CN114836579B - 中枢神经系统感染性病原体的多重荧光定量pcr检测引物组合 - Google Patents
中枢神经系统感染性病原体的多重荧光定量pcr检测引物组合 Download PDFInfo
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Abstract
本发明公开了一种中枢神经系统感染性病原体的多重荧光定量PCR检测引物组合,其包括检测脑膜炎奈瑟菌、疱疹病毒6型、伤寒立克次氏体、猿猴空泡病毒40、伤寒沙门氏菌、解脲脲原体、弓形虫、BK多瘤病毒的特异性引物和探针;该方法具有特异性强、灵敏度高、反应快速且应用范围广的优点,大幅度提升了检测的速度并节约成本,可广泛应用于脑炎脑膜炎等各种中枢神经系统感染性疾病常见病原体的检测,提高病原体的检出率,对临床诊断中枢神经系统感染性疾病意义重大。
Description
技术领域
本发明属于病原菌体测技术领域,具体涉及一种中枢神经系统感染性病原体的多重荧光定量PCR检测引物组合。
背景技术
中枢神经系统(CNS)感染性疾病是由细菌、病毒、真菌等病原体侵犯CNS的脑膜、实质、血管导致的急性或慢性炎症(非炎症性)疾病。
中枢神经系统感染的途径包括血源性感染、直接感染和间接感染。血源性感染是最常见的感染途径,它是指病原体先穿过粘膜屏障进入血液再穿透血脑屏障进入中枢神经系统。直接感染是指神经外科手术或开放性颅脑外伤导致血脑屏障开放,当进行开颅手术的时,病原体感染的几率会上升,或是造成颅内邻近组织感染如中耳炎、额窦炎,感染后病原体会蔓延侵入CNS。逆行感染是指一些嗜神经病毒比较容易感染呼吸道、消化道或皮肤黏膜, 然后沿神经末梢逆行感染,导致中枢神经系统感染性疾病。确定了CNS感染性疾病常见的8种病原体有脑膜炎奈瑟菌(Neisseria meningitidis)、疱疹病毒6型(Human HerpesVirus 6,HHV6)、伤寒立克次氏体(Rickettsia typhi)、猿猴空泡病毒40(SimianVacuolating Virus SV40)、伤寒沙门氏菌(Bacterium typhosum)、解脲脲原体(Ureaplasma urealyticum)、弓形虫(Toxoplasma Gondii)、BK多瘤病毒(BKPolyornaviruses,BKV)等会导致严重的后果。
由于该些病原体感染的途径较广泛,所以导致的疾病普遍具有较高的发病率和死亡率,因此及时发现和治疗对于患者的生存至关重要。目前临床上常用的检测方法包括培养法、镜检法、免疫学方法等具有一定的局限性,检测灵敏度低或检测时间长等。生理生化检测必须对新鲜的脑脊液进行检测,检测的灵敏度很低,只能大致判断中枢感染类型,临床上一般会将脑脊液涂片检查和分离培养方法结合使用,虽然检测成本低,但灵敏度极差,很容易延误病情,耽误病人的治疗。免疫学检测检测容易出现假阳性,在临床上诊断具有局限性。实验室常用的诊断方法包括PCR检测、基因芯片技术、二代测序技术和荧光定量PCR检测。这些方法中PCR检测检测通量低且容易造成假阳性反应,二代测序技术与基因芯片技术所用成本很高,不能大规模的应用与临床。
发明内容
针对现有技术的不足,本发明提供了一种中枢神经系统感染性病原体的多重荧光定量PCR检测引物组合,其包括检测脑膜炎奈瑟菌、疱疹病毒6型、伤寒立克次氏体、猿猴空泡病毒40、伤寒沙门氏菌、解脲脲原体、弓形虫、BK多瘤病毒的特异性引物和探针;检测时采用多重荧光定量PCR方法,检测中还使用到GAPDH基因的特异引物探针及多重荧光定量PCR检测的其他常规试剂;本发明检测引物组合具有检测灵敏度高、特异性强、重复性良好,对仪器设备要求不高、操作简便、所需时间短等优点,因此具有较大的应用价值。
