CN114507746A - 用于高产毒艰难类梭菌快速鉴定和分型的探针、引物组及方法 - Google Patents
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Abstract
本发明提供了一种用于高产毒艰难类梭菌快速鉴定和分型的探针组、引物组,同时提供了利用上述探针组和引物组进行高产毒艰难类梭菌快速鉴定和分型的方法。本发明能快速准确鉴定出RT027/ST1型高产毒艰难类梭菌,还能初步鉴定出非027型高产毒艰难类梭菌及其他产毒型艰难类梭菌,对CDI的临床诊断有重要意义。
Description
技术领域
本发明涉及一种用于高产毒艰难类梭菌快速鉴定和分型的探针、引物组及方法。
背景技术
艰难类梭菌(Clostridioides difficile,C. difficile)是一种革兰染色阳性,能产生芽孢的专性厌氧菌。C. difficile是医院抗生素相关性腹泻(Antibiotic-associated diarrhea,AAD)的主要病原菌,可以导致轻到重度的腹泻、结肠炎、伪膜性肠炎、中毒性巨结肠等,严重者甚至可导致死亡。
近年来,国内外艰难类梭菌感染(Clostridiodes difficile infection,CDI)的发病率和死亡率逐渐增高,每年致全球数万人死亡,治疗费用高达数十亿美元。2013年和2019年,美国CDC(Center for Disease Control and Prevention)将艰难类梭菌感染列为“微生物导致公共健康威胁”的紧迫级病原菌。艰难类梭菌致病在很大程度上归因于这两种毒素——肠毒素A(TcdA)和细胞毒素B(TcdB)。CDI的主要发病过程是通过芽孢经粪-口途径进入人体定植在结肠,当长期使用广谱抗生素或质子泵抑制剂等导致肠道菌群失衡后,艰难类梭菌大量繁殖产生TcdA和TcdB,破坏肠道细胞骨架,使得细胞通透性增高,最终导致腹泻等疾病症状。除此之外,近年来艰难类梭菌高产毒株NAP1/RT027/B1在欧美多个国家暴发流行,其不仅产生TcdA和TcdB,还可以产生二元毒素(cdtA和cdtB),导致CDI的发病率和病死率均大幅增高,引起了全社会对CDI的广泛关注。
CDI的快速而准确的诊断,对指导治疗和阻止病原菌的传播至关重要。目前,针对不同的检测目标,CDI的实验室检测方法有多种。检测粪便中的游离毒素的方法有酶免疫测定法(EIA)和粪便细胞毒性测定法(CTA);测定艰难类梭菌的特异抗原有EIA方法测谷氨酸脱氢酶(GDH);检测产毒艰难类梭菌菌株的方法有产毒艰难类梭菌培养(TC)和核酸扩增试验(NAAT)。其中,粪便CTA和TC分别是检测毒素或有毒菌株的金标准,但是由于技术问题和检测时间过长,这两种方法都没有得到常规使用。NAAT方法是基于实时荧光PCR、环介导等温扩增技术或微阵列技术等分子检测技术进行的艰难类梭菌检测。NAATs检测的靶基因种类很多,包括tcdB、tcdA以及tcdC基因(将∆117作为高产毒株RT027/ST1流行毒株的标记)或二元毒素(cdtA/B)基因。NAATs方法非常敏感(平均敏感性为96%(95% CI = 0.93-0.98)),具有较高的阴性预测值。而且,其检测速度较快,可以在一小时内得到结果,因此常把它作为CDI筛选试验。然而,NAATs方法仍存在潜在问题,如由于基因变异,tcdB或 tcdA基因片段可能导致假阴性。而使用tcdC(∆117)作为对高产毒株RT027/ST1型艰难类梭菌的特异性检测位点,也存在一定争议。市场现有的RT027/ST1型高产毒艰难类梭菌诊断试剂盒费用相对较为昂贵,不适用于临床CDI的大规模筛查或实验室的大批量艰难类梭菌检测。因此,针对RT027/ST1高产毒艰难类梭菌特异基因序列设计新的引物和探针,开发准确快速、经济适用的高产毒株RT027/ST1型艰难类梭菌检测试剂盒对我国CDI的防治有重要意义。
