CN111154898A - 鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法 - Google Patents
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Abstract
本发明提供了一种鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法,选择16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因进行多个PCR扩增,能快速准确地鉴别结核分枝杆菌,有效排除牛分枝杆菌和BCG弱毒株(卡介苗)的干扰。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种用于鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法,能快速有效地鉴定人结核分枝杆菌。
背景技术
结核病是严重危害人类以及畜牧业健康的传染性疾病。据世界卫生组织(WHO)估计,目前全球已有20亿人感染结核分枝杆菌(MTB),平均每1秒钟就有1个新感染者出现,每年新发结核病人达800万~1000万,300万人死于结核病,是死亡率最高的感染性疾病。引发人和动物结核感染的病原体为结核分枝杆菌,在病原学上结核分枝杆菌包括结核分枝杆菌复合体(Mycobacterium tuberculosis complex,MTC)和非结核分枝杆菌(Nontuberculousmycobacteria,NTM)。MTC由人型结核分枝杆菌(Mycobacterium tuberculosis,MTB)、牛分枝杆菌、非洲分枝杆菌、田鼠分枝杆菌等组成。NTM则指除MTC和麻风分枝杆菌以外的其它分枝杆菌。其中人型和牛型结核分枝杆菌可以造成人和反刍类动物的交叉感染,MTB是引起人类结核病的主要病原体,约占90%-95%,其次则为牛分枝杆菌。因此,研究结核分枝杆菌在人及动物群体中的感染特性,不但对于维护群众健康,减少疾病的传播机率,同时也为农业及畜牧业的健康发展,具有重要意义,而控制结核的传播和耐药的关键就是早期准确诊断。由于不同种类的分枝杆菌引起结核病的临床症状、影像学特征极为相似,但其天然宿主、临床用药却大不相同,所以对于结核患者的致病分枝杆菌有效鉴定在结核病的诊断、治疗及流行病学调查极为关键。现阶段结核病的常用诊断方法包括涂片染色镜检、结核菌素实验、分离培养以及生化鉴定、抗原诊断和基因诊断等。涂片通过抗酸染色后镜检是临床诊断结核常用的方法,其操作简便,诊断速度快,但不能进行菌种鉴定无法区分自然感染者与卡介苗免疫者以及判断早期感染结核患者。对于病原菌分离培养及生化鉴定是临床上普遍采用的结核病诊断及种类初步鉴定的可靠方法,但其操作复杂,样本检测周期长(1-2个月),结果易受检测者的主观因素影响。免疫学诊断则由于现有的结核杆菌分泌抗原通过抗原抗体反应进行检测,但其检测结果特异性较差、假阳性率高。
目前基因诊断在结核分枝杆菌检测中具有一定优势,其灵敏度高检测周期短,且利用特异性的DNA或RNA可进行结核分枝杆菌种属鉴定。因此,高效、灵敏和精确的分子生物学技术有望成为结核病早期诊断和菌种鉴定的方法。基因诊断常用的分子指标有16SrRNA,IS6110,38kDa,hsp65,Rv1510和mtp40等。这些基因在诊断结核病方面有各自的优势,但其单独的检测和鉴定方面均局限性。例如16S rRNA可将多种分枝杆菌鉴定至种,但却不能区分结核分枝杆菌复合体以及其他分枝杆菌。IS6110为多拷贝序列,灵敏度高,能够检测和鉴别结核分枝杆菌复合体,但目前已发现其在某些MTB菌株中缺失。因此,迫切需要一个能够特异而灵敏的检测人型结核分枝杆菌的分子技术。
发明内容
本发明的目的是提供一种用于鉴定(检测)结核分枝杆菌、牛分枝杆菌和卡介苗的方法,用于鉴定人体分泌物或者培养物中是否存在结核分枝杆菌。
本发明的第一方面提供了一种用于鉴定(检测)人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法,其中鉴定(检测)过程的技术路线如图1所示,该鉴定(检测)过程通过6次复合PCR的方式分步骤进行鉴定。
