CN102912036B - 快速检测和鉴定生物物体的方法 - Google Patents
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Abstract
检测和鉴定未知生物体,包括细菌、病毒等的方法,采用了能与生物体的核酸的保守序列区域杂交的并含有能独特分辨法生物体的可变序列区域的引物。结果是一种“碱基组成签名”(BCS),然后与碱基组成签名数据库匹配,由此鉴定该生物体。
Description
本申请是国际申请日为2002年3月4日的国际申请PCT/US2002/006763进入中国、申请号为02809122.1的题为“快速检测和鉴定生物物体的方法”的发明专利申请的分案申请。
政府支持的声明
本发明得到美国政府DARPA/SPO合同BAA00-09的支持。美国政府可在发明中拥有一定权利。
技术领域
本发明涉及快速检测和鉴定环境、临床或其它样品中的生物体的方法。该方法提供了检测和特征分析任何生物体,包括细菌和病毒的独特碱基组合签名(BCS)。这种独特的BCS可用于快速鉴定该生物体。
背景技术
快速和明确的微生物鉴定对各种工业、医学、环境、质量和研究原因是需要的。传统上,微生物学实验室的功能是通过直接检查和培养样品来鉴定传染性疾病的病原体。自从1980年代中期以来研究者们反复证明了分子生物学技术在实践上的用途,许多这些技术形成了临床诊断试验的基础。这些技术中的一些包括核酸杂交分析、限制性酶分析、遗传序列分析和核酸的分离及纯化(参见,如J.Sambrook,E.F.Fritsch和T.Maniatis,分子克隆:实验室手册,第二版,冷泉港实验室出版社,冷泉港,纽约,1989)。这些方法一般费时且冗长。另一选择是聚合酶链式反应(PCR),或其它扩增方法,根据所用侧翼引物扩增特异性靶DNA序列。最后,检测和数据分析可将杂交转换成分析结果。
其它检测生物体的技术包括高分辨率质谱(MS)、低分辨率MS、荧光、放射性碘化、DNA芯片和抗体技术。这些技术中没有一个是完全令人满意的。
质谱提供了被分析分子的详细信息,包括很高的质量准确性。它也是一种可容易地自动化的方法。然而,高分辨率MS单独不能用来对未知的或生物工程改造的生物体进行分析,或运用于存在高背景水平生物体(“混乱的”背景)的环境中。低分辨率MS不能检测某些已知的生物体,如果它们的光谱线足够弱或足够接近样品中其它活生物体的光谱线。具有特异性探针的DNA芯片只能够测定具体的预计的生物体的存在与否。因为有几十万种良性细菌,一些细菌在序列上十分类似于有威肋的细菌,即使有10,000个探针的芯片陈列仍缺乏检测具体生物体所需的宽度。
抗体面对的多样性限制比陈列更严峻。如果抗体设计是针对高度保守的靶子以提高多样性,假报警问题就会是主要问题,还是因为危险生物体十分类似于良性生物体。抗体只能检测相对不混乱环境中的已知生物体。
一些研究中组报道了用高分辨率电喷射离子化-傅立叶变换-离子回旋共振质谱法(ESI-FT-ICRMS)检测PCR产物。准确测定确切的质量与至少一种核苷的数量的知识相结合,可计算出大约100个碱基对的PCR双链体产物的总碱基组成。(Aaserud等.,J.Am.Soc.MassSpec.7:1266-1269,1966;Muddiman等.,Anal.Chem.69:1543-1549,1997;Wunschel等.,Anal.Chem.70:1203-1207,1998;Muddiman等.,Rev.Anal.Chem.17:1-68,1998)。电喷射离子化-傅立叶变换-离子回旋共振(ESI-FT-ICR)MS可通过平均分子质量来确定500碱基对双链体,PCR产物的质量(Hurst等.,RapidCommun.Mass.Spec.10:377-382,1996)。已报道了采用基质-辅助的激光解吸离子化飞行时间(MALDI-TOF)质谱来特征鉴定PCR产物。(Muddiman等.,RapidCommun.Mass.Spec.13:1201-1204,1999)。然而,用MALDI只能观察75个核苷以上的DNA的降解限制了此方法的应用。
美国专利5,849,492描述了挽回系统发育信息DNA序列的方法,包括搜索两个高度保守片断包围的基因组DNA的高度趋异区段,设计了用PCR扩增此高度趋异区域的通用引物,用此通用引物经PCR技术扩增该基因组DNA,然后测是比基因序列以确定该生物体的身份。
美国专利5,965,363公开了用质谱技术分析扩增的靶核酸来筛检核酸多态性的方法,和提高这些方法的质量分辨率及质量准确性的方法。
WO99/14375描述了通过质谱分析预选的DNA串联核苷酸重复等位基因所用的方法、PCR引物和试剂盒。
WO98/12355公开了通过质谱分析测定靶核酸的质量的方法,通过切割靶核酸以减少其长度,产生靶单链并用MS测定单链变短的靶子的质量。还公开了制备用于MS分析的双链靶核酸的方法,包括扩增靶核酸,使一条链结合于固相载体,释放第二条链,并随后释放第一条链,接着用MS分析。也提供了制备靶核酸的试剂盒。
PCTWO97/33000公开了检测靶核酸中突变的方法,通过使靶子非随机断裂成一组单链非随机长度的片断,并用MS测定它们的质量。
美国专利5,605,798报道了快速和高度准确的以质谱仪为基础的方法,检测生物样品中特定核酸的存在用于诊断目的。