发明内容
本发明的目的是提供一种用于高产毒艰难类梭菌快速鉴定和分型的探针组、引物组,同时提供了利用上述探针组和引物组进行高产毒艰难类梭菌快速鉴定和分型的方法。
本发明采用如下技术方案
一种用于高产毒艰难类梭菌快速鉴定和分型的探针组,其包括pilW基因探针、二元毒素B基因探针、毒素B基因探针以及热休克蛋白基因探针;
所述pilW基因探针的序列如SEQIDNo.1所示;
所述二元毒素B基因探针的序列如SEQIDNo.2所示;
所述毒素B基因探针的序列如SEQIDNo.3所示;
所述热休克蛋白基因探针的序列如SEQIDNo.4所示。
一种用于高产毒艰难类梭菌快速鉴定和分型的引物组,其包括扩增pilW基因的引物对1F/R、扩增二元毒素B基因的引物对2F/R、扩增毒素B基因引物对3F/R以及扩增热休克蛋白基因的引物对4F/R;
所述引物对1F/R的序列如SEQIDNo.5和SEQIDNo.6所示;
所述引物对2F/R的序列如SEQIDNo.7和SEQIDNo.8所示;
所述引物对3F/R的序列如SEQIDNo.9和SEQIDNo.10所示;
所述引物对4F/R的序列如SEQIDNo.11和SEQIDNo.12所示。
一种包含上述探针组和/或引物组进行高产毒艰难类梭菌快速鉴定和分型的试剂盒。
一种利用上述探针组和引物组进行高产毒艰难类梭菌快速鉴定和分型的方法,利用四重实时荧光定量PCR检测pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因的保守区域,快速鉴定高产毒艰难类梭菌并分析菌株的毒力情况。
高产毒艰难类梭菌快速鉴定和分型的方法中,(a)若pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因均为阳性表示检出RT027/ST1型高产毒艰难类梭菌;(b)若二元毒素B基因、毒素B基因以及热休克蛋白基因均为阳性而pilW基因为阴性表示检出非027型高产毒菌株;(c)若仅有毒素B基因以及热休克蛋白基因为阳性,表示检出产毒型艰难类梭菌;(d)若仅有热休克蛋白基因阳性表示检出非产毒型艰难类梭菌。
高产毒艰难类梭菌快速鉴定和分型的方法具体包括如下步骤:
(I)用四重实时荧光定量PCR法对上述提取的DNA进行pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因检测;
(II)结果判读。
进一步的,其还包括标本处理步骤:
(1)向待检粪便标本中加入无水乙醇,静置1h后3200r/min离心15min,离心结束后弃去标本中的无水乙醇,用灭菌接种环挑取试管底部粪便标本三区划线接种于CDMN平板,37℃厌氧培养36-48h;
(2)挑取上述CDMN平板上长出的典型艰难类梭菌菌落涂片革兰染色镜检,鉴定为艰难类梭菌后挑取单菌落于强化布氏血琼脂上传代培养,37℃厌氧培养36-48h;
(3)将强化布氏血琼脂平板上的菌落进行DNA提取。
进一步的,步骤(1)中,粪便和无水乙醇的比例为1∶1。
进一步的,步骤(3)中,将强化布氏血琼脂平板上的菌落尽数刮进无菌水中,按照细菌DNA提取试剂盒说明进行DNA提取。
进一步的,步骤(I)中,四重实时荧光定量PCR法的反应总体系为20μL,包括Premix Ex TaqTM10μL,上、下游引物及探针各0.2μL,ddH2O 5.6μL,模板DNA 2μL;反应条件为:95℃预变性3min;95℃变性5s,61℃退火延伸30s,共进行39个循环。