所述特异性引物为针对脑膜炎奈瑟菌的SEQ ID NO:1和SEQ ID NO:2、针对疱疹病毒6型的SEQ ID NO:4和SEQ ID NO:5、针对伤寒立克次氏体的SEQ ID NO:7和SEQ ID NO:8、针对猿猴空泡病毒40的SEQ ID NO:10和SEQ ID NO:11、针对伤寒沙门氏菌的SEQ ID NO:13和SEQ ID NO:14、针对解脲脲原体的SEQ ID NO:16和SEQ ID NO:17、针对弓形虫的SEQ IDNO:19和SEQ ID NO:20;针对BK多瘤病毒的SEQ ID NO:22和SEQ ID NO:23;
所述探针为针对脑膜炎奈瑟菌的SEQ ID NO:3、针对疱疹病毒6型的SEQ ID NO:6、针对伤寒立克次氏体的SEQ ID NO:9、针对猿猴空泡病毒40的SEQ ID NO:12、针对伤寒沙门氏菌的SEQ ID NO:15、针对解脲脲原体的SEQ ID NO:18、针对弓形虫的SEQ ID NO:21;针对BK多瘤病毒的SEQ ID NO:24。
使用上述多重荧光定量PCR检测引物组合的方法如下:
1、样品核酸(DNA)提取,样品为脑脊液;
2、以步骤(1)核酸为模板,采用靶向8种病原体的特异性引物和探针(检测8种病原体及内参基因的特异性引物和探针的核苷酸序列如SEQ ID NO:1- SEQ ID NO:24所示),经过多重实时荧光定量PCR进行检测,以GAPDH基因为内参(特异性引物和探针的核苷酸序列如SEQ ID NO:25- SEQ ID NO:27所示),根据Ct值进行结果判定;多重荧光定量PCR检测试剂检测病原体的扩增反应体系如下:2×Pro Taq HS Probe Premix 20μL、4种病原体及内参GAPDH基因上下游引物和探针各1μL、DNA模板4μL、ddH2O补足至40μL。反应程序为:95℃预变性30s;95℃变性3s,58℃退火并延伸30s,40个循环,在每个循环的退火延伸阶段收集荧光信号;
在检测中检测脑膜炎奈瑟菌、疱疹病毒6型、伤寒立克次氏体、猿猴空泡病毒40的特异性引物和探针同时使用;检测伤寒沙门氏菌、解脲脲原体、弓形虫、BK多瘤病毒的特异性引物和探针同时使用;以GAPDH基因为内参。
3、检测阳性结果判读如下:(1)内参(GAPDH基因)Ct值≤36,阴性对照组、无模板对照组无Ct值;如不符合须再次进行多重实时荧光定量PCR检测,或重新提取核酸进行多重实时荧光定量PCR检测;(2)病原体Ct值≤36.0,若Ct值>36.0,需要针对该病原体进行单重实时荧光定量PCR验证;(3)扩增曲线呈标准“S”型且无异常波动。
本发明与现有技术相比,本发明具有如下优点和技术效果:
本发明提供的多重荧光定量PCR检测引物组合应用在多重荧光定量PCR检测中,实验结果显示每个病原体的特异性引物和探针与其他病原体之间均无交叉反应,针对各个病原体的特异性良好;灵敏度评价发现脑膜炎奈瑟菌、伤寒立克次氏体、猿猴空泡病毒40、伤寒沙门氏菌、弓形虫检测限达到10copies/μL量级;HHV6、解脲脲原体、BK多瘤病毒的检测限达到100copies/μL量级,灵敏度很高;对该方法进行重复性评价发现各组病原体的特异性引物和探针在批间和批内的变异系数(CV)均小于5,重复性良好;通过检测17例脑脊液临床样本进行MqPCR准确性评价,结果发现MqPCR方法准确性良好。
附图说明
图1为脑膜炎奈瑟菌的单重qPCR特异性试验结果;
图2为疱疹病毒6型的单重qPCR特异性试验结果;
图3为伤寒立克次氏体的的单重qPCR特异性试验结果