WO98/21066描述了通过质谱测定特定靶核酸序列的方法。公开了用PCR扩增和质谱检测来检测生物样品中靶核酸存在的方法,也报道了通过用含有限制性酶切位点和标记的引物扩增样品中的靶子,延伸和切割扩增的核酸,并检测延伸产物的存在来检测靶核酸的方法,其中存在不同于野生型质量的DNA片断表明有突变。通过质谱方法测定核酸序列的方法也有报道。
WO97/37041、WO99/31278和美国专利5,547,835描述了用质谱测定核酸序列的方法。美国专利5,622,824、5,872,003和5,691,141描述了用于外切核酸酶-介导的质谱测序的方法、系统和试剂盒。
因此,需要一种特异和快速的生物体检测和鉴定的方法,其中不需要给核酸测序。本发明符合此需求。
发明内容
本发明的一个实施方案是一种鉴定未知生物体的方法,该方法包括(a)使该生物体的核酸接触至少一对寡核苷酸引物,此引物能与该核酸序列杂交并侧接有一可变的核酸序列;(b)扩增此可变的核酸序列产生扩增产物;(c)测定此扩增产物的分子量;(d)将此分子量与在多种已知生物体上进行步骤(a)-(c)所获得的一种或多种扩增产物的分子量相比较,其中一种匹配可鉴定该未知的生物体。在此优选实施方案的一个方面,能与至少一对寡核苷酸引物杂交的序列高度保守。优选扩增步骤包括聚合酶链式反应。或者,扩增步骤包括连接酶链式反应或链置换扩增。在此优选实施方案的一个方面,生物体是细菌、病毒、细胞或孢子。有利的是此核酸是核糖体RNA。在另一方面,此核酸编码RNA酶P或RNA-依赖的RNA聚合酶。优选扩增产物在分子量测定前离子化。该方法还可包括在使该核酸接触至少一对寡核苷酸引物前,从生物体中分离得到核酸的步骤。该方法还可包括使用一对不同的寡核苷酸引物进行步骤(a)-(d)的步骤,并将结果与在步骤d)的不同的多种已知生物体上进行步骤(a)-(c)获得的一种或多种扩增产物的分子量相比较。优选一种或多种分子量包含在分子量数据库中。在此优选实施方案的另一个方面,扩增产物通过电喷射离子化、基质-辅助的激光解吸或快速原子轰击而离子化。有利的是,分子量通过质谱测定。优选质谱是傅立叶变换离子回旋共振质谱法(FT-ICR-MS)、离子陷阱、四极、磁扇面、飞行时间(TOF)、Q-TOF或三重四极。该方法还可包括在存在具有与腺苷、胸苷、鸟苷或胞苷不同的分子量的腺嘌呤、胸苷、鸟苷或胞苷类似物时进行步骤(b)。在一方面,互寡核苷酸引物在引物中各个三联体位置1和2上含有碱基类似物或替换碱基,其中此碱基类似物或替换碱基能以比天然碱基更高的亲和力与其互补物相结合。优选该引物在引物中各个三联体位置3上含有通用碱基。此碱基类似物或替换碱基可以是2,6-二氨基嘌呤、丙炔T、丙炔G、吩嗪或G-夹子。优选此通用碱基是肌苷、胍、尿苷、5-硝基吲哚、3-硝基吡咯、dP或dK或1-(2-脱氧-β-D-呋喃核糖)-咪唑-4-酰胺。
本发明另一个实施方案是一种鉴定未知生物体的方法,该方法包括(a)使该生物体的核酸接触至少一对寡核苷酸引物,此引物能与该核酸序列杂交并侧接有一可变的核酸序列;(b)扩增此可变的核酸序列产生扩增产物;(c)测定扩增产物的碱基组成;(d)将此碱基组成与在多种已知生物体上进行步骤(a)-(c)获得的扩增产物的一种或种种碱基组成相比较,其中一种匹配可鉴定该未知生物体。在此优选实施方案的一个方面,能与至少一对寡核苷酸引物杂交的序列高度保守。优选扩增步骤包括聚合酶链式反应。或者,扩增步骤包括连接酶链式反应或链置换扩增。在此优选实施方案的一个方面,生物体是细菌、病毒、细胞或孢子。有利的是,此核酸是核糖体RNA。在另一方面,此核酸编码RNA酶P或RNA-依赖的RNA聚合酶。优选扩增产物在分子量测定前离子化。该方法还可包括在使该核酸接触至少一对寡核苷酸引物前,从生物体中分离得到核酸的步骤。该方法还可包括使用一对不同的寡核苷酸引物进行步骤(a)-(d)的步骤,并将结果与在步骤d)的不同的多种已知生物体上进行步骤(a)-(c)获得的一种或多种扩增产物的碱基组成相比较。优选一种或多种碱基组成包含在碱基组成数据库中。在此优选实施方案的另一个方面,扩增产物通过电喷射离子化、基质一辅助的激光解吸或快速原子轰击而离子化。有利的是,分子量通过质谱测定。优选质谱是傅立叶变换离子回旋共振质谱法(FT-ICR-MS)、离子陷阱、四极、磁扇面、飞行时间(TOF)、Q-TOF或三重四极。该方法可进一步包括在存在具有与腺苷、胸苷、鸟苷或胞苷不同的分子量的腺嘌呤、胸苷、鸟苷或胞苷类似物时进行步骤(b)。在一方面此寡核苷酸引物在引物中各个三联体位置1和2上含有碱基类似物或替换碱基,其中此碱基类似物或替换碱基能以比天然碱基更高亲和力与和其互补物相结合。优选该引物在引物中各个三联体位置3上含有通用碱基。此碱基类似物或替换碱基可以是2,6-二氨基嘌呤、丙炔T、丙炔G、吩嗪或G-夹子。优选此通用碱基是肌苷、胍、尿苷、5-硝基吲哚、3-硝基吡咯、dP或dK获1-(2-脱氧-β-D-呋喃核糖)-咪唑-4-酰胺。
本发明也提供检测个体中单个核苷多态性的方法,该方法包括以下步骤(a)分离个体的核酸;(b)使该核酸与寡核苷酸引物接触,此引物能与侧接有一含有潜在多态性的区域的该核酸杂交;(c)扩增此区域产生扩增产物;(d)测定扩增产物的分子量;(e)将该分子量与个体中已知具有多态性的区域的分子量相比较,其中如果两分子量相同,则该个体具有此多态性。
