CN101812505B - 16S rRNA基因为靶基因的TaqMan probe实时荧光定量PCR检测体系的建立方法 - Google Patents
16S rRNA基因为靶基因的TaqMan probe实时荧光定量PCR检测体系的建立方法 Download PDFInfo
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Abstract
本发明是应用新兴分子生物学技术之一的Real-time PCR对动物源性嗜衣原体(Chlamydophilapsittaci、C.abortus、C.caviae、C.pecorum、C.felis)实施的快速检测与诊断方法。本检测体系采用以动物源性嗜衣原体的16S rRNA的保守区域为基础设计了一对引物及相应的TaqMan探针。该体系能对来自动物的粪便和泄殖腔的棉拭子及人的气管洗脱液中存在的动物源性嗜衣原体基因进行检测,并且检测敏感度比普通的PCR高出10倍。Real-time PCR具有无需进行后PCR的处理,可避免因进行琼脂糖凝胶电泳而可能造成的交叉污染,从而提高检测的准确率和可信度。
Description
技术领域:
本发明属于分子生物学领域,具体地涉及一种以动物源性嗜衣原体的16S rRNA为靶基因的TaqMan probe实时荧光定量PCR检测体系的建立方法。
背景技术:
衣原体是专性细胞内寄生病原体,不同于其它***,表现在其独特的双向生存周期,即一个细胞外的生存形式——原生小体和一个细胞内复制的形式——网状体,二者之间可相互转化。1999年,衣原体科被分成2个属9个种。其中,衣原体属有3个种,包括沙眼衣原体、猪衣原体和鼠肺炎沙眼衣原体;嗜衣原体属有6个种,即猪流产嗜衣原体、豚鼠嗜衣原体、猫嗜衣原体、兽类嗜衣原体、肺炎嗜衣原体和鹦鹉热嗜衣原体。
这些微生物是许多人畜共患病的病原体,如沙眼衣原体可导致性传播疾病和沙眼;肺炎衣原体会导致人体引起诸如肺炎、支气管炎等呼吸***疾病,也被认为是哮喘和动脉粥样硬化等慢性疾病的病因之一;猪流产嗜衣原体、豚鼠嗜衣原体、猫嗜衣原体、兽类嗜衣原体和鹦鹉热嗜衣原体广泛分布于各种动物中,可寄生于哺乳类动物宿主引起流产、肠炎、肺炎、多发性关节炎及生殖道、消化道疾病;鹦鹉热嗜衣原体可感染禽鸟类与哺乳动物引起非典型性肺炎和流产,它也可通过隐性感染或患病动物传播给人体,引发严重的非典型肺炎。
众所周知,血清学试验、微量免疫荧光法(MIF)、补体结合试验(CF)和酶联免疫吸附试验(ELISA)是检测衣原体感染的常规诊断方法。由于这些方法存在交叉反应从而使其缺乏特异性,而聚合酶链反应(PCR)已成为检测动物衣原体感染的一种确实有效的方法。该方法具有易使用,能快速获得结果,同时适合于处理大量的检体样本等诸优点,但是它无法避免后PCR的交叉污染。实时荧光定量PCR是一种理想的替代方法,由于它无需进行后PCR处理从而不易引起交叉污染,而且杂交探针也可提高检测的特异性,因此它已被证实是一种更确实有效的微生物检测方法。编码16S rRNA小亚基的基因经常被当作PCR检测的靶基因位点,因为它有大量的数据库,在临床检测诊断中具有探索和利用价值。
由于动物源性嗜衣原体可引起动物和人类的多种疾病,动物以流产、肺炎、肠炎、结膜炎、多发性关节炎及脑脊髓炎等多种病症为主,而人类以发热、头痛和非典型性肺炎为主要症状,同时有报道称与人类的动脉粥样硬化和冠心病也有关。许多人和动物在感染后常呈隐性经过和无症状带菌状态,反复感染和与其他病原菌的混合感染也较为多见,这增加了临床诊断工作的难度。
目前,国外学者已成功应用嗜衣原体的主要外膜蛋白、热休克蛋白、脂多糖、16S和23S rRNA基因的全长序列为靶基因序列,通过运用PCR、DNA杂交技术及Real-time PCR等技术实施衣原体的检测。而国内主要依据病原体分离和血清学检查对衣原体进行检测。病原分离主要包括鸡胚接种、细胞培养和动物回归试验,其操作程序复杂,而且费时,且结果判定常带有主观性从而使这些方法在检测时受到限制。尤其当由这些病原体引起的爆发时,常由于缺乏快速、准确、敏感的检测方法而贻误疾病的控制,给人类的健康与经济带来巨大的损失。
发明内容:
本发明的目的是确立一种快速、准确、灵敏度高的基于动物源性嗜衣原体16SrRNA的TaqMan probe实时荧光定量PCR的检测体系。在对其与鹦鹉热嗜衣原体的常用PCR检测体系进行了敏感性和特异性比较的同时,证实该方法可在临床上应用。
