、准确性高、重复性好的特点,对感染患者尤其是急危重症患者而言,快速准确的鉴别诊断病原体对辅助治疗、用药和改善预后具有重要意义。
附图说明
图1:嗜麦芽窄食单胞菌S.mal探针引物测试临床样本的琼脂糖凝胶电泳图
图2:部分嗜麦芽窄食单胞菌S.mal探针引物测试临床样本的Sanger测序结果与比对图
图3:嗜麦芽窄食单胞菌Smal N1与Smal N2探针引物琼脂糖凝胶电泳图
图4:部分Sanger测序结果
图5:多重荧光PCR、单重荧光PCR扩增曲线图
图6:新探针引物预混结果对比
具体实施方式
以下结合附图和实施例对本发明进行进一步的阐述,下述说明仅是为了解释本发明,并不对其内容进行限定。
本发明中所用实验材料、试剂仪器如下:
实验试剂:磁珠法大体积游离核酸提取试剂盒(Tiangen:DP710-T2或QIAampCirculating Nucleic Acid Kit(50):55114);探针和引物合成(南京金斯瑞生物公司);HieffUniversal TaqMan multiplex qPCR master mix(翌圣:11211ES08)。
实验仪器:荧光定量PCR仪(Bio-Rad:CFX384);微孔板迷你离心机(其林贝尔:BE-6100)。
实施例1探针引物设计及测试
1)探针引物设计
通过对美国国立生物技术信息中心(NCBI)网站中鲍曼不动杆菌、肺炎克雷伯菌、铜绿假单胞菌和嗜麦芽窄食单胞菌基因组中特异性序列比对,找到高度保守的核苷酸序列,并结合文献,设计鲍曼不动杆菌、肺炎克雷伯菌、铜绿假单胞菌和嗜麦芽窄食单胞菌的特异性多重荧光PCR引物和探针。其验证测试实验引物组设计如下表1所示:
表1四种高频检出病原体的引物组
2)多重qPCR与单重qPCR对比
选择混合临床阳性样本进行多重qPCR与单重qPCR对比实验,结果如下表2所示:
表2多重qPCR与单重qPCR对比结果
N/A:未检测到Cq值;/:未进行检测
注:多重qPCR每种菌设置一个NTC,单重qPCR每种菌设置5个NTC
结果显示,多重qPCR和单重qPCR结果整体无明显差异,而多重qPCR和单重qPCR中NTC均出现了嗜麦芽窄食单胞菌Cq值,但胶图结果显示qPCR产物并非目的片段,可能为非特异性扩增。
3)临床样本测试
我们选取了8个临床进样本进行测试,判定方法是:
鲍曼不动杆菌和肺炎克雷伯菌:待测样本孔有Cq值和明显的S型扩增曲线,说明该孔对应菌种为阳性检出;待测样本孔无Cq值和明显的S型扩增曲线,说明该孔对应菌种为阴性检出。
嗜麦芽窄食单胞菌和铜绿假单胞菌:当NTC无Cq值时,待测样本孔有Cq值和明显的S型扩增曲线,说明该孔对应菌种为阳性检出;当NTC有Cq值时,待测样本满足Cq NTC-Cq样本>1,说明该孔对应菌种为阳性检出;当NTC无Cq值时,待测样本孔无Cq值和明显的S型扩增曲线,说明该孔对应菌种为阴性检出;当NTC有Cq值时,待测样本无Cq值或满足Cq NTC-Cq样本≤1,说明该孔对应菌种为阴性检出。
具体检测结果如下表3所示:
表3嗜麦芽窄食单胞菌临床样本验证结果统计
并对以上多重qPCR和临床其它方法验证的结果不一致的3例样本的扩增产物,进行琼脂糖凝胶电泳和Sanger测序进一步验证,部分结果见图1和图2。发现这3例样本均无法比对到嗜麦芽窄食单胞菌的基因组上,表明嗜麦芽窄食单胞菌的引物存在非特异性扩增,将考虑重新设计嗜麦芽窄食单胞菌的引物探针。
实施例2引物优化及验证
1)引物优化
因嗜麦芽窄食单胞菌的检测结果不符合预期,故重新设计嗜麦芽窄食单胞菌引物探针。
嗜麦芽窄食单胞菌新探针引物,如下表4所示:
表4嗜麦芽窄食单胞菌新探针引物
2)引物测试
选择混合临床阳性样本进行嗜麦芽窄食单胞菌多重qPCR与单qPCR重对比测试,结果如下表5所示:
表5嗜麦芽窄食单胞菌多重qPCR与单重qPCR对比结果
N/A:未检测到Cq值
qPCR产物琼脂糖凝胶电泳结果见图3,部分Sanger测序结果如图4。
结果显示,探针引物1的多重qPCR的Cq值略高于单重qPCR,但无明显差异;探针引物2的多重qPCR结果略低于单重qPCR;经琼脂糖凝胶电泳和Sanger测序验证,探针引物1和探针引物2的qPCR产物均为嗜麦芽窄食单胞菌目的片段;NTC中的嗜麦芽窄食单胞菌可能为环境污染;探针引物1的特异性比探针引物2好,Cq值整体偏低,但NTC的Cq值也较低;探针引物2扩增出的非特异性产物较多。故选择探针引物Smal N1进行后续测试。