在此优选实施方案的一个方面,使引物与高度保守的序列杂交。优选多态性与其疾病相关连。或者,多态性是一种血型抗原。在此优选实施方案的一个方面,扩增步骤是聚合酶链式反应。或者,扩增步骤是连接酶链式反应或链置换扩增。优选扩增产物在质量测定前离子化。一方面,扩增产物通过电喷射离子化、基质-辅助的激光解吸或快速原子轰击而离子化。有利的是,分子量通过质谱测定。优选质谱法是傅立叶变换离子回旋共振质谱法(FT-ICR-MS)、离子陷阱、四极、磁扇面、飞行时间(TOF)、Q-TOF或三重四极。
附图说明
图1A-1I显示保守区域例子的共有序列图,此保守区域来自16SrRNA(图1A-1B)(SEQIDNO:3)、23SrRNA(3’-半,图1C-1D;5’-半,图1E-F)(SEQIDNO:4)、23SrRNA结构域I(图1G)、23SrRNA结构域IV(图1H)和16SrRNA结构域III(图1I),它们适合用于本发明。带箭头的线是设计用于PCR引物对所针对的区域的例子。各引物对的标记代表该共有序列图上被扩增区域的起始和终止碱基的编号。大写字母的碱基95%以上保守;小写字母的碱基90-95%保守;实心圆80-90%保守;空心圆低于80%保守。各引物对的标记代表该共有序列图上被扩增区域的起始和终止碱基的编号。
图2显示图1C中所示的16SrRNA结构域III的典型的引物扩增区域。
图3显示RNA酶P中保守区域的示意图。大写字母的碱基90%以上保守;小写字母的碱基80-90%保守;实心圆代表的碱基70-80%保守;空心圆代表的碱基低于70%保守。
图4是碱基组成签名测定的示意图,用核苷类似物“标志”来确定碱基组成签名。
图5显示炭疽芽胞杆菌(Bacillusanthracis)具有或没有质量标志硫代磷酸A(A*)区域的去卷积质谱。这两个质谱的区别在于含该质量标记的序列测定的分子量大于未修饰的序列。
图6显示金黄色葡萄球菌(S.aureus16S_1337F)和炭疽杆菌(B.anthr.16S_1337F)的PCR产物的碱基组成签名(BCS)质谱,,扩增采用了相同的引物。两条链的差别仅为两个(AT→CG)替换并且根据它们的BCS可清楚地鉴别。
图7显示两种序列(炭疽芽胞杆菌的A14对蜡样芽胞杆菌的A15)之间的一个差别可容易地用ESI-TOF质谱检测。
图8是炭疽杆菌芽孢杆菌包膜蛋白sspE56聚体加校准的ESI-TOF。此信号明确地鉴定了其它芽胞杆菌和炭疽杆菌。
图9是炭疽杆菌合成的16S_1228双链体(反向和正向链)的ESI-TOF。此技术可容易的鉴别正向和反向链。
图10是合成的炭疽杆菌16S_133746碱基对双链体的ESI-FTICR-MS。
图11是炭疽杆菌saspB基因的56聚体寡核苷酸(3次扫描)的ESI-TOF-MS,采用内部质量标准。此内部质量标准用星号表示。
图12是用5mMTBA-TFA缓冲液配的内部标准品的ESI-TOF-MS,显示含三丁铵三氟醋酸盐的带电条带降低了最大丰度的带电状态,从[M-8H+]8-降到[M-3H+]3-。
具体实施方式
本发明提供了非PCR生物量检测模式,优选高分辨率MS,与采用“智能引物”的以PCR为基础的BCS技术相结合,此智能引物能与衍生自某生物体的核酸的保守序列区域杂交并包含能独特地鉴定该生物体的可变序列区域。高分辨率MS技术用于测定被扩增序列区域的分子量和碱基组成签名(BCS)。然后将此独特的“碱基组成签名”(BCS)输入最大可能性检测算法中,用于匹配同一被扩增区域中碱基组成签名的数据库。本方法结合了以PCR为基础的扩增技术(提供特异性)和分子量检测模式(提供速度且不需要测定被扩增的靶序列的核酸序列),用于生物体检测和鉴定。
本方法与现有方法相比能非常迅速和精确地检测和鉴定生物体。此外,即使当样品物质不纯时,此快速检测和鉴定也是可行的。因此,此方法在各种领域中是有用的,包括但不限于,环境测试(如检测和区别水或其它样品中的病原和非病原菌)、细菌战争(可立即鉴定生物体和适当治疗)、药物遗传分析和医学诊断(包括根据突变和多态性作出癌症诊断;药物抗性和易感性试验;筛检和/或诊断遗传疾病和状况;诊断传染病和状况)。此方法影响了正在进行的关于毒性、病原性、药物抗性和基因组测序的生物医学研究,提供了与现有方法相比大为改进的灵敏性、特异性和可靠性,假阳性率更低的方法。
本方法可用于检测和分类任何生物体,包括细菌、病毒、真菌和毒素。作为一个例子,当该生物体是一种生物威胁,所得的信息可用于确定对策所需的实践信息,包括毒素基因,病原分离和抗生素抗性基因。此外,此方法可用于鉴定天然事物或仔细考虑的基因工程事物,包括染色体片断交换、分子繁殖(基因改组)和冒出的传染病。
细菌具有一套共同的绝对需要的基因。约250个基因存在于所有种类的细菌中(Proc.Natl.Acad.Sci.U.S.A.93:10268,1996;Science270:397,1995),包括小基因组,如支原体(Mycoplasma)、脲原体(Ureaplasma)和立克次体(Rickettsia)。这些基因编码的蛋白,参与翻译、复制、重组和修复、转录、核苷酸代谢、氨基酸代谢、脂类代谢、能量产生、吸收、分泌等。这些蛋白质的例子是DNA聚合酶IIIβ、延伸因子TU、热激蛋白groEL、RNA聚合酶B、磷酸甘油酸激酶、NADH脱氢酶、DNA连接酶、DNA拓扑异构酶和延伸因子G。操纵子也可用本方法靶向。操纵子的一个例子是肠道致病性大肠杆菌的bfp操纵子。多种核心染色体基因可用于在属或种水平上分类细菌来确定某生物体是否具有潜在威胁。此方法也可用于检测病原性标记(质粒或染色体)和抗生素抗性基因来证实某生物体的潜在威胁和指导对策。