本发明的有益效果在于:本发明提供了新的引物组和探针组,实现了RT027/ST1型高产毒艰难类梭菌的特异性鉴定,同时还可对非027型高产毒艰难类梭菌、产毒型艰难类梭菌进行快速鉴定,在CDI快速诊断方面有重要的应用价值。
附图说明
图1、RT027型和RT017菌株基因预测的分泌蛋白和膜相关蛋白的比较。
图2、线上RT027/ST1型菌株pilW基因特异性位点突变(165 C→T)。
图3、本地菌株pilW基因扩增结果。
图4、本地RT027/ST1型菌株pilW基因特异性位点突变(165 C→T)。
图5、RT027/ST1型高产毒艰难类梭菌实验结果(pilW + cdtB + tcdB + HSP +)。
图6、非RT 027型高产毒艰难类梭菌实验结果(pilW - cdtB + tcdB + HSP +)。
图7、产毒型艰难类梭菌实验结果(pilW - cdtB - tcdB + HSP +)。
图8、非产毒型艰难类梭菌实验结果(pilW - cdtB - tcdB - HSP +)。
具体实施方式
下面结合附图对本发明进行详细的说明。
选取高产毒艰难类梭菌RT027/ST1型菌株和普通产毒艰难类梭菌RT017/ST37型菌株进行全基因组测序分析,并进行蛋白的定位预测。
结果显示,高产毒RT027/ST1型菌株的分泌性蛋白有158个分泌性蛋白,903个膜相关蛋白;普通产毒艰难类梭菌RT017/ST37型菌株有145个分泌性蛋白,888个膜相关蛋白。
分别比较RT027/ST1型和RT017/ST37菌株的分泌性蛋白和膜相关蛋白,发现有个43分泌性蛋白和89个膜相关蛋白特异存在于RT027/ST1细菌;有30个分泌性蛋白和74个膜相关蛋白特异存在于RT017/ST37菌株(图1)。
在膜相关蛋白中,我们发现菌毛蛋白基因pilW的基因特异的存在于RT027/ST1型细菌,而在RT017/ST37菌株则没有。
为了进一步验证菌毛蛋白基因pilW否在RT027/ST1和RT017/ST37菌株中具有特异性,使用pilW的基因序列在NCBI数据库中进行BLAST序列比对。选择比对得到的与检索序列一致性较高的83个基因(Identity score>85)进行比对分析。结果发现25株RT027/ST1菌株中有24株菌的pilW基因存在特异性位点突变(165 C→T)(图2)。而其他菌株中,仅有ST188型菌株存在此突变。
鉴于尚未有关于ST188型艰难类梭菌感染的报道,而RT027/ST1型高产毒菌株又是临床上导致严重CDI感染的主要致病菌,我们认为该突变位点对于艰难类梭菌高产毒菌株(RT027/ST1)检测和鉴定具有重要意义。更重要的是,在随机选择的83个基因序列中,并没有发现RT017/ST37型菌株,结合全基因测序结果,我们推测该基因在RT017/ST37型不存在。
为了进一步确认pilW基因在RT017/ST37菌株中的特异性缺失以及pilW基因位点突变(165 C→T)对RT027/ST1菌株的鉴定作用,我们设计了pilW基因的引物(前引物:ACTCTAGTGGAATTATTAGTAGTAATTGCTATAA;后引物:ATCTAGACCACTTTTGCTTATTTCTCCTT),对本地的实际分离的不同型别菌株(ST1,ST2,ST3,ST37,ST35,ST54)进行普通PCR扩增并进行琼脂糖凝胶电泳和测序。反应总体系为50μL,包括 2×Taq Master Mix混合液25μL,上、下游引物(10 μmol/L)各2μL, ddH2O 19μL,模板DNA 2μL。反应条件为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸1min,共进行29个循环;最后72℃再延伸10min。将所得PCR产物1%琼脂糖凝胶进行电泳并成像。质量合格后,对扩增产物进行测序和序列比对。
