CN114990260B - 用于检测中枢神经系统感染性病原体的多重荧光定量pcr检测试剂 - Google Patents
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Abstract
本发明公开了一种用于检测中枢神经系统感染性病原体的多重荧光定量PCR检测试剂,其特征在于:包括检测EB病毒、流感嗜血杆菌、巨细胞病毒、单纯疱疹病毒2型、水痘带状疱疹病毒、钩端螺旋体、B‑溶血性链球菌、诺卡菌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
本发明属于生物技术领域,针对引发中枢神经系统感染性疾病常见8种病原体,建立特异性强、灵敏度高、反应快速的多重荧光定量PCR检测方法。
背景技术
中枢神经系统(Central Nervous System,CNS)感染性疾病指的是细菌、病毒、真菌等病原体侵犯CNS的脑膜、实质、血管导致的慢性或急性炎症或非炎症性的疾病。中枢神经系统感染性疾病有三种不同的分类,临床上依据中枢神经系统感染部位的不同可分为:(1)脑炎、脊髓炎或脑脊髓炎,病原体主要侵犯脑和(或)脊髓实质,其中最常见的疾病为脑炎,据报道感染性脑炎的发病率为1.5-7例/100000人/年。(2)脑膜炎、脊膜炎或脑脊膜炎,主要侵犯脑和(或)脊髓软膜,其中脑膜炎是最常见的CNS感染性疾病,脑膜有三层,由外向内为硬脑膜、蛛网膜和软脑膜。脑膜炎可累及硬脑膜、蛛网膜和软脑膜,目前临床上的脑膜炎实际上是指软脑膜炎。脑膜炎是一类特别严重的疾病,需要及时治疗,如果治疗不及时,可能会造成死亡或留下一些后遗症。(3)脑膜脑炎指的是脑膜与脑实质合并感染。脑膜脑炎是一种病毒性疾病,发生在蛛网膜和软脑膜之间。确定了CNS感染性疾病常见的8种病原体有EB病毒(Epstein-Barr Virus,EBV)、猿猴空泡病毒40(Simian Vacuolating VirusSV40)、2型单纯疱疹病毒(Herps Simplex Virus 2,HSV-2)、水痘带状疱疹病毒(Varicella-Zoster Virus,VZV)、巨细胞病毒(Cytomegalovirus,CMV)、诺卡菌(Nocardiosis)、流感嗜血杆菌(Haemophilus influenzae)、B-溶血性链球菌(β-Streptococcus haemolyticus)、钩端螺旋体(Leptospira)等会导致严重的后果。
病毒感染是人类中枢神经系统感染的主要原因,在儿童群体中感染率最高。CNS的真菌感染可能是毁灭性的并且难以治疗,相关死亡率超过90%。细菌感染可能会致命或在幸存者中留下严重的CNS后遗症。快速准确的检测中枢神经系统的病原体对疾病的治疗是非常必要的。目前常用的脑脊液病原体的检测方法存在一些弊端,比如脑脊液病原体分离培养法培养时间长(2-5天),很多病原体要求的培养条件非常严格,可能会延误病情,错过最佳治疗时期,且检测灵敏度低。免疫学检测和普通PCR的方法对病原体的检测也都有灵敏度低的特点。近几年新兴的基因芯片法和mNGS的方法虽然检测灵敏度很高,但价格昂贵,且需要专业人员操作。
发明内容
针对现有技术的不足,本发明提供了一种用于检测中枢神经系统感染性病原体的多重荧光定量PCR检测试剂,其包括检测EB病毒、流感嗜血杆菌、巨细胞病毒、单纯疱疹病毒2型、水痘带状疱疹病毒、钩端螺旋体、B-溶血性链球菌、诺卡菌的特异性引物和探针;包括GAPDH基因的引物和探针;还包括多重荧光定量PCR检测的其他常规试剂;本发明检测试剂具有检测灵敏度高、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EB病毒的SEQ ID NO:1和SEQ ID NO:2、针对流感嗜血杆菌的SEQ ID NO:4和SEQ ID NO:5、针对巨细胞病毒的SEQ ID NO:7和SEQ ID NO:8、针对单纯疱疹病毒2型的SEQ ID NO:10和SEQ ID NO:11、针对水痘带状疱疹病毒的SEQ ID NO:13和SEQ ID NO:14、针对钩端螺旋体的SEQ ID NO:16和SEQ ID NO:17、针对B-溶血性链球菌的SEQ ID NO:19和SEQ ID NO:20;针对诺卡菌的SEQ ID NO:22和SEQ ID NO:23;
所述探针为针对EB病毒的SEQ ID NO:3、针对流感嗜血杆菌的SEQ ID NO:6、针对巨细胞病毒的SEQ ID NO:9、针对单纯疱疹病毒2型的SEQ ID NO:12、针对水痘带状疱疹病毒的SEQ ID NO:15、针对钩端螺旋体的SEQ ID NO:18、针对B-溶血性链球菌的SEQ ID NO:21;针对诺卡菌的SEQ ID NO:24。
使用上述多重荧光定量PCR检测试剂的方法如下:
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,经过多重实时荧光定量PCR进行检测,以GAPDH基因为内参,根据Ct值进行结果判定;
在检测中检测 EB病毒、流感嗜血杆菌、巨细胞病毒、单纯疱疹病毒2型的特异性引物和探针同时使用;检测水痘带状疱疹病毒、钩端螺旋体、B-溶血性链球菌、诺卡菌的特异性引物和探针同时使用;以GAPDH基因为内参。
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;GAPDH内参基因序列如SEQ ID NO.25-27所示。
采用本发明多重荧光定量PCR检测试剂检测病原体的扩增反应体系如下:2×ProTaq 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。
3、检测阳性结果判读包括如下内容:(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:
