CN103525909A - 鲍曼不动杆菌实时荧光环介导等温扩增试剂盒 - Google Patents
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Abstract
本发明属于医学领域,特别涉及一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒,其试剂盒中含有二条外侧引物、二条内测引物及一条环引物,通过使用试剂盒中特异性引物,利用实时荧光环介导等温扩增技术扩增靶序列的特定区域,在一系列质控和阴性、阳性对照的辅助下,从分子水平上对鲍曼不动杆菌核酸进行检测。本发明的试剂盒具有简便、快速、灵敏度高和特异性强等特点,具有良好的应用前景。
Description
技术领域
本发明属于医学领域,特别涉及一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒。
背景技术
近年来,不动杆菌属细菌在全世界范围医院内广泛传播流行,成为医院获得性感染的重要致病菌。目前已经发现不动杆菌属至少有33个基因种,其中鲍曼不动杆菌是引起医院获得性感染的最重要的基因种。另外,醋酸钙不动杆菌、鲍曼不动杆菌、不动杆菌基因种3和不动杆菌基因种13TU在遗传和表型方面都十分相似,传统的生化鉴定方法很难区分这4种不动杆菌,故临床上常常把它们统称为醋酸钙-鲍曼不动杆菌复合群。然而,这4种不动杆菌的流行病学和临床特征存在着一定的差异。因此,快速鉴定鲍曼不动杆菌并正确区分醋酸钙-鲍曼不动杆菌复合群,对预防鲍曼不动杆菌爆发流行和指导临床治疗具有重要的意义。
目前,临床上鉴定鲍曼不动杆菌主要依靠细菌培养和一系列的生化反应检测。虽然广泛使用于临床,但存在多种缺陷:⑴繁琐耗时,一般需要48-72h,如果做药敏试验,所需的时间更长;⑵要求送检快,否则细菌易出现死亡或被空气污染导致假阴性或假阳性;⑶难以区分醋酸钙-鲍曼不动杆菌复合群。
随着分子生物学和基因诊断技术的迅速发展,尤其是作为生物技术里程碑的聚合酶链式反应(polymerase chain reaction,PCR)技术的出现以及细菌核酸测序技术的不断完善,在基因水平诊断细菌,已成为病原菌诊断的新的研究方向。用于鲍曼不动杆菌鉴定的分子生物学方法已有一些研究报道,以PCR为基础的鉴定方法如多重PCR,实时定量PCR具有敏感、快速等优点,但存在许多不足:⑴需要昂贵的仪器和试剂;⑵对检测人员有较高的技术要求;⑶容易引起交叉污染,出现假阳性等。另外,还有DNA-DNA杂交、16S rRNA基因限制性酶切分析、扩增片段长度多态性指纹图谱、16与23S间区直接测序分析等,但这些方法相对于临床实验室而言,有的费用过于昂贵,有的操作过于繁琐,难以推广应用。
实时荧光环介导等温扩增技术(Real-time fluorescence loop mediatedisothermal amplification,RealAmp)则使床边病原体检测成为可能。我们利用一台扩增单元与荧光检测单元相结合的小型便携式仪器即可完成鲍曼不动杆菌的检测。RealAmp除具有LAMP的特点外,还有以下优点:⑴在仪器液晶显示屏上可直接判读结果,或者在电脑上实时监测反应;⑵DNA模板无需纯化等。
发明内容
本发明的目的在于提供一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒,其使床边病原体检测成为可能。
本发明是这样实现的,一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒,其试剂盒中含有二条外侧引物、二条内测引物及一条环引物,这五条特异引物分别为:
外侧引物为:
F3:5'-CTTCTGTTAATAGGTCTAAGCG-3'
B3:5'-TTAAATACCCCTGCTCATCA-3'
内测引物为:
FIP:5'-CGGCGATCATCCCCATGAAAGCTCATTTTAATTTTATGGGCAA-3'
BIP:5'-GCTCCGAATAGCTCTGTTGAGTGCTGAGACTTTTGTAATTCTGAT-3'
环引物为:
LB:5'-CTGGCCTCACAGTTTATGGTCA-3'
本发明最大的优点为该试剂盒使床边病原体检测成为可能,还具有如下优点:适合于实验室快速检测鲍曼不动杆菌且特异性强、等温高效、敏感性高及操作简便。
附图说明