图4为猿猴空泡病毒40的单重qPCR特异性试验结果;
图5为伤寒沙门氏菌的单重qPCR特异性试验结果;
图6为解脲脲原体的单重qPCR特异性试验结果;
图7为弓形虫的单重qPCR特异性试验结果;
图8为BK多瘤病毒的单重qPCR特异性试验结果;
图9为内参基因的单重qPCR特异性试验结果;
图10 为脑膜炎奈瑟菌、疱疹病毒6型、伤寒立克次氏体、猿猴空泡病毒40的多重qPCR特异性试验结果;
图11为伤寒沙门氏菌、解脲脲原体、弓形虫、BK多瘤病毒的多重qPCR特异性试验结果;
图12为脑膜炎奈瑟菌、疱疹病毒6型的多重qPCR灵敏性试验结果;
图13为伤寒立克次氏体、猿猴空泡病毒40的多重qPCR灵敏性试验结果;
图14为伤寒沙门氏菌、解脲脲原体的多重qPCR灵敏性试验结果;
图15为弓形虫、BK多瘤病毒的多重qPCR灵敏性试验结果;
图16为内参GADPH基因的多重qPCR灵敏性试验结果。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下通过具体实施方式来进一步说明本发明的技术方案,但本发明并非局限在实施例范围内;以下实施例中采用的材料不限于上述列举,可用其他同类材料替代,仪器未注明具体条件的,按照常规条件,或按照制造厂商所建议的条件,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂,使用常规方法。
实施例1:引物和探针的设计
1、在NCBI(National Center for Biotechnology Information,美国国家生物技术信息中心)网站中下载病原体基因参考序列如下:脑膜炎奈瑟菌ctrA编码基因、疱疹病毒6型large tegument protein编码基因、伤寒立克次氏体outer membrane protein B(ompB)编码基因、猿猴空泡病毒40 small T antigen编码基因、伤寒沙门氏菌staG编码基因、解脲脲原体ureB编码基因、弓形虫重复序列TgMc132a编码基因、BK多瘤病毒VP1编码基因各20条;使用Mega 7软件对核苷酸序列进行对齐,使用Primer Select软件设计引物与探针,并需要满足以下条件:
(1)Tm值:一般探针Tm值较引物Tm高8-10℃,其中探针Tm值一般为60℃以上;
(2)GC含量:一般不低于40%;
(3)不产生引物二聚体,发夹结构软件评估结果为OK;
(4)扩增片段大小一般小于200bp;
2、初步设计完成的引物探针核苷酸序列,再次使用NCBI网站中的BLAST检索功能,进行比对,选择特异性高的引物、探针序列;
靶向8种中枢神经系统感染性疾病病原体及内参GAPDH基因的特异性引物和探针的核苷酸序列如SEQ ID NO:1 -SEQ ID NO:27所示,见下表;
;
4、质粒的构建
将8种病原体的特异性序列、内参基因GAPDH的序列与pUC57载体连接,合成质粒标准品,其中弓形虫、脑膜炎奈瑟菌合成在一个质粒上,伤寒立克次氏体、SV40合成在一个质粒上,BKV和HHV6合成到一个质粒上,伤寒沙门氏菌、解脲脲原体、内参GAPDH单独各合成一个质粒,质粒构建由中美泰和生物技术北京有限公司完成;通过紫外分光光度计进行浓度测定,根据各个质粒长度和浓度计算出质粒的拷贝数;
各病原体的的特异性序列如下表所示:
拷贝数结果如下表所示;
将质粒按照10倍稀释法梯度稀释,共设置六个梯度分别为106、105、104、103、102、10copies/μL量级。
实施例2:qPCR扩增及特异性、灵敏度、重复性试验
1、单重荧光定量PCR