理论上理想的生物体检测器应能鉴定、定量测定和报告到达传感器的各生物体的全核酸序列。病原体核酸组分的全序列应提供关于威胁的所有相关信息,包括它的身份和药物抗性或病原性标记的存在。这个理想还未达到。然而,本发明提供了采用碱基组成签名(BCS)获得具有用相同实际值信息的简单策略。虽然基因片断的碱基组成不如序列本身那样富含信息,但如果适当选择短的分析物序列片断,就不需要分析该基因的全序列。可制备参考序列数据库,其中将各序列指数化成独特的碱基组成签名,从而某序列的存在可精确地从其签名的存在来推知。碱基组成签名的优点是它们可以整块平行方式,采用多重PCR(在PCR中两对或多种引物对同时扩增靶序列)和质谱定量测定。这些多对引物扩增的区域能独特地鉴定最有感胁的和普遍的背景细菌和病毒。此外,簇-特异引物对能分辨重要的本地簇(如炭疽群)。
在本发明的上下文中,“生物体”是任何生物体,活的或死的,或衍生自这种生物体的核酸。生物体的例子包括但不限于细胞(包括但不限于人临床样品、细菌细胞和其它病原体)、病毒、毒素基因和生物调节化合物)。样品可以是活的或死的或处在生长状态(例如,生长繁殖细菌或孢子)且可以是被包裹的或经生物工程改造的。
如本文所用,“碱基组成签名”(BCS)是能独特地鉴定靶基因和来源生物体的核酸序列所选片断的精确碱基组成。BCS可认为是特定基因的独特指数。
如本文所用,“智能引物”是能与侧接有一价入可变区的序列区域相结合的引物。在一个较佳实施方案中,这些侧接有可变区的序列区域在不同种类的生物体中高度保守。例如,此序列区域可在所有种类芽孢杆菌中高度保守。术语“高度保守”,意味着这些序列区域显示约80-100%的相同性,更优选约90-100%、最优选约95-100%相同性能扩增16S和23SrRNA区域的智能引物的例子见图2。箭头代表能与侧接有16SrRNA结构域III可变区的高度保守区域相结合的引物。被扩增的区域是“1100-1188”下的茎环结构。
如本文所用,“匹配”指任何统计学上有显著性的概率确定或结果。
本法明检测方法的一个主要优点是不需要具体细菌种类或者菌属的特异性引物,如芽胞杆菌(Bacillus)或链霉菌(Streptomyces)。此引物能识别横跨数百个细菌种类的高度保守区域,包括但不限于,本文所述的种类。因此,同一引物对可用于鉴定任何期望的细菌,因为它会与侧接有一可变区,此可变区对一个菌种是特异的或对若干菌种是共同的保守区域结合,从而能使该介入序列的核酸扩增和测定它的分子量及碱基组成。例如,16S_971-1062、16S_1228-1310和16S_1100-1188区域在约900种细菌中98-99%保守(16S=16SrRNA,数字表示核苷酸位置)。在本发明的一个实施方案中,本方法所用的引物能结合一个或多个这些区域或其部分。
本发明提供了非PCR生物量检测模式,优选高分辨率MS,与采用“智能引物”的以核酸扩增为基础的BCS技术相组合,该智能引物能与保守区域杂交并含有能独特鉴定该生物体的可变区域。虽然采用PCR是优选的但其它核酸扩增技术也可使用,包括连接酶链式反应(LCR)和链置换扩增(SDA)。高分辨率MS技术可在高度混杂的环境中从背景谱线中分离出生物体的线。然后将被分辨的谱线翻译成BCS,输入到最大可能性检测算法中,与一种或多种已知的BCS谱线作匹配。较佳的是,生物体BCS谱与许许多多生物体的BCS的数据库的一种或多种相匹配。优选采用最大可能性检测算法进行此种匹配。
在一个较佳实施方案中,碱基组成签名以整块平行方式定量测定,采用聚合酶链式反应(PCR),优选多重PGR和质谱(MS)方法。必须存在足够量的核酸用于MS检测生物体。本领域技术人员熟知多种制备大量纯化核酸或其片断的技术。PCR要求能与侧接有待扩增靶序列的区域相结合的一对或多对寡核苷酸引物。这些引物引导了DNA不同链的合成,合成发生时一个引物朝向另一个引物的方向。将引物、待扩增的DNA、热稳定的DNA聚合酶(如Taq聚合酶)、四种脱氧核苷三磷酸和缓冲液相混合起动DNA合成。加热变性此溶液,然后冷却使新加入的引物退火,接着进行又一轮的DNA合成。此过程通常重复约30个循环,导致靶序列的扩增。
“智能引物”明确了待扩增和分析的靶序列。在一个实施方案中,靶序列是核糖体RNA(rRNA)基因序列。现在可得到许多最小的微生物基因组的全序列,故有可能鉴定一套定义为“最小生命”的基因和鉴定能独特分辨各基因和生物体的组成签名。编码核心生命功能,如DNA复制、转录、核糖体结构、翻译和转运的基因广泛分布于细菌基因组中且是BCS分析的优选区域。核糖体RNA(rRNA)基因包含有提供有用碱基组成签名的区域。像许多参与核心生命功能的基因,rRNA基因包含横跨细菌结构域的非常保守的序列,散布着对各菌种更特异的高度不同的区域。可利用这些不同区域来构建碱基组成签名的数据库。策略包括建立rRNA基因小亚基(16S)和大亚基(23S)序列的以结构为基础的排列对比。例如,目前核糖体RNA数据库中已有超过13,000种序列由RobinGutell,UniversityofTexasatAustin创建和维持,并且公众可在theInstituteforCellularandMolecularBiology网页(www.rna.icmb.utexas.edu/)中得到。公众也可得到由UniversityofAntwerp,比利时网页(www.rrna.uia.ac.be)创建和维持的rRNA数据库。