图1是表示实时荧光环介导等温扩增技术(Real-time fluorescence loopmediated isothermal amplification,RealAmp)试剂盒的扩增时间和靶DNA数量之间的关系图。图中X轴表示反应时间,Y轴表示荧光强度;1:100ng/μL;2:10ng/μL;3:1ng/μL;4:100pg/μL;5:10pg/μL;6:1pg/μL;PC:阳性对照;NC:阴性对照。
图2是表示RealAmp试剂盒和PCR检测鲍曼不动杆菌ATCC19606的敏感性比较图。(A)RealAmp试剂盒检测鲍曼不动杆菌ATCC19606的敏感性。图中X轴表示反应时间,Y轴表示荧光强度;1:1×107CFU/mL;2:1×106CFU/mL;3:1×105CFU/mL;4:1×104CFU/mL;5:1×103CFU/mL;6:1×102CFU/mL;7:1×101CFU/mL;NC:阴性对照;(B)PCR检测鲍曼不动杆菌ATCC19606的敏感性。1:1×107CFU/mL;2:1×106CFU/mL;3:1×105CFU/mL;4:1×104CFU/mL;5:1×103CFU/mL;6:1×102CFU/mL;7:1×101CFU/mL;NC:阴性对照。
图3是表示RealAmp试剂盒检测醋酸钙-鲍曼不动杆菌复合群图。图中X轴表示反应时间,Y轴表示荧光强度;1:鲍曼不动杆菌ATCC19606;2:不动杆菌基因种13TU;3:不动杆菌基因种3;4:醋酸钙不动杆菌;PC:阳性对照;NC:阴性对照。
具体实施方式
一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒,其试剂盒中含有二条外侧引物、二条内测引物及一条环引物,这五条特异引物分别为:
外侧引物为:
F3:5'-CTTCTGTTAATAGGTCTAAGCG-3'
B3:5'-TTAAATACCCCTGCTCATCA-3'
内测引物为:
FIP:5'-CGGCGATCATCCCCATGAAAGCTCATTTTAATTTTATGGGCAA-3'
BIP:5'-GCTCCGAATAGCTCTGTTGAGTGCTGAGACTTTTGTAATTCTGAT-3'
环引物为:
LB:5'-CTGGCCTCACAGTTTATGGTCA-3'
试剂盒中的五条引物是通过以下方式制得:
一:在线软件设计试剂盒中的五条引物
以鲍曼不动杆菌中编码聚-β-(1-6)-乙酰氨基葡萄糖(poly-β-(1-6)-N-acetylglucosamine,PGA)的pgaD基因(GeneBank登录号:FJ866500,CP003856,CP003500,CP001937,CP002522,CP001921,CP000863,CP001172,CP001182,CU459141,CP000521,NZ_GG704572)的保守序列为目的片段,通过计算机上的在线软件Primer Explorer(http://PrimerExplorer.jp/e/v4_manual/Index.html)设计5条特异引物,分别为外侧引物(F3和B3)、内侧引物(FIP和BIP)及环引物LB(见表1)。具体过程如下:
1、从http://www.ncbi.nlm.nih.gov/nucleotide/中下载鲍曼不动杆菌ATCC17978的pgaD基因全序列,保存为FASTA格式。
2、上传pgaD基因全序列:⑴点击计算机上的在线软件Primer Explorer V4上的“Browser”按钮,上传鲍曼不动杆菌的pgaD基因全序列到在线软件PrimerExplorer V4启动窗口;⑵选择“Normal”选项;⑶点击“Primer Design”按钮进行引物设计;
3、设计外侧引物(F3和B3)和内侧引物(FIP和BIP):⑴点击“DetailSettings”按钮,选择“Parameter Condition”下拉菜单中的“AT rich”选项,同时选择“Ignore range”按钮;⑵点击“Generate”按钮,生成1000套引物;⑶在“Sorting Rule”的下拉菜单中选择“Easy”,点击“Display”按钮,显示12套引物列表;
4、显示结果:引物列表窗口显示每一套引物的ID,二聚体形成的自由能及引物F2、F3、F1c、B1c、B2和B3所对应的位置;