该方法系一种基于嗜衣原体16S rRNA序列设计相应的引物及探针,并以嗜衣原体标准菌株由来的DNA作为模板而确立的TaqMan probe实时荧光定量PCR检测体系,并在检测特异性与敏感性等方面进行了评价,最后应用该检测体系对鸟类临床样本进行了检测,以期评价该检测体系在临床检测方面的应用可能性。
本发明的目的是通过下述的技术方案得以实现的:
一种TaqMan probe实时荧光定量PCR检测体系的确立,以动物源性嗜衣原体的16S rRNA为靶基因,包括标准菌株的培养和DNA的分离纯化、TaqMan probe实时荧光定量PCR检测体系与检测步骤的确立。
标准菌株的培养步骤是选用动物衣原体和嗜衣原体的标准菌株用HeLa 229和Hep-2细胞进行培养,培养后使用Puregene DNA分离纯化试剂盒(Funakoshi,Japan)从没感染力的衣原体EB中分离纯化基因组DNA。
TaqMan probe检测体系确立的步骤包括:
(1)根据鹦鹉热嗜衣原体C.psittaci 6BC(Accession No.AB001778)的16S rRNA片段序列,利用Primer express.2.0设计如下序列的引物和探针:
AC89F16S:5’-GGCGGAAGGGTTAGTAATACATAGATAA-3’
AC218R16S:5’-AAGGTCCGAAGATCCCCTTCT-3’;
Probe:
5’(FAM)-CCGCTAATACCGAATGTGGTATGTTTAGGC-(TAMRA)3’;
(2)TaqMan probe real-time PCR检测体系确立时所进行的实时荧光定量PCR反应均由7500real-time PCR仪实施完成,包括反应体系、反应条件,反应体系是经过对上下游引物和探针的浓度优化后确定建立25μl反应体系;反应条件是由引物和探针的Tm值确立并对其进行优化。
TaqMan probe real-time PCR检测体系的检测包括检测体系的特异性试验、敏感性试验,同时将该检测体系与一步法PCR检测方体系进行特异性与敏感性比较;特异性试验是将10pg的从动物源性嗜衣原体的没有感染力的EB中分离纯化的DNA加入反应体系中进行检测,同时将同量的其他衣原体和相关菌株作为对照,根据扩增曲线图,以判定其检测特异性;敏感性试验是将计算得到的标准菌株C.psittaciNJ-1的浓度作梯度稀释,并将Ct<38设为该检测体系的下限进行灵敏性试验;随后, 将该检测体系与一步法PCR检测体系进行特异性与敏感性的比较。
本发明的方法是基于动物源性嗜衣原体16S rRNA的Real-time PCR检测体系的确立,可概括为以下步骤:
1)以嗜衣原体16S rRNA序列为靶基因序列进行引物和探针的设计;
2)标准菌株的培养和DNA的分离纯化;
3)TaqMan probe real-time PCR检测体系的确立;
4)特异性试验;
5)该检测体系对标准菌株最低检测敏感性的确定,以及与常用一步法PCR检测体系的比较。
附图说明:
图1为鹦鹉热嗜衣原体C.psittaci 6BC(Accession No.AB001778)的16S rRNA片段序列,Alignment of conserved DNA sequences on 16S rRNA region ofChlamydophila and Chlamydia;
图2为本发明Real-time PCR检测体系特异性试验,Specificity test ofthe real-timePCR;
1.C.psittaci NJ-1 2.C.caviae OKM-2 3.C.felis Fe/C-56
4.C.pecorum Maeda 5.C.psittaci Helajika 6.C.abortus B577;
图3为本发明Real-time PCR检测体系敏感性试验,Sensitivity test of theReal-time PCR detection system,①100pg②10pg③1pg④0.1pg⑤0.01pg。
具体实施方式:
下面结合附图,用本发明的实施例来进一步阐述本发明,但并不以此来限定本发明。
实施例1:
1.相关菌株DNA的提取:
1.1本发明所应用到的衣原体和嗜衣原体的标准菌株如下所示:
C.psittaci Cal10,C.psittaci Helajika,C.psittaci Bud-1,C.psittaci NJ1,C.abortusB577,C.caviae OKM-2,C.felis Fe/C-56,C.muridarum MoPn,C.pecorum IPA,C.pecorum Maeda,C.pneumoniae TW183,C.pneumoniae Kkpn-1,C.pneumoniae AR39,C.pneumoniae TWAR,C.suis CS 0301,C.trachomatis A/G,trachomatis B/Tw,C.trachomatis Ba/Ap,C.trachomatis C/TW,C.trachomatis Da/UW,C.trachomatis D/UW,C.trachomatis E/UW,C.trachomatis F/UW,C.trachomatis G/UW,C.trachomatisH/UW,C.trachomatis I/UW,C.trachomatis Ia/UW,C.trachomatis J/UW,C.trachomatisK/UW,C.trachomatis L1,C.trachomatis L2,C.trachomatis L2a,C.trachomatis L3。
以上标准菌株均使用Hela 229和Hep-2细胞进行培养。除此之外还有非衣原体菌株作为阴性对照,具体如下:
Acinetobacter baumannii,Acinetobacter lwoffi,Alcaligenes faecalis,Alcaligenesxylosoxydans,Bacillus subtilis,Chryseobacterium indologenes,Ehrlichia canis,Ehrlichia chaffeensis,Escherichia coli,Flavobacterium odoratum,Haemophilusinfluenzae,Klebsiella pneumoniae,Legionella pneumophila,Listeria monocytogenes,Pseudomonas aeruginosa,Pseudomonas fluorescens,Psudomonas stutzeri,Rickettsiaconorii,Rickettsiajaponica,Rickettsia prowazekii,Rickettsia typhii,Salmonella arizonae,Salmonella choleraesuis,Salmonella enteritidis,Salmonella typhimurium,Staphylococcus aureus,Staphylococcus spidermidis,Streptcoccus pyogenes,Streptococcus pneumoniae,Yersinia enterocolitica.
1.2标准菌株DNA的提取:
嗜衣原体和衣原体相关标准菌株的基因组DNA是使用Puregene DNA分离纯化试剂盒(Funakoshi,Japan)从没有感染力的EB中分离纯化得到。具体提取方法按制造商提供的说明书所列的操作步骤进行即可。
1.3临床样本:
用于本检测体系的临床样本为鸟类的粪便样本共239例,由以下单位提供:鸟园135例,鸟类宠物店104例。
1.4临床样本的DNA分离纯化,按QIAamp DNA stool Mini kit(Qiagen,Germany)说明要求进行,其具体步骤如下:
1)将180-220mg粪便加入2ml的离心管中,置于冰上。
1)加入1.6mlASL,震荡数分钟直至粪便充分混匀。
2)将混匀的样本置于70℃加热5min,在加热的同时随时震荡混匀,随后以15,000rpm转速离心2min;
3)吸取1.4ml上清液加入至另一2ml离心管。
4)加入Inhibit EX片,持续震荡一分钟,直至药片被充分混匀。在室温中放置1min,使PCR抑制剂被充分吸附。
5)以15,000rpm转速离心4min,将粪便颗粒和被吸附的PCR抑制剂被充分沉淀。
6)离心结束后,吸取上清置于另一个新的1.5ml的离心管中,随后高速离心数分钟
7)将20μl的蛋白酶K加入另一个新的1.5ml离心管中,
8)从7中吸取300μl上清加入蛋白酶K的离心管中。
9)随后加入300μlAL缓冲液震荡数秒钟(不可以直接将蛋白酶K加入AL缓冲液中)
10)置于70℃孵育10min。
11)将10的混合液充分混匀后进行微量离心,随后再加入300μl乙醇(96-100%),震荡混匀,再次微量离心。
12)将QIAamp柱进行标记,同时将11步中的裂解液加入QIAamp柱,不可将边缘弄湿。盖上盖子,高速离心2min。将QIAamp柱转移至另一新的1.5ml离心管,将有滤液的离心管丢弃。
13)打开盖子加入500μl AW1缓冲液,高速离心1分钟。将QIAmap柱转移至另一 新的1.5ml离心管,将有过滤液的离心管丢弃。