已分析了这些数据确定了用作为碱基组成签名的区域。这些区域的特征是:a)在感兴趣的具体生物种类中,作为序列扩增引物位置的上游和下游核苷酸序列的相同性在80-100%之间,优选>95%;b)介入的可变我显示在诸种类中相同性不大于约5%;c)保守区域之间,分隔约30至1000个核苷酸,优选不超过50-250个核苷酸更优选不超过约60-100个核苷。
由于它们总体上保守,侧翼的rRNA引物序列可作为优良的“通用”引物结合位点来扩增大多数(如果不是所有)菌种感兴趣的区域。几套引物之间的该介入区域在长度和/或组成上不同,因此提供了独特的碱基组成签名。
设计的此“智能引物”的优点是通用性,可能最大程度减少需合成的引物数量,可用一对引物检测许多生物种类。这些引物对可用于扩增这些物种中的不同区域。因为物种中这些保守区域中的任何变化(由于密码子第三个位置的不稳定)可能发生在DNA三联体的第三个位置,可设计寡核苷酸引物使对应这个位置的核苷酸是可结合一种以上核苷酸的碱基,本文称为“通用碱基”。例如,在这种“不稳定的”配对中,肌苷(I)能结合U、C或A;鸟嘌呤(G)能结合U或C,尿苷(U)能结合U或C。其它通用碱基的例子包括硝基吲哚如5-硝基吲哚或3-硝基吡咯(Loakes等,NucleosidesandNucleotides14:1001-1003,1995)、简并核苷dP或dK(Hill等)、含5-硝基吲唑的无环核苷类似物(VanAerschot等.,NucleosidesandNucleotides14:1053-1056,1995)或嘌呤类似物1-(2-脱氧-β-D-呋喃核糖)-咪唑-4-酰胺(Sala等.,Nucl.Acids.Res.24:3302-3306,1996)。
在本发明的另一个实施方案中,为补偿“不稳定”碱基的较弱结合,设计了寡核苷酸引物使各个三联体的第一和第二个位置为比未修饰的核苷酸亲和力更高的核苷酸类似物所占据。这些类似物的例子是能结合胸腺嘧啶的2,6-二氨基嘌呤、能结合腺嘌呤的丙炔T和丙炔C和吩嗪,包括能结合G的G-夹子。丙炔描述见美国专利5645958、5830653和5494908其整个内容纳入本文参考文献。吩嗪的描述见专利5,502,177、5,763,588和6,005,096,它们的完整内容纳入本文参考文献。G-夹子描述见美国专利号6,00,992和6,028,183,它们的完整内容纳入本文参考文献。
能被现有方法检测的细菌生物战物质包括炭疽芽孢杆菌(炭疽热)、耶尔森鼠疫杆菌(鼠疫性肺炎)、土拉热弗朗西斯菌(土拉热)、猪布鲁菌、流产布鲁菌、马耳他布鲁菌(波状热)、鼻疽伯克霍尔德氏菌(鼻疽)、类鼻疽伯克霍尔德氏菌(类鼻疽)、伤寒沙门菌(伤寒症)、斑疹伤寒立克次体(流行性斑疹伤寒症)、普氏立克次体(地方性斑疹伤寒)和伯纳特立克次体(Q热)、荚膜红细菌、肺炎衣原体、大肠杆菌、志贺痢疾菌、弗氏志贺菌、蜡样芽胞杆菌、肉毒梭菌、伯纳特立克次体、绿脓假单胞菌、嗜肺军团菌和霍乱弧菌。
除了16S和23SrRNA,其它适合用于本发明检测的细菌靶区域包括5SrRNA和RNA酶P(图3)。
生物战真菌生物战物质包括immitis球孢子菌球孢子菌病。
能通过本发明的方法检测的生物战毒素基因包括肉表毒素、T-2霉菌素素、篦麻毒素、葡萄球菌肠毒素B、志贺菌毒素、相思豆毒素、黄曲霉毒素、产气荚膜梭菌ε毒素、芋螺毒素、蛇形菌素、河豚毒素和蛤蚌毒素。
生物战病毒威胁物质主要是RNA病毒(正链和负链),除天花外。每种RNA病毒是一个相关病毒的家族(准种)。这些病毒变异迅速且作为工程发行改造株的潜力(天然或仔细考虑的)很高。RNA病毒聚簇形成诸家族,它们在病毒基因组上具有保定的RNA结构域(如病毒粒子组分,辅助蛋白)和编码核心病毒蛋白的保守管家基因,包括单链正链RNA病毒、RNA依赖的RNA聚合酶、双连RNA解旋酶、胰凝乳蛋白酶样和木瓜蛋白酶样蛋白酶及转甲基酶。
(-)-链RNA病毒的例子包括沙粒病毒(如sabia病毒、拉沙热、Machupo、阿根廷出血热、flexal病毒)、木雅病毒(如汉坦病毒属、纳伊罗病毒、白蛉病毒、汉坦病毒、刚果-克里米亚半岛出血热、里夫特裂谷热)和单负病毒(mononegavirales)(如纤丝病毒、副粘病毒、埃博拉病毒、马尔堡病毒、马麻疹病毒)。