结果显示,本地的分离的RT017/ST37菌株中确实缺失了pilW基因(图3)。而且,pilW基因位点突变(165 C→T)在本地分离的菌株中对RT027/ST1菌株也具有特异性(图4)。基于以上研究结果,我们设计的pilW基因的引物可准确的从众多ST型中鉴定出ST1型菌株。
在此基础上,我们利用pilW序列设计了引物和探针。与此同时,我们基于二元毒素B、毒素B及热休克蛋白基因序列的保守区域设计了用于检测二元毒素B(cdtB)和毒素B(tcdB)的引物和探针,加上热休克蛋白(HSP)作为内标基因,共同组成四重实时荧光定量PCR(Quadruple RT-PCR)。
表1 四重实时荧光定量PCR的引物及探针序列
多重荧光定量PCR检测:用PCR法对上述提取的DNA进行pilW,cdtB,tcdB,HSP基因检测(引物及探针序列见表1)。反应总体系为20μL,包括 Premix Ex TaqTM (Takara BioInc)10μL,上、下游引物及探针 (50 μmol/L)各0.2μL, ddH2O 5.6μL,模板DNA 2μL。反应条件为:95℃预变性3min;95℃变性5s,61℃退火延伸30s,共进行39个循环。反应结束后可直接根据各靶标曲线的扩增情况进行结果判读。
对于具体的样本而言,可选择预处理的方法对样品进行处理:
(a)富集芽孢法培养艰难类梭菌:向待检粪便标本中加入无水乙醇(粪便和无水乙醇的比例为1∶1),静置1h后3200r/min离心15min,离心结束后弃去标本中的无水乙醇,用灭菌接种环挑取试管底部粪便标本三区划线接种于CDMN平板,37℃厌氧培养36-48h.
(b)挑取上述CDMN平板上长出的典型艰难类梭菌菌落涂片革兰染色镜检,艰难类梭菌为革兰阳性杆菌,部分菌体可于菌体次级端形成芽孢,鉴定为艰难类梭菌后挑取单菌落于强化布氏血琼脂上传代培养,37℃厌氧培养36-48h。
CDMN培养基的配置方法为:称取 34.5g 艰难类梭菌基础琼脂粉末,加入到 500ml双蒸水中,121℃、 1.034×105Pa 高压蒸汽灭菌 15min,冷却到 50℃左右,将预先用 2ml无菌双蒸水溶解的艰难类梭菌 CDMN 选择性添加剂加入培养基,再按照 6%的比例加入新鲜无菌脱纤维羊血,摇晃混匀后,在无菌环境中加入到一次性 70mm 培养皿中,待其冷却凝固后放入4℃冰箱备用。
强化布鲁氏血琼脂培养基的配置方法为:称取布鲁氏琼脂干粉 34.4g,加入到800ml 双蒸水中,同时加入0.8ml氯化血红素(5mg/ml),0.8ml维生素 K1 工作液(1mg/ml),121℃、 1.034×105 Pa 高压蒸汽灭菌 15min,冷却至 50℃左右时,按照 6%的比例加入新鲜无菌脱纤维羊血,摇晃混匀后,在无菌环境下加入到一次性 70mm 培养皿中,待其冷却凝固后放入4℃冰箱备用。
(c)DNA提取:将上述强化布氏血琼脂平板上的菌落尽数刮进无菌水中,按照细菌DNA提取试剂盒说明进行DNA提取,提取出来的DNA若不能及时进行检测可冻存于-20℃。
大部分高产毒艰难类梭菌的二元毒素B为阳性,几乎所有的产毒型艰难类梭菌毒素B为阳性。Quadruple RT-PCR在快速鉴定高产毒艰难类梭菌、诊断CDI的同时,还能分析菌株的毒力表达情况。
(1)若 pilW 、 cdtB 、 tcdB 、HSP 均为阳性表示检出RT027/ST1型高产毒艰难类梭菌;
(2)若 cdtB 、 tcdB 、 HSP 为阳性而pilW为阴性表示检出其他高产毒菌株(如RT078型);