5、选择最佳的外侧引物(F3和B3)和内侧引物(FIP和BIP):根据BLAST比对引物特异性、F2和B2的3’末端及F1c和B1c的5’末端的稳定性、二聚体发生的趋势、Tm值和GC含量等来选择最佳外侧引物和内侧引物;
6、设计LB环引物:⑴点击“Primer Information”按钮,保存最佳外侧引物(F3和B3)和内侧引物(FIP和BIP)信息用于设计环引物;⑵在计算机在线软件Primer Explorer V4启动窗口上传先前保存的“Primer Information”文件,然后点击“Primer Design”按钮;⑶保持参数为默认值,点击“Generate”按钮,生成42条LB环引物,未生成LF环引物;⑷点击“Display”按钮,显示环引物列表;
7、选择最佳LB环引物:根据环引物3’末端的稳定性来选择最佳LB环引物。
二、确定RealAmp试剂盒的特异性和敏感性
(一)标准菌株及源自临床痰标本的分离株
鲍曼不动杆菌ATCC19606由第三军医大学大坪医院野战外科研究所惠赠。醋酸钙不动杆菌、不动杆菌基因种3和不动杆菌基因种13TU各1株由浙江大学医学院附属第一医院呼吸科惠赠。所有菌株生长在LB培养基中,37℃过夜培养。使用TaKaRa MiniBEST细菌基因组DNA提取试剂盒提取鲍曼不动杆菌ATCC19606的基因组DNA。为了评估扩增时间和DNA数量之间的关系,用灭菌蒸馏水10倍梯度稀释鲍曼不动杆菌ATCC19606的基因组DNA,使其浓度分别为100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL和1pg/μL。
(二)痰标本处理及DNA提取
1、在痰液中加入3倍体积4%NaOH旋紧瓶盖强烈震荡,室温放置40min后轻柔混匀,吸取1ml液化后的痰液至注明编号的1.5ml Ep管中,盖紧盖子,12000rpm离心10min,收集沉淀。
2、用1ml1×TE缓冲液洗涤沉淀一次,12000rpm离心10min,收集沉淀。
3、加入40ul灭菌蒸馏水于沉淀中,并混匀。
4、将上述Ep管置于金属干浴锅中,100℃煮沸15min后立即置于冰上10min。
5、12000rpm离心10min,取上清用于RealAmp和PCR检测。
(三)确定RealAmp试剂盒的特异性
为了确定试剂盒的特异性,我们提取临床常见的铜绿假单胞菌、金黄色葡萄球菌、白色念珠菌等22株菌的DNA为模板进行检测(见表2)。RealAmp反应体系如下:各0.2μM的F3和B3外侧引物,各1.6μM的FIP和BIP内侧引物,0.8μM的LB环引物,8U的Bst DNA聚合酶(New England Biolabs,Ipswich,MA),0.5μL的1:100倍稀释的SYBR green I(Invitrogen),2x反应液(含40mM Tris-HCl pH8.8、20mM KCl、16mM MgSO4、20mM(NH4)2SO4、0.2%Tween-20、0.8M Betaine和2.8mM dNTPs)和2μL提取的模板DNA,反应终体积为25μL,反应在0.2mL PCR管中进行。RealAmp反应条件如下:0.2mL PCR管置于小型便携式仪器ESE-Quant Tube Scanner(QIAGEN Lake Constance GmbH,Stockach,Germany)的反应孔中,设置参数为:每隔30s收集一次荧光信号,63℃反应60min。该仪器重约1kg,大小为74mm×178mm×188mm。带有锂离子可充电电源,无需外部电源即可使用。RealAmp的反应产物检测和结果判读:无需电泳,在小型便携式仪器的液晶显示屏上即可直接判读结果,或者将仪器与电脑连接,可在电脑上实时监测反应曲线,并判读结果。
(四)确定RealAmp试剂盒的敏感性