(+)-链RNA病毒的例子包括小核糖核酸病毒(如柯萨奇病毒、艾柯病毒、人柯萨奇病毒A、人艾柯病毒、人肠道病毒、人脊髓灰质炎病毒、甲肝病毒、人parechovirus、人鼻病毒)、星状病毒(如人星状病毒)、钙病毒(calcivimses)(如千叶病毒(chiba)、chitta病毒、人钙病毒、诺沃克病毒)、nidovirales(如人冠状病毒、人torovirus)、黄病毒(如登革热病毒1-4、日本脑炎病毒、森林出血热病毒、Murray河谷脑炎病毒、Rocio病毒、圣路易斯脑炎病毒、西尼罗河病毒、黄热病病毒、丙肝病毒)和披膜病毒(如屈曲病毒、东部马脑炎病毒、Mayaro病毒、O’nyong-nyong病毒、罗斯河冰毒、委内瑞拉马脑炎病毒、风疹病毒、戊型肝炎病毒)。丙型肝炎病毒有340个核苷酸的5’-非翻译区域,编码9种蛋白质的开放读框有3010个氨基酸和240个核苷的3’-非翻译区域。5’-UTR和3’-UTR在丙型肝炎病毒中99%保守。
在一个实施方案中,靶基因是RNA依赖的RNA聚合酶或由(+)-链RNA病毒编码的解旋酶,或(-)-链RNA病毒的RNA聚合酶。(+)-链RNA病毒是双链RNA且能通过采用RNA依赖的RNA聚合酶和正链作为模板的RNA-指导的RNA合成来复制。解旋酶解旋RNA双链使得单链RNA复制。这些病毒包括来自小核糖核酸病毒家族的病毒(如脊髓灰质炎病毒、柯萨奇病毒、艾柯病毒)、披膜病毒(如甲病毒、黄病毒、风疹病毒)、沙粒病毒(如淋巴细胞性脉络丛脑膜炎病毒、拉沙热病毒)、cononaviridae(如人呼吸病毒)和甲肝病毒。编码这些蛋白的基因包含可变区和侧接该可变区的高度保守区域。
在一个较佳实施方案中,对生物体产生PCR产物的检测方案有三个特征。首先,该技术可同时检测和区分多种(一般约6-10种)PCR产物。第二,该技术提供了从可能的引物位置独特鉴定生物体的BCS。最后,该检测技术快速,使多个PCR反应平行进行。
在一个实施方案中,此方法可用于检测细菌种类中抗生素抗性和/或毒素基因的存在。例如,含有四环素抗性质粒和编码一种或两种炭疽毒素(px01和/或px02)的质粒的炭疽芽胞杆菌可用抗生物抗性引物对和毒素基因引物对检测。如果炭疽杆菌四环素抗性是阳性,可用一种不同的抗生素,如quinalone。
以质谱(MS)为基础的PCR产物检测提供了所有这些特点和另外的优点。MS本身是一种平行的检测方案,无需放射性或荧光标记,因为具有独特碱基组成的各扩增产物可通过其分子量鉴定。本领域在质谱上的当前状况是可以容易地分析小于毫微微摩尔量的物质以提供关于样品分子成分的信息。物质分子量的精确测定可迅速获得,无论样品分子量是几百还是超过十万原子质量单位(amu)或道尔顿。完整的分子型离子可用使样品转变成气相的多种电离技术之一从扩增产物中产生。这些电离技术包括但不限于,电喷射离子化(ES)、基质-辅助的激光解吸离子化(MALDI)和快速原子轰击(FAB)。例如,核酸的MALDI,和用于核酸MALDI的矩阵的例子一起,描述见WO98/54751(Genetrace,Inc.)。
在离子化上,由于形成了带不同电荷的离子,可从一个样品中观察到几个峰。平均单个质谱中获得的多个分子量读数提供了对该生物体子量的估计。电喷射离子化质谱(ESI-MS)对分子量非常高的多聚体,如蛋白质和分子量超过10kDa的核酸特别有用,因为它产生了该样品的多种电荷分子的分布,而不引起显著量的断裂。
本发明方法中所用的质量检测器包括但不限于,傅立叶变换离子回旋共振质谱法(FT-ICR-MS)、离子陷阱、四极、磁扇面、飞行时间(TOF)、Q-TOF或三重四极。
通常可用于本发明的质谱技术包括但不限于,串联质谱、红外多光子解离和热解气相色谱质谱(PGC-MS)。在本发明的实施方案中,生物体检测系统采用没有PCR的热解GC-MS以生物体检测模式连续操作,快速检测生物量的增加(例如,饮用水或细菌战争介质粪便污染的上升)。为使等待最短,使样品流连续直接流入PGC-MS的燃烧室。当检测到生物量的增加,PCR过程自动启动。生物体的存在引起100-7,000Da大分子片断的水平上升,这些片断在PGC-MS质谱中可观察到。将所见质谱与阈值水平相比,当测定的生物量水平超过预定的阈值时,启动本文上述的生物体分类过程(结合PCR和MS,优选FT-ICR-MS)。任选地此种检测到的生物量水平也可启动报警和其它过程(停止通风气流、物理分离)。
大DNAs分子量的精确测定受到每条链PCR反应的阳离子加合、自然界丰富的13C和15N同位素的同位素峰的分辨和离子带电状态排布的限制。阳离子可用流经芯片的在线透析去除,在电场梯度与液流正交的情况下,使含PCR产物的溶液接触含醋酸铵的溶液。后两个问题通过以分辨能力>10,000操作和加入同位素衰竭的核苷三磷酸到DNA中可解决。仪器的分辨能力也是考虑因素。分辨能力为10,000时,84聚体PCR产物的[M-14H+]14-带电状态的模式信号特征表现弱且此带电状态的排布或确切质量测定是不可能的。分辨能力为33,000时,可看见各同位素组分的峰。分辨能力为100,000时,可分辨相对于基线的同位素峰且离子带电状态的排布是简单易懂的。为获得[13C,15N]-衰竭的三磷酸,例如,可通过在耗尽的培养基上生培养收集核苷的(Batey等.,Nucl.Acids.Res.20:4515-4523,1992)。
虽然完整核酸区域的质量测定被认为足以确定大多数生物体串,但联质谱(MSn)技术可提供关于分子相同性或序列的更明确的信息。串联MS包括联合使用质量分析的两个或更多阶段,当分离和检测步骤都以质谱为基础时。第一个阶段用于选择样品的离子或组分,从中进一步获得结构信息。用如黑体辐射、红外多光子解离或碰撞活化然后使所选的离子断裂,。例如,电喷射离子化(ESI)产生的离子可用IR多光子解离断裂。活化导致糖苷键与磷酸骨架分离,产生两种系列的碎片离子,称为ω-系列(内部切割后含有完整的3’末端和5’磷酸)和α-碱基系列(含有完整的5’末端和3’呋喃)。