14)打开盖子加入500μl AW2缓冲液。高速离心3分钟。将QIAmap柱转移至另一新的1.5ml离心管,将有过滤液的离心管丢弃。再将QIAamp柱转移至一新的1.5ml离心管以甩干QIAmap柱,将有过滤液的离心管丢弃。
15)将QIAmap柱转移至一新的并已作标记的1.5ml离心管,向QIAmap柱的膜上小心添加25μl AE缓冲液,室温放置1分钟,然后高速离心1分钟,洗脱所得的DNA供分子生物学检测备用。
注:粪便样本采集后需用10%SPG缓冲液进行预处理;最后进行DNA洗脱时将说明书中的100μl AE改用25μl进行;并将温浴时间延长。
2.TaqMan probe real-time PCR检测体系的建立:
2.1TaqMan probe实时荧光定量PCR引物与探针的设计与合成:
根据鹦鹉热嗜衣原体C.psittaci 6BC(Accession No.AB001778)的16S rRNA片段序列(附图1所示),利用Primer express.2.0设计引物和探针,目的片段长度130bp。序列情况如下:
AC89F16S:5’-GGCGGAAGGGTTAGTAATACATAGATAA-3’
AC218R16S:5’-AAGGTCCGAAGATCCCCTTCT-3’;
Probe:
5’(FAM)-CCGCTAATACCGAATGTGGTATGTTTAGGC-(TAMRA)3’。
2.2Taqman probe real-time PCR检测体系的建立:
在该检测体系确立过程中所进行的Real-time PCR反应均由ABI 7500real-timePCR仪实施完成。
2.2.1TaqMan probe real-time PCR反应体系的确立:
经过对上下游引物和探针的浓度优化后确立了25μl反应体系:900nM的上下游引物AC89F16S/AC218R16S,12.5μl的2×TaqMan universal PCR反应混合液,TaqMan probe 250nM,模板DNA 2.5μl,最后用灭菌超纯水将反应体系补足 25μl。
2.2.2TaqMan probe real-time PCR反应程序的确定:
由引物和探针的Tm值,确立并对其进行了优化(数据略)该反应过程的退火温度温度为62℃,其反应过程为:50℃2min,95℃预变性10min,然后40个循环扩增:95℃15s,62℃1min。
3.TaqMan probe实时荧光定量PCR检测体系的检测和评价:
3.1该检测体系的特异性试验:
该检测体系以嗜衣原体和衣原体的标准菌株和其他相关菌株进行特异性检测评价。具体是将10pg从动物源性嗜衣原体的没有感染力的EB中分离纯化的DNA加至实时定量PCR的反应体系中进行检测,同时以衣原体和其他菌株作为对照,从所得到的扩增曲线图,确定其检测特异性,结果表明该检测体系的probe能检出Chlamydophilapsittaci、C.abortus、C.caviae、C.pecorum、C.felis种动物源性嗜衣原体(如图2所示)。
3.2该检测体系的敏感性试验:
将计算得到的标准菌株C.psittaci NJ-1的浓度作十倍梯度稀释,结果将每个反应体系的鹦鹉热嗜衣原体的DNA量分别确定为0.01,0.1,1,10,100pg,随后进行敏感性试验,以Ct<38个循环为检测下限作为该检测体系的检测极限。通过扩增曲线图可知本检测体系可检测到0.01pg相当的鹦鹉热嗜衣原体基因组DNA(如图3与表1所示),与一步法PCR的检测敏感度相比高出10倍。
3.3临床样本的检测:
利用上述确立的检测体系对临床样本进行检测(临床样本的来源、数目及DNA的提取按上述方法),并与常规的一步法PCR进行比较,其结果如表2所示,该检测体系检测到4例阳性样本,而常规PCR检测体系则均为阴性,表明该检测体系明显优于普通PCR检测体系。
表1.该实时荧光定量PCR检测体系与一步法PCR检测限度的比较
表2.该实时荧光定量PCR检测体系与一步法PCR在鸟类临床粪便样本检测上的比较
Claims (2)
1.一种建立TaqMan probe实时荧光定量PCR检测体系的方法,以动物源性嗜衣原体的16S rRNA为靶基因,包括标准菌株的培养和DNA的分离纯化、TaqManprobe检测体系的确立,TaqMan probe检测体系的检测步骤;
所述标准菌株的培养步骤是选用动物衣原体和嗜衣原体的标准菌株用HeLa229和Hep-2细胞进行培养,培养后使用Puregene DNA分离纯化试剂盒Funakoshi,Japan,从没感染力的衣原体EB中分离纯化基因组DNA;