为了确定试剂盒的敏感性并比较RealAmp和PCR的敏感性,用LB培养基10倍梯度稀释过夜培养液从1×107CFU/mL to101CFU/mL。CFU(colony formingunit)是经培养所得菌落形成单位的英文缩写。RealAmp反应体系如下:各0.2μM的F3和B3外侧引物,各1.6μM的FIP和BIP内侧引物,0.8μM的LB环引物,8U的Bst DNA聚合酶(New England Biolabs,Ipswich,MA),0.5μL的1:100倍稀释的SYBR green I(Invitrogen),2x反应液(含40mM Tris-HCl pH8.8、20mM KCl、16mM MgSO4、20mM(NH4)2SO4、0.2%Tween-20、0.8M Betaine和2.8mM dNTPs)和2μL提取的模板DNA,反应终体积为25μL,反应在0.2mLPCR管中进行。RealAmp反应条件如下:0.2mL PCR管置于小型便携式仪器ESE-Quant Tube Scanner(QIAGEN Lake Constance GmbH,Stockach,Germany)的反应孔中,设置参数为:每隔30s收集一次荧光信号,63℃反应60min。PCR反应体系如下:1×PCR缓冲液(10mM Tris-HCl pH8.3,50mM KCl),0.2mMdNTPs,1.5mM MgCl2,各400nM的F3和B3外侧引物,0.5U的Taq聚合酶(Takara Co.Ltd.,Japan),和2μL的DNA模板。PCR反应条件如下:94℃预变性5min,94℃变性30s,52℃退火30s,72℃延伸45s,共计30个循环,最后72℃延伸10min。RealAmp的反应产物检测和结果判读:无需电泳,在小型便携式仪器的液晶显示屏上即可直接判读结果,或者将仪器与电脑连接,可在电脑上实时监测反应曲线,并判读结果。另外,取5μL PCR产物用于2%琼脂糖凝胶电泳观察结果。
(五)RealAmp测定临床痰标本分离的菌株验证试剂盒的敏感性和特异性
为了验证试剂盒的敏感性和特异性,我们收集2012年8月~2012年10月由中山大学附属第一医院和广州医科大学呼吸疾病国家重点实验室分离的临床痰标本162份。所有痰标本必须进行涂片革兰染色镜检,评价痰标本质量,并记录所见细菌等病原体情况。2h内经镜检白细胞数>10/低倍镜和鳞状上皮细胞数<25/低倍镜判定为合格标本。这162份痰标本经VITEK-2自动细菌鉴定仪鉴定到种,其中90份痰标本有鲍曼不动杆菌生长,72份痰标本没有鲍曼不动杆菌生长,痰标本中的细菌含量在1×103-1×108CFU/mL(见附表1)。所有从痰标本中分离到的鲍曼不动杆菌经16S rRNA基因测序确定,然后用常规PCR和RealAmp进行检测。
三、结果
(一)RealAmp试剂盒的扩增时间和靶DNA数量之间的关系
结果显示随着靶DNA数量的减少,RealAmp扩增时间延长(见图1)。
(二)试剂盒的敏感性
用RealAmp检测鲍曼不动杆菌ATCC19606,结果显示试剂盒的检测下限为1×103CFU/mL,而PCR的检测下限为1×104CFU/mL(见图2),说明该试剂盒的灵敏度好。RealAmp的扩增时间在15-60min之间。临床痰样本的细菌含量越低,RealAmp的扩增时间越长。
(三)试剂盒的特异性
我们选择8株属于不动杆菌属的菌株和18株属于其他非不动杆菌属的菌株(见表2)。结果表明,用RealAmp检测方法可以从18株属于其他非不动杆菌属的菌株中区分鲍曼不动杆菌(见表2),说明该试剂盒的特异性强。此外,RealAmp试剂盒还可以区分鲍曼不动杆菌、醋酸钙不动杆菌和不动杆菌基因种3(见图3)。
(四)RealAmp测定临床痰标本分离的菌株验证试剂盒的敏感性和特异性
RealAmp与VITEK2自动细菌鉴定仪和常规PCR的敏感性和特异性比较的结果见表3。在VITEK2自动细菌鉴定仪鉴定的90个阳性标本中,RealAmp检测结果显示89个为阳性(+),PCR检测结果显示84个为阳性(+);在VITEK2自动细菌鉴定仪鉴定的72个阴性标本中,RealAmp检测结果显示54个为阴性(-),PCR检测结果显示60个为阴性(-)。与VITEK2自动细菌鉴定仪相比,RealAmp试剂盒的敏感性和特异性分别为98.9%(95%CI:94.0–99.8%)和75.0%(95%CI:63.9–83.6%);与PCR相比,RealAmp试剂盒的敏感性和特异性分别为100%(95%CI:95.6–100%)和90.0%(95%CI:79.9-95.3%)。
四、结论
上述试验证明鲍曼不动杆菌的RealAmp试剂盒具有敏感性高、特异性强、快速简单和操作容易等特点,适合于实验室和床边快速检测鲍曼不动杆菌。
表1:RealAmp试剂盒中的外侧引物、内侧引物及环引物的序列
Table1Sequences of primers F3,B3,FIP,BIP and LB used in the kit ofRealAmp assay.