(3)若仅有tcdB 、HSP 为阳性表示检出产毒型艰难类梭菌,提示患者体内可能发生CDI;
(4)若仅有HSP 为阳性表示检出非产毒型艰难类梭菌。
利用上述方法对不同类型的艰难类梭菌进行验证实验,结果如图5~图8所示。验证实验表明,由RT027/ST1型艰难类梭菌特有的pilW,高产毒艰难类梭菌表达的二元毒素B(cdtB),产毒艰难类梭菌表达的毒素B(tcdB),内参基因(HSP)组成的四重RT-PCR(pilW+cdtB+tcdB+HSP)可用于特异性地鉴定高产毒RT027/ST1型艰难类梭菌,与此同时,还能检出其他高产毒株和产毒株,在CDI快速诊断方面有重要的应用价值。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述,但并不限于此,本领域的技术人员很容易根据上述实施例领会本发明的精神,并作出不同的引申和变化,但只要不脱离本发明的精神,都在本发明的保护范围之内。
SEQUENCE LISTING
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Claims (6)
1.一种用于高产毒艰难类梭菌快速鉴定和分型的探针组,其特征在于,包括pilW基因探针、二元毒素B基因探针、毒素B基因探针以及热休克蛋白基因探针;
所述pilW基因探针的序列如SEQIDNo.1所示;
所述二元毒素B基因探针的序列如SEQIDNo.2所示;
所述毒素B基因探针的序列如SEQIDNo.3所示;
所述热休克蛋白基因探针的序列如SEQIDNo.4所示。
2.一种用于高产毒艰难类梭菌快速鉴定和分型的引物组,其特征在于,其包括扩增pilW基因的引物对1F/R、扩增二元毒素B基因的引物对2F/R、扩增毒素B基因引物对3F/R以及扩增热休克蛋白基因的引物对4F/R;
所述引物对1F/R的序列如SEQIDNo.5和SEQIDNo.6所示;
所述引物对2F/R的序列如SEQIDNo.7和SEQIDNo.8所示;
所述引物对3F/R的序列如SEQIDNo.9和SEQIDNo.10所示;
所述引物对4F/R的序列如SEQIDNo.11和SEQIDNo.12所示。
3.一种包含如权利要求1所述的探针组和/或权利要求2所述的引物组进行高产毒艰难类梭菌快速鉴定和分型的试剂盒。
4.一种利用如权利要求1所述的探针组和权利要求2所述的引物组进行高产毒艰难类梭菌快速鉴定和分型的方法,其特征在于,利用四重实时荧光定量PCR检测pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因的保守区域,快速鉴定高产毒艰难类梭菌并分析菌株的毒力表达情况。
5.根据权利要求4所述的高产毒艰难类梭菌快速鉴定和分型的方法,其特征在于,(a)若pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因均为阳性表示检出RT027/ST1型高产毒艰难类梭菌;(b)若二元毒素B基因、毒素B基因以及热休克蛋白基因均为阳性而pilW基因为阴性表示检出其他高产毒菌株;(c)若仅有毒素B基因以及热休克蛋白基因为阳性,表示检出产毒型艰难类梭菌;(d)若仅有热休克蛋白基因阳性表示检出非产毒型艰难类梭菌。
6.根据权利要求5所述的高产毒艰难类梭菌快速鉴定和分型的方法,其特征在于,其具体包括如下步骤:
(I)用四重实时荧光定量PCR法对提取的艰难类梭菌DNA进行pilW基因、二元毒素B基因、毒素B基因以及热休克蛋白基因检测;
(II)结果判读。
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