本发明所述的鉴定(检测)人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法,包括以下步骤:使用16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因进行多个PCR扩增进行鉴定。
在本发明的一个优选实施方式中,所述鉴定(检测)人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法包括以下步骤:
(1)培养菌体,使用CTAB(十六烷基三甲基溴化铵)提取结核分枝杆菌DNA。
(2)配制PCR反应体系,扩增使用20μL的体系;所述反应体系包含16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因。
(3)PCR反应体系放入PCR仪进行扩增反应。
(4)扩增后的PCR产物用1%琼脂糖凝胶电泳检测。
(5)根据扩增片段的大小进行结果分析。
在本发明的另一个优选实施方式中,步骤(1)中所述培养菌体的具体方法为:收集结核病患者的痰液或者分离培养菌,痰液可直接煮沸离心取上清使用。结核分枝杆菌培养使用改良罗氏培养基。
在本发明的另一个优选实施方式中,步骤(2)中所述反应体系中包含2x PCR Mix10μL,浓度为10μΜ的上游和下游引物各2μL,结核分枝杆菌DNA模板1μL,最后加ddH2O5μL;所述上游和下游引物参照表1根据目的基因进行选择。
在本发明的另一个优选实施方式中,步骤(3)中所述PCR扩增反应的反应程序设置为预变性94℃5分钟,变性94℃30秒,根据不同引物设置退火温度53-64℃30秒,根据不同扩增片段大小设置延伸时间(按1分钟/1kb计算),延伸温度为72℃;循环30次后终延伸温度为72℃,时间为10分钟,最后4℃保存。
在本发明的另一个优选实施方式中,步骤(3)中所述不同引物按照表1设置退火温度,按扩增片段的大小计算并设置延伸时间。
表1不同目的基因的引物序列、退火温度及扩增片段大小
在本发明的另一个优选实施方式中,步骤(4)中所述扩增后的PCR产物用1%琼脂糖凝胶电泳检测的具体方法为:取2000bp Marker,5μL PCR产物加到点样孔中、以120V电压电泳25分钟,等电泳结束后,在紫外线凝胶成像仪下观察结果。
本发明的另一方面提供了上述检测方法用于鉴定人结核分枝杆菌的用途。
本发明中,使用复合PCR的方式针对多个不同的基因,同时进行扩增根据电泳条带区分不同菌种。复合PCR技术设计目的是区分结核分枝杆菌复合群和非结核分枝杆菌,在结核分枝杆菌复合群中区分人结核分枝杆菌、牛分支杆菌和卡介苗弱毒株(BCG)。本发明选择16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因进行多个PCR扩增。首先用细菌通用16S rRNA和分枝杆菌特异性16S rRNA(Myco.)基因来检测分枝杆菌属病原菌。其中IS6110基因为结核杆菌复合群特异性基因,其能从分支杆菌中区分结核分支杆菌复合群和非结核分枝杆菌;Rv2659c基因中有128bp为人结核分枝杆菌特异性基因,其能在结核分支杆菌复合群中筛选人源结核杆菌。通过基因组比对发现,结核分枝杆菌基因组中的一些片段在牛分枝杆菌或卡介苗(Bacillus Calmette Guerin,BCG)基因组中缺失,这些缺失片段被称为差异区域(region of differences,RD)。在结核杆菌复合群中Rv1510基因为牛分支杆菌缺失基因,通过Rv1510基因可以在结核杆菌复合群中筛选牛分枝杆菌。在牛分枝杆菌中RD1区ESAT6基因为BCG弱毒株的缺失基因,因此根据ESAT6基因可从牛分枝杆菌中排出BCG弱毒株。基于上述的检测技术,我们可以快速有效地对结核分枝杆菌进行准确鉴定。
本发明的另一个方面提供了一种用于鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的PCR试剂盒,所述试剂盒包含PCR反应液;所述PCR反应液包括鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的五个目标基因的引物,分别为16SrRNA、IS6110、Rv2659c、ESAT6和Rv1510。