TaqMan probe检测体系的确立步骤包括:(1)根据鹦鹉热嗜衣原体C.psittaci 6BC的16S rRNA Accession No.AB001778片段序列,利用Primer express.2.0设计如下序列的引物和探针:
AC89F16S:5’-GGCGGAAGGGTTAGTAATACATAGATAA-3’
AP218R16S:5’-AAGGTCCGAAGATCCCCTTCT-3’;
Probe:
5’(FAM)-CCGCTAATACCGAATGTGGTATGTTTAGGC-(TAMRA)3’;
(2)TaqMan probe real-time PCR检测体系的建立的实时荧光定量PCR反应均由7500real-time PCR仪实施完成,包括反应体系、反应条件,反应体系是经过对上下游引物和探针的浓度优化后确定为25μl反应体系;反应条件是由引物和探针的Tm值确立并对其进行优化。
2.如权利要求1所述的方法,其特征在于TaqMan probe real-time PCR检测体系的检测,包括检测体系的特异性试验、敏感性试验,同时将该两试验与一步法PCR检测体系进行比较;特异性试验是将10pg从动物源性嗜衣原体的没有感染力的EB中分离纯化的DNA加入实时荧光定量PCR反应体系中进行检测,同时以同量的其他衣原体和相关菌株的DNA作为对照,得到扩增曲线图,以确定其检测特异性;敏感性试验是将计算得到的标准菌株C.psittaci NJ-1的浓度作梯度稀释,以Ct值<38个循环为下限检测该体系的灵敏性。
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CHARLOTTE A.GAYDOS et al..Phylogenetic Relationship of Chlamydia pneumoniae to Chlamydia psittaci and Chlamydia trachomatis as Determined by Analysis of 16s Ribosomal DNA Sequences.《International Journal of Systematic Bacteriology》.1993,第43卷(第3期),610-612. |
CHARLOTTE A.GAYDOS et al..Phylogenetic Relationship of Chlamydia pneumoniae to Chlamydia psittaci and Chlamydia trachomatis as Determined by Analysis of 16s Ribosomal DNA Sequences.《International Journal of Systematic Bacteriology》.1993,第43卷(第3期),610-612. * |
Justin Hardick et al..Real-Time PCR for Chlamydia pneumoniae Utilizing the Roche Lightcycler and a 16S rRNA Gene Target.《Journal of Molecular Diagnostics》.2004,第6卷(第2期),132-136. * |
Karin D. E. Everett et al..Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards.《International Journal of Systematic Bacteriology》.1999,第49卷(第2期),415-440. * |
Melissa A. Wooters et al..A real-time quantitative polymerase chain reaction assay for the detection of Chlamydia in the mouse genital tract model.《Diagnostic Microbiology and Infectious Disease》.2009,第63卷(第2期),140-147. * |
张阳根等.肺炎衣原体研究进展.《实验与检验医学》.2008,第26卷(第5期),530,537-538. * |
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