实施例3检测灵敏性验证
将鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌的扩境目的片段合成质粒,并稀释于不同的10~108copies/mL的标准溶液,再将相同浓度的各个细菌的标准溶液按照等体积混合后分别采用上述确定的引物、反应条件进行荧光定量PCR检测,并计算得到灵敏度。经过检测,四种细菌检测灵敏度分别为10copies/mL。
实施例4临床样本检测
用本发明的多重荧光PCR方法检测临床10份感染患者的样本,样本类型包括肺泡灌洗液、痰液和穿刺液。结果见表6,结果显示本发明所建立的方法与mNGS测序结果完全一致,本发明准确、可靠。
表6临床样本检测结果
以上所述实施例仅为本发明的几种优选实施方式,并不能因此理解为对本发明专利范围的限制。应当指出的是,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (8)
1.一种多重qPCR检测细菌的探针引物组,其特征在于,用于单反应同时检测鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌的四重荧光PCR反应,包括:
用于对鲍曼不动杆菌进行检测的正反向引物对,核苷酸序列如SEQ ID NO.1-2所示;
用于对肺炎克雷伯菌进行检测的正反向引物对,核苷酸序列如SEQ ID NO.4-5所示;
用于对嗜麦芽窄食单胞菌进行检测的正反向引物对,核苷酸序列如SEQ ID NO.13-14所示;
用于对铜绿假单胞菌检测的正反向引物对,核苷酸序列如SEQ ID NO.10-11所示;
所述的探针引物组中还包括分别对扩增产物进行报告的探针组合;所述的探针组合包括:
用于对鲍曼不动杆菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.3所示;
用于对肺炎克雷伯菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.6所示;
用于对嗜麦芽窄食单胞菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.15所示;
用于对铜绿假单胞菌扩增产物进行报告的探针,核苷酸序列如SEQ ID NO.12所示。
2.根据权利要求1所述的多重qPCR检测细菌的探针引物组,其特征在于,所述探针的5’端修饰有报告基团,所述报告基团为FAM、HEX、ROX和Cy5;四种探针采用不同的报告基团修饰。
3.根据权利要求2所述的多重qPCR检测细菌的探针引物组,其特征在于,所述探针的3’端修饰有淬灭基团,所述淬灭基团为BHQ1和BHQ2; SEQ ID NO.3所示核苷酸序列与SEQ IDNO.6所示核苷酸序列采用淬灭基团BHQ1修饰,SEQ ID NO.15所示核苷酸序列与SEQ IDNO.12所示核苷酸序列采用淬灭基团BHQ2修饰。
4.权利要求1所述的探针引物组在制备四重荧光PCR反应检测鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌的试剂盒中的用途,其特征在于,包括如下步骤:采用探针引物组进行多重定量PCR反应对鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌进行鉴定。
5.根据权利要求4所述的用途,其特征在于,包括如下步骤:1)从样品中提取核酸;2)以提取的核酸为模板,用探针引物组进行多重荧光定量PCR扩增反应并收集荧光信号;3)根据荧光信号判定样品中是否含有鲍曼不动杆菌、肺炎克雷伯菌、嗜麦芽窄食单胞菌和铜绿假单胞菌。
6.根据权利要求5所述的用途,其特征在于,所述的样本包括肺泡灌洗液、痰液、血液、脑脊液、心包积液、胸水、尿液、脓液、拭子或者组织。
7.根据权利要求6所述的用途,其特征在于,上述步骤2)中的多重荧光定量PCR扩增反应体系为:
;
多重荧光qPCR扩增反应程序为:95℃预变性5min;95℃变性15s,58.5℃退火30s,共45个循环。
8.一种试剂盒,其特征在于,其中包含有权利要求1所述的探针引物组。
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