将浓度为103 copies/μL的质粒标准品分别取10μL混合为模板,使用艾科瑞生物公司Pro Taq HS预混型探针法qPCR试剂盒进行单重荧光定量PCR检测,分别用每种病原体的特异性引物和探针检测8种病原体的混合质粒模板,检测结果如图1-9所示;从结果能够看出8种病原体及内参基因均有扩增曲线。
2、多重qPCR特异性试验
由于多重荧光定量PCR需要在一个体系中检测4种病原体及1种内参基因,所以多重荧光定量PCR反应体系为40μL,其中4种病原体及1种内参基因的引物探针均加入1μL,模板为4μL:
扩增反应程序为:95℃预变性30s;95℃变性3s,58℃退火并延伸30s,40个循环,在每个循环的退火延伸阶段收集荧光信号。
将浓度为103 copies/μL量级的质粒标准品分别取10μL混合为模板,使用艾科瑞生物公司Pro Taq HS预混型探针法qPCR试剂盒进行多重荧光定量PCR检测,分别用每组4种病原体的引物和探针检测8种病原体的混合质粒模板,结果如图10-11;从以上结果可以看出各组病原体之间均无交叉反应,说明多重荧光定量PCR特异性良好。
3、多重qPCR灵敏性试验
用MqPCR的方法对梯度106、105、104、103、102、10 copies/μL的质粒模板进行检测,确定MqPCR检测方法能检测到的最低质粒浓度,结果如图12-16所示,从图12-13中可以看出HHV6的检测限达到100copies/μL量级,脑膜炎奈瑟菌、伤寒立克次式体、猿猴空泡病毒40检测限达到10copies/μL量级;图14-15结果伤寒沙门氏菌、弓形虫检测限达到10copies/μL量级;解脲脲原体、BK多瘤病毒的检测限均达到100copies/μL量级;图16结果显示内参GADPH基因的检测限达到10 copies/μL量级。
4、多重qPCR重复性试验
为了验证MqPCR检测方法的重复性,用103copies/μL量级质粒作为模板进行实验,分别进行组内和组间重复性实验;以每组病原体的特异性引物和探针对质粒模板进行检测,同一时间重复进行三次,观察记录其Ct值,每周一进行重复性检测,连续进行三周,观察记录其Ct值,重复性结果下表:
。
实施例3:脑脊液的检测
1、样本的采集
脑脊液采集用腰椎穿刺的方法,临床医师首先核对患者姓名与检验申请姓名是否一致,然后对患者进行腰椎穿刺,腰椎穿刺成功后留取脑脊液标本于无菌试管中,脑脊液标本留取后应立即送检,这些脑脊液样本在运输过程中储存在大约4℃,然后保存在-80℃直至分析。
2、基因组DNA提取
使用病毒基因组DNA/RNA提取试剂盒提取脑脊液DNA与RNA,具体步骤如下:
(1)用移液器将20 μL Proteinase K加入一个干净的1.5 mL离心管中;
(2)向离心管中加入200μL脑脊液样本;
(3)加入200μL Carrier RNA工作液(为缓冲液GB与Carrier RNA溶液的混合液,配制方法按照公式计算:
n×0.22 mL=y mL; y mL×28μL /mL=zμL
n=同时提取的样品个数,y=需要加入缓冲液GB的体积,z=需要加入Carrier RNA溶液的体积)。盖上管盖,涡旋振荡15s混匀,使样品和Carrier RNA工作液需要彻底混匀,保证其裂解充分;
(4)在56℃孵育15 min,简短离心以收集附着在管壁及管盖的液体;
(5)加入250μL无水乙醇,此时可能会出现絮状沉淀。盖上管盖并涡旋振荡15s,彻底混匀。在室温(15-25℃)放置5min;
(6)简短离心以收集附着在管壁及管盖的液体;
(7)仔细将离心管中的溶液和絮状沉淀全部转移至RNase-Free吸附柱CR2(吸附柱放在收集管中),盖上管盖,8000rpm离心1min,弃废液,将吸附柱放回收集管中。