然后将质量分析的第二个阶段用于检测和测定这些产物离子所得片断的质量。这种片段化后的离子选择可以进行多次以基本上完全剖析样品分子序列。
如果有两个或多个碱基组成或质量类似的靶子或如果一次扩增反应产生的产物质是与两种或多种生物体参考标准品相同,它们可采用质量修饰的“标志”来区分。在本发明的实施方案中,可在扩增时加入与未修饰的碱基具有不同分子量的核苷酸类似物或“标志”(如5-(三氟甲基)脱氧胸苷三磷酸)而提高质量的区分。这些标志的描述见PCTWO97/33000。这更限制了与任何质量相符合可能的碱基组成的数量。例如,5-(三氟甲基)脱氧胸苷三磷酸可用于在分离的核酸扩增反应中代替dTTP。测定常规扩增产物和标记产物之间的质量转变可用于定量测定每条单链中胸苷核苷酸的数量。因为二链是互补的,也可测定每条链中腺苷核苷酸的数量。
在另一个扩增反应中,测定每条链中G和C残基的数量,可采用例如胞苷类似物5-甲基胞嘧啶(5-meC)或丙炔C。联合A/T反应和G/C反应,然后测定分子量,这提供了独特的碱基组成。此方法总结于图4和表1。
表1
质量标志硫代磷酸A(A*)用于区分炭疽芽胞杆菌簇。炭疽芽胞杆菌(A14G9C14T9)平均MW为14072.26,炭疽芽胞杆菌(A1A*13G9C14T9)平均分子量为14281.11且硫代磷酸A平均分子量为+16.06,用ESI-TOF-MS测定。去卷积谱见图5。
在另一个实施例中,假定每条链测定的分子量分别为为30,000.115Da和31,000.115Da,dT和dA残基测定的数目为(30,28)和(28,30)。如果分子量精确到100ppm,每条链有7种dG+dC的可能组合。然而,如果测定的分子量精确到10ppm,仅有2种dG+dC的组合,在1ppm精确性时每条链仅有一种可能的碱基组成。
可将质谱仪的信号输入到最大可能性检测和分类算法,如广泛用于雷达信号处理那样。该检测处理采用了在质量-碱基计数空间中观察到的匹配的BCS过滤,允许检测并减去已知的无害生物体的签名,和检测未知的生物体威胁。将新观察到的生物体与已知生物体作比较,即将它们的BCS和已知生物体的BCS及已知病原性增强的形式,如插入了抗生素抗性基因或毒素基因的生物体的BCS作比较,来估计威胁水平。
处理可用Bayesian分类器,采用从观察到的信号和平均背景水平产生的对数可能性比值来结束。此程序强调性能预测,预测对于包括天然产生的生物体和环境污染物的复杂背景情况以检测可能性对假报警可能性作图达到顶点。匹配的过滤器由信号值的优选预期值组成,考虑到引物对组是用于各生物体的。基因组序列数据库(如GenBank)可用于确定的质量碱基计数匹配的过滤器。该数据库包含已知的威胁物质和良性背景生物体。已知背景生物体的最大可能性检测采用匹配的过滤器和对噪音协方差的运行-小结估计来实施。估计背景信号的强度并与匹配的过滤器一起用来形成随后被减去的签名。将最大可能性过程应用于该“清除”数据,方式类似于使用匹配过滤器于生物体和使用噪音协方差的运行-小结估计于被清除数据。
在一个实施方案中,通过测定多个核心基因的信号来“三角划分”各生物体的策略,可用于减少假阴性和假阳性信号,以及可重新构建源来的生物体或杂交的或工程改造的生物体。在鉴定多个核心基因后,从核酸序列数据库中产生排列对比。然后分析排列对比物中的保守区和可变区,鉴定侧接有可变区的可能的引物结合位置。接着,选出用于签名分析的扩增靶区域,根据具体基因组的差异(即碱基组成)区分生物体。例如,在缺乏炭疽芽胞杆菌基因组的预期签名时,检测到炭疽杆菌的三部分毒素基因的签名(Bowen,J.E和C.P.Quinn,J.Appl.Microbiol.1999,87,270-278)提示有遗传工程改造事件。
本方法也可用于迅速和准确地检测单个核苷酸多态性(SNPs)或多个核苷酸多态性。SNP定义为各个体相互不同的基因组中单个碱基对位置。差别可表达为缺失、插入或替换,常与疾病状态相连。由于它们发生于每100-1000个碱基,SNPs在人基因组中是最常见的遗传标记结合类型。
例如,镰状细胞贫血来自于A-T转换,其编码缬氨酸而不是谷氨酸残基。可设计寡核苷酸引物,使它们结合SNP位点侧翼序列,然后进行才核苷酸扩增和扩增产物的质量测定。因为无镰状细胞贫血个体所得产物的分子量不同于此病个体产物的分子量,此方法可用于区分这两种个体。因此,此方法可用于检测个体中任何已知SNP,从而诊断或确定对疾病或病况易感性的增加。
在一个实施方案中,抽取个体的血液,分离外周血单个核细胞(PBMC)并宜用高通量筛选方法同时测试,一种或多种SNP,根据SNP区域侧翼的已知序列使用合适引物。生物技术信息国家中心保持公众可获得SNP数据库(www.ncbi.nlm.nih.gov/SNP/)。