本发明建立了8种中枢神经系统感染性病原体的多重荧光定量PCR检测方法,对该方法进行特异性评价发现每组病原体与其他组外病原体之间均无交叉反应,各组病原体的特异性良好;灵敏度评价发现除巨细胞病毒的灵敏度均达到100copies/μL量级外另外其他7种病原体的灵敏度均达到10 copies/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20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EB病毒的单重qPCR特异性试验结果;
图2为流感嗜血杆菌的单重qPCR特异性试验结果;
图3为巨细胞病毒的的单重qPCR特异性试验结果
图4为单纯疱疹病毒2型的单重qPCR特异性试验结果;
图5为水痘带状疱疹病毒的单重qPCR特异性试验结果;
图6为钩端螺旋体的单重qPCR特异性试验结果;
图7为B-溶血性链球菌的单重qPCR特异性试验结果;
图8为诺卡菌的单重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EB病毒、流感嗜血杆菌、巨细胞病毒、单纯疱疹病毒2型的多重qPCR特异性试验结果;
图11为水痘带状疱疹病毒、B-溶血性链球菌、钩端螺旋体、诺卡菌的多重qPCR特异性试验结果;
图12为EB病毒、流感嗜血杆菌的多重qPCR灵敏性试验结果;
图13为巨细胞病毒、单纯疱疹病毒2型的多重qPCR灵敏性试验结果;
图14为水痘带状疱疹病毒、B-溶血性链球菌的多重qPCR灵敏性试验结果;
图15钩端螺旋体、诺卡菌的多重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,本领域技术人员应当掌握使用常规材料及仪器的相关知识。
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:EB病毒Virion glycoprotein gL编码基因、流感嗜血杆菌Hypothetical protein编码基因、巨细胞病毒Regulatory proteinIE1编码基因、单纯疱疹病毒2型helicase-primase helicase subunit编码基因、水痘带状疱疹病毒transcriptional regulator ICP4 pseudogene编码基因、B-溶血性链球菌surface immunogenic protein编码基因、钩端螺旋体outer membrane protein LipL32编码基因及诺卡菌16S ribosomal RNA编码基因各20条;
2、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。
3、引物与探针BLAST评估: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(SEQ ID NO:28和SEQ ID NO:36)、内参基因GAPDH的序列与pUC57载体连接,合成质粒标准品,其中EB病毒、巨细胞病毒合成在一个质粒上,诺卡菌、钩端螺旋体合成在一个质粒上,单纯疱疹病毒2型和GAPDH合成到一个质粒上,B族溶血性链球菌、流感嗜血杆菌、水痘带状疱疹病毒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;
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12-15所示,从图12-13中可以看出巨细胞病毒、EB病毒、流感嗜血杆菌、单纯疱疹病毒2型的检测限达到10copies/μL量级;图14-15结果显示水痘带状疱疹病毒、B-溶血性链球菌、钩端螺旋体的检测限均达到10copies/μL量级,诺卡菌的检测限均达到10copies/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10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10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病原体感染性疾病病原体及内参GAPDH基因的多重实时荧光定量PCR具有良好的应用价值。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
序列表
<110> 昆明理工大学
<120> 用于检测中枢神经系统感染性病原体的多重荧光定量PCR检测试剂
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catggtctac atgttccggt atgattccac ccatggcaaa ttccatggca ctgtcaaggc 180
tgagaacgag aagcttgtca acaatggaaa tcccatcacc atcttc 226
Claims (1)
1.一种用于检测中枢神经系统感染性病原体的多重荧光定量PCR检测试剂,其特征在于:包括检测EB病毒、流感嗜血杆菌、巨细胞病毒、单纯疱疹病毒2型、水痘带状疱疹病毒、钩端螺旋体、B-溶血性链球菌、诺卡菌的特异性引物和探针;
所述特异性引物为针对EB病毒的SEQ ID NO:1和SEQ ID NO:2、针对流感嗜血杆菌的SEQ ID NO:4和SEQ ID NO:5、针对巨细胞病毒的SEQ ID NO:7和SEQ ID NO:8、针对单纯疱疹病毒2型的SEQ ID NO:10和SEQ ID NO:11、针对水痘带状疱疹病毒的SEQ ID NO:13和SEQID NO:14、针对钩端螺旋体的SEQ ID NO:16和SEQ ID NO:17、针对B-溶血性链球菌的SEQID NO:19和SEQ ID NO:20;针对诺卡菌的SEQ ID NO:22和SEQ ID NO:23;
所述探针为针对EB病毒的SEQ ID NO:3、针对流感嗜血杆菌的SEQ ID NO:6、针对巨细胞病毒的SEQ ID NO:9、针对单纯疱疹病毒2型的SEQ ID NO:12、针对水痘带状疱疹病毒的SEQ ID NO:15、针对钩端螺旋体的SEQ ID NO:18、针对B-溶血性链球菌的SEQ ID NO:21;针对诺卡菌的SEQ ID NO:24。
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