F3:上游外侧引物;B3:下游外侧引物;
FIP:上游内侧引物;BIP:下游内侧引物;
LB:下游环引物;bp:碱基对。
表2:分析RealAmp试剂盒的特异性所使用的菌株和检测结果
Table2Strains used and the results of RealAmp assays.
a:源自美国标准菌库的标准菌株;
b:浙江大学附属第一医院呼吸科惠赠菌株;
c:广州医科大学呼吸疾病国家重点实验室保存菌株;
RealAmp:实时荧光环介导等温扩增技术;
-:RealAmp检测的阴性结果;
+:RealAmp检测的阳性结果。
表3:RealAmp试剂盒与VITEK2自动细菌鉴定仪和常规PCR检测临床痰标本分离菌株的敏感性和特异性比较
Table3Sensitivity and Specificity of the RealAmp assay compared to VITEK2system and PCR assay.
*:经16S rRNA基因测序确定不动杆菌种;
VITEK2system:VITEK-2自动细菌鉴定仪;
RealAmp:实时荧光环介导等温扩增技术;
PCR:聚合酶链反应。
附表1:临床痰标本分离菌株的基本信息
Supplemental Data Table1The information of the clinical samples in thisstudy.
+:PCR或RealAmp检测的阳性结果;
-:PCR或RealAmp检测的阴性结果;
N:无致病菌生长;
ND:未做检测。
Claims (1)
1.一种鲍曼不动杆菌实时荧光环介导等温扩增试剂盒,其特征在于该试剂盒中含有二条外侧引物、二条内测引物及一条环引物,这五条特异引物分别为:
外侧引物为:
F3:5'-CTTCTGTTAATAGGTCTAAGCG-3'
B3:5'-TTAAATACCCCTGCTCATCA-3'
内测引物为:
FIP:5'-CGGCGATCATCCCCATGAAAGCTCATTTTAATTTTATGGGCAA-3'
BIP:5'-GCTCCGAATAGCTCTGTTGAGTGCTGAGACTTTTGTAATTCTGAT-3'
环引物为:
LB:5'-CTGGCCTCACAGTTTATGGTCA-3' 。
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CN110819728A (zh) * | 2019-08-28 | 2020-02-21 | 北京大学首钢医院 | 一种结合多交叉置换扩增和金纳米检测的检测技术及应用 |
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Cited By (4)
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CN104774961A (zh) * | 2015-04-29 | 2015-07-15 | 郭旭光 | 检测无乳链球菌的引物、试剂盒和方法 |
CN110819728A (zh) * | 2019-08-28 | 2020-02-21 | 北京大学首钢医院 | 一种结合多交叉置换扩增和金纳米检测的检测技术及应用 |
WO2021250139A1 (en) * | 2020-06-09 | 2021-12-16 | Certus Molecular Diagnostics Ag | Isothermal real-time pcr method for determining presence of a pre-determined nucleic acid sequence of a bacterium of the mollicutes class in a sample |
WO2021250138A3 (en) * | 2020-06-09 | 2022-02-10 | Certus Molecular Diagnostics Ag | Isothermal real-time pcr method for determining presence of a pre-determined nucleic acid sequence in human samples |
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