(8)小心打开吸附柱盖子,加入500μL缓冲液GD(使用前请先检查是否已加入无水乙醇),盖上管盖,8000 rpm离心1min,弃废液,将吸附柱放回收集管;
(9)小心打开吸附柱盖子,加入600μL漂洗液PW〈使用前请先检查是否已加入无水乙醇),盖上管盖,静置2min,8000rpm离心1min,弃废液,将吸附柱放回收集管;
(10)重复步骤9;
(11)小心打开吸附柱盖子,加入500μL无水乙醇,盖上管盖,8000rpm离心1min,弃废液;
(12)将吸附柱放回收集管中,12000rpm离心3min,使吸附膜完全变干,弃废液;
(13)将吸附柱放入一个RNase-Free离心管(1.5mL)中,小心打开吸附柱的盖子,室温放置3 min,使吸附膜完全变干。向吸附膜的中间部位悬空滴加50μL RNase-Freedd H2O,盖上盖子,室温放置5 min;12000 rpm离心1 min;
(14)核酸收集于离心管,标记好信息,放于-80℃保存;
3、从医院收集9例阳性脑脊液样本(细菌培养或一代测序验证的阳性病原体)和8例阴性脑脊液样本(细菌培养或一代测序未检出病原体),通过多重实时荧光定量PCR技术检测,并且将多重实时荧光定量PCR技术与细菌培养、一代测序结果进行对比,结果如下表所示;
由上表可知,通过本发明中枢神经系统感染性病原体的多重荧光定量PCR检测试剂,发现本发明可以检测出细菌培养或一代测序检测不到的病原体,即可以对细菌培养或一代测序的结果起到补充作用;说明本发明建立的8种中枢神经系统感染性疾病病原体的多重实时荧光定量PCR具有良好的应用价值。
序列表
<110> 昆明理工大学
<120> 中枢神经系统感染性病原体的多重荧光定量PCR检测引物组合
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tatgtcagga tcatcaaatc aattc 25
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Claims (1)
1.一种中枢神经系统感染性病原体的多重荧光定量PCR检测引物组合,其特征在于:包括检测脑膜炎奈瑟菌、疱疹病毒6型、伤寒立克次氏体、猿猴空泡病毒40、伤寒沙门氏菌、解脲脲原体、弓形虫、BK多瘤病毒的特异性引物和探针;
所述特异性引物为针对脑膜炎奈瑟菌的SEQ ID NO:1和SEQ ID NO:2、针对疱疹病毒6型的SEQ ID NO:4和SEQ ID NO:5、针对伤寒立克次氏体的SEQ ID NO:7和SEQ ID NO:8、针对猿猴空泡病毒40的SEQ ID NO:10和SEQ ID NO:11、针对伤寒沙门氏菌的SEQ ID NO:13和SEQ ID NO:14、针对解脲脲原体的SEQ ID NO:16和SEQ ID NO:17、针对弓形虫的SEQ IDNO:19和SEQ ID NO:20;针对BK多瘤病毒的SEQ ID NO:22和SEQ ID NO:23;
所述探针为针对脑膜炎奈瑟菌的SEQ ID NO:3、针对疱疹病毒6型的SEQ ID NO:6、针对伤寒立克次氏体的SEQ ID NO:9、针对猿猴空泡病毒40的SEQ ID NO:12、针对伤寒沙门氏菌的SEQ ID NO:15、针对解脲脲原体的SEQ ID NO:18、针对弓形虫的SEQ ID NO:21;针对BK多瘤病毒的SEQ ID NO:24。
Priority Applications (1)
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