附图说明
图1为PCR扩增后筛选结核分枝杆菌的流程图;
图2为16S rRNA基因扩增结果;其中:图2A为16S rRNA细菌通用引物(1500bp)扩增结果;M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株;图2B为16S rRNA分支杆菌属特异性引物(212bp)扩增结果;M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株。
图3为IS6110基因(416bp)扩增结果;其中:M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株。
图4为Rv1510基因(1033bp)扩增结果;其中:M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株。
图5为ESAT-6基因(220bp)扩增结果;其中:M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株。
图6为Rv2659c基因(90bp)扩增结果;其中:M为DL 2000Marker;1为结核分枝杆菌H37Ra菌株;2为结核分枝杆菌BCG菌株(卡介苗);3为牛分枝杆菌552号菌株;4为非结核分枝杆菌739号菌株;5为李氏杆菌菌株。
图7为结核分枝杆菌罗氏培养基生长情况。
实施例
实施例1人结核分枝杆菌、牛分枝杆菌和卡介苗的多重PCR检测
(1)制备试样
使用如下所示的细菌,通过下述方法提取、纯化DNA,得到DNA试样。
a)结核分枝杆菌H37Ra菌株
b)结核分枝杆菌BCG菌株(卡介苗)
c)牛分枝杆菌552号菌株
d)非结核分枝杆菌739号菌株;
e)李氏杆菌菌株
具体步骤如下:
李氏杆菌作为对照使用LB培养基进行培养。提取后的DNA溶解在无菌水中,使用NanoDrop 2000对所得DNA浓度进行测定。
采用改良的CTAB法提取结核分枝杆菌的总DNA,具体步骤如下:
1、用接种环刮取一定量的菌体于离心管中(已加入400μL TE缓冲液),80℃灭活30分钟,然后室温离心4000rpm 10分钟,去除上清,用450μL GTE溶液重悬结核菌,再加入50μL的10mg/ml溶菌酶,充分混匀后于37℃过夜。
2、加入2:1体积的10%SDS(100μL)和10mg/ml蛋白酶K(50μL),轻轻混匀,在55℃下孵育20-40分钟。加入10μL RNaseA,37℃孵育10-30分钟。再加入200μL的5M NaCl,轻轻混匀。
3、在65℃下提前预热CTAB(新鲜配制的CTAB),加160μL的CTAB溶液,轻轻混匀后65℃孵育10分钟。
4、加入等体积的24:1(v/v)氯仿/异戊醇溶液,混匀后离心12000r/min,5分钟。离心后,吸取上清并转移到另一个新的离心管中。重复该步骤抽提一次。
5、将上清移到一个新的2ml离心管中,加入3mol/L的醋酸钠20μL,再加入0.7倍异丙醇,轻轻颠倒,此时应当看到絮状的沉淀。将离心管置于-70℃冰箱过夜。
6、过夜后取出离心管待完全融化温度达到室温后,室温离心12000r/min,10分钟。小心吸尽上清后,加1ml 70%乙醇洗DNA,轻轻混匀,来回颠荡数次后,12000r/min,离心10分钟。最后再次吸出上清保留微量DNA沉淀,将其斜放置于干净的地方,空气干燥15分钟,等到管中DNA沉淀由白色变为透明。
7、加入40μL 60℃预热的无菌水溶解DNA,4℃过夜溶解后,1%琼脂糖凝胶电泳检测样品质量和浓度。
8、GTE溶液配制:25Mm Tris-HCl PH 8.0,10mM EDTA,50mM葡萄糖混匀在121℃高压灭菌15分钟,常温保存备用。
9、CTAB溶液配制:先称取4.1g NaCl溶解于90ml水中,待完全溶解后加入10gCTAB,在65℃下溶解。
(2)PCR
配制PCR反应体系,扩增使用20μL的体系。其中包含2x PCR Mix 10μL,浓度为10μΜ的上游和下游引物各2μL,结核分枝杆菌DNA模板1μL,最后加ddH2O 5μL。
配制好后放入PCR仪进行扩增反应。PCR反应程序设置为预变性94℃5分钟,变性94℃30秒,根据不同引物(参照表1)设置退火温度53-64℃30秒,根据不同扩增片段大小(参照表1)设置延伸时间72℃(1分钟1kb),循环30次后终延伸72℃10分钟,最后4℃保存。
(3)检测
扩增后的PCR产物用1%琼脂糖凝胶电泳检测。取2000bp Marker,5μL PCR产物加到点样孔中、以120V电压电泳25分钟,等电泳结束后,在紫外线凝胶成像仪下观察结果。
(4)结果
所得的电泳结果如图2-6所示。
图2为16S rRNA基因扩增结果,其中,图2A为16S rRNA细菌通用引物(1500bp)扩增结果;图2B为16S rRNA分支杆菌属特异性引物(212bp)扩增结果;图3为IS6110基因(416bp)扩增结果;图4为Rv1510基因(1033bp)扩增结果;图5为ESAT-6基因(220bp)扩增结果;图6为Rv2659c基因(90bp)扩增结果。各泳道数字分别表示分别使用以下试样时的结果:
M:DL 2000Marker
1:结核分枝杆菌H37Ra菌株
2:结核分枝杆菌BCG菌株(卡介苗)
3:牛分枝杆菌552号菌株
4:非结核分枝杆菌739号菌株
5:李氏杆菌菌株
最后根据扩增片段的大小参照图1的PCR扩增后筛选结核分枝杆菌的流程图进行结果分析:只有结核分枝杆菌在16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510扩增结果中均观察到相应的荧光条带,由此可以准确检测结核分枝杆菌,有效排除牛分支杆菌和卡介苗弱毒株(BCG)的干扰。
实施例2待测菌株制备
本检测方法所用待测菌株的收集待方式包括两种。第一种方式,对临床疑似结核病人的痰液进行收集,将收集的痰液在100℃高温加热煮沸10分钟,再将其12000rpm/min高速离心10分钟,最后取上清作为PCR模板待用。第二种方式,若痰液收集菌量过少无法达到PCR检测标准,则使用改良罗氏培养基进行扩增培养,培养后的菌株进行如下操作。
改良罗氏培养基多用于分枝杆菌的分离培养、保存菌株以及菌种鉴定。其培养分支杆菌精确可靠,特异性较高。罗氏培养基中含有分枝杆菌生长繁殖所需的基础物质、营养物质和抑制杂菌生长的抑菌剂。基础物质包括天门冬素、磷酸二氢钾、硫酸镁、柠檬酸镁、丙三醇和水分,营养物质主要为鸡蛋,它们能为细菌的生长繁殖提供无机盐、碳源、氮源和水分,镁、钾元素有调节菌体渗透压的作用,使分枝杆菌细胞膜具有良好的通透性。蛋黄中含有磷、磷脂和一些盐类,是很好的营养物质,鸡蛋白能中和脂肪酸的毒性并有缓冲作用。磷酸二氢钾为缓冲剂,可调节培养基的pH值。孔雀绿可抑制标本中部分杂菌的生长。结核分枝杆菌罗氏培养基生长如图7所示。
Claims (8)
1.一种鉴定人结核分枝杆菌、牛分枝杆菌和卡介苗的技术方法,其特征在于,所述方法包括以下步骤:使用16SrRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因进行多个PCR扩增进行鉴定。
2.根据权利要求1所述的技术方法,其特征在于,所述方法包括以下步骤:
(1)培养菌体,使用CTAB(十六烷基三甲基溴化铵)提取结核分枝杆菌DNA;
(2)配制PCR反应体系,扩增使用20μL的体系;所述反应体系包含16S rRNA、IS6110、Rv2659c、ESAT6和Rv1510五个目的基因;
(3)PCR反应体系放入PCR仪进行扩增反应;
(4)扩增后的PCR产物用1%琼脂糖凝胶电泳检测;
(5)根据扩增片段的大小进行结果分析。
3.根据权利要求2所述的技术方法,其特征在于,步骤(1)中所述培养菌体的具体方法为:收集结核病患者的痰液或者分离培养菌,痰液可直接煮沸离心取上清使用;结核分枝杆菌培养使用改良罗氏培养基。
5.根据权利要求2所述的技术方法,其特征在于,步骤(3)中所述PCR扩增反应的反应程序设置为预变性94℃5分钟,变性94℃30秒,根据不同引物设置退火温度53-64℃30秒,根据不同扩增片段大小设置延伸时间(按1分钟/1kb计算),延伸温度为72℃;循环30次后终延伸温度为72℃,时间为10分钟,最后4℃保存。
7.根据权利要求1-6任一所述技术方法的用途,其特征在于,用于鉴定人结核分枝杆菌。
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