本发明的方法也可用于血型检定。编码A、B或O血型的基因可能有四种单个核苷多肽性而不同。如果此基因包含序列CGTGGTGACCCTT(SEQIDNO:5),产生抗原A。如果此基因包含序列CGTCGTCACCGCTA(SEQIDNO:6),产生抗原B。如果此基因包含序列CGTGGT-ACCCCTT(SEQIDNO:7),产生血型O(“-”表示缺失)。这些序列可通过设计侧接这些区域的一对引物,然后进行扩增和质量测定来区分。
虽然本发明依据一些较佳实施方案作了具体描述,下列实施例仅用于阐述发明,而不意味着限制本发明。
实施例1
核酸分离和PCR
在一个实施方案中,分离生物体的核酸并用标准方法作PCR扩增然后以质谱测定BCS。分离核酸,例如可用洗涤剂裂解细菌细胞、离心和乙醇沉淀。核酸分离方法描述见例如CurrentProtocolsinMolecularBiology(Ausubel等)和分子克隆:实验室手册(Sambrook等)。随后用标准方法扩增核酸,如PCR,采用能结合该核酸保守区域的引物,其含有下述的介入性可变序列。
实施例2
质谱
FTICR仪器:FTICR仪器以7特斯拉活化比屏蔽超导磁体和修饰的BrunkerDaltonicsApexII70e离子光学和真空室为基础。此分光光度计界面接触LEAPPAL自动取样器和用于高通量筛选的定制流体控制系统。直接分析96孔或384孔微量滴定板的样品,速度约1样品/分钟。Bruker数据-获取平台由实验室-构建的辅助NT数据站支持,此数据站控制着自动取样器和含有能产生复杂的rf-激发波形(频率扫描、过滤噪音、存储波形反傅立叶变换(SWIFT)等)的随意波形发生器,用于高级串联MS实验。对于20-30聚体寡核苷酸,其典型性能特征包括超过100,000(FWHM)的质量分辨能力、低ppm质量测定误差和在50到5000m/z之间的可操作m/z范围。
改进的ESI源:在样品-限制的分析中,分析物溶液以150nL/分钟输送到装在3-D显微操纵器上的30mmi.d.熔融二氧化硅ESI发射器。ESI离子光学仪由一个加热的金属毛细管、一个rf唯一六级、一个铲削器锥和一个辅助门控电极组成。6.2cm的rf唯一六级包括1mm直径的杆,以5MHz频率在380Vpp电压下操作。可使用实验室-构建的电-机械快门来防止电喷射束进入进口毛细管,除非通过数据站的TTL脉冲触发而处于“打开”位置。当处于“关闭”位置时,稳定的电喷射束维持在ESI发射器和快门表面之间。快门臂背面含有弹性封条,可调节位置用进口毛细管形成真空密封。当去除密封时,快门叶片和毛细管进口之间1mm的空隙使外部离子贮存地保持恒定压力,无论快门处在开或关闭位置。当触动快门时,离子的“时间薄片”得以进入进口毛细管并随后聚集在外部离子贮存池中。离子快门的快速反应时间(<25ms)提供了可复制的用户设定的间隔,其间离子可注入并聚集在外部离子贮存池中。
红外多光子解离装置在10.6μm下操作的25瓦CWCO2激光仪界面接触分光光度计得以进行红外多光子解离(IRMPD)作寡核苷酸测序和其它串联MS应用。将铝光学台座放置离活化屏蔽超导磁体约1.5m,这样激光束与磁体中轴在一直线上。采用标准IR-相容镜和运动性镜设置,校准未聚焦的3mm激光束使之直接穿过FTICR捕获离子室的捕获电极中的3.5mm孔,并纵向穿过外部离子引导的六极区域,最后撞击铲削器锥。此过程使IRMPD以m/z选择方式在捕获离子室中传导(如以下的SWIFT分离感兴趣的物种),或在外部离子贮存池的高压区域中以宽带模式传导,在贮存池中与中性分子碰撞使IRMPD-产生的亚稳态片断离子稳定,导致增加的片断离子产生和序列覆盖。
实施例3
生物体的鉴定
表1显示对超过9套引物和大约30种生物体计算的分子量数据库的横截面.9套引物衍生自rRNA排列对比。RRNA共有序列排列对比区域的例子见图1A-1C。带箭头的线是设计用于PCR的智能引物对的区域。这些引物对在细菌序列数据库(目前超过10,000种生物体)中>95%保守。介入区域在长度和/或组成上是可变的,因此提供了各生物体的碱基组成“签名”(BCS)。选择引物对使扩增区域总长度小于约80-90核苷。各引物对的标记代表共有序列图上扩增区域的起始和结束碱基编号。
表1中包括的短细菌数据库横截面是许多熟知的病原体/生物战物质(以粗体/红色字体表示)如炭疽芽胞杆菌或耶尔森鼠疫杆菌以及一些普遍见于自然环境中的细菌如链霉菌。甚至紧密相关的生物体可通过适当选择引物彼此相区别。例如,两种低G+C生物体,炭疽芽胞杆菌和金黄色葡萄球菌,可通过采用16S_1337或23S_855(4Da的ΔM)确定的引物对相互区分。
表2
计算的分子量1的数据库横截面
1.用于横跨各种引物对(纵列)选择生物体(行)的PCR扩增区域的分子量分布。病原体以粗体表示。空的小室表示目前不完整或缺失的数据。
图6显示使用ESI-FT-ICRMS测定精确的质量。显示源于金黄色葡萄球菌(上部)和炭疽芽胞杆菌(下部)16SrRNA的1337位置的46聚体PCR产物的谱。这些数据来自此谱的区域,含有各自5’-3’链的[M-8H+]8-带电状态信号。两条链的区别在于两个(AT→CG)替换,测定的质量分别为14206.396和14208.373±0.010Da。衍生自炭疽芽胞杆菌产物的正向和反向链质量的可能碱基组成列于表3中。
表3炭疽杆菌产物的可能碱基组成
计算的质量 | 误差 | 碱基组成 |
14208.2935 | 0.079520 | A1 G17 C10 T18 |
14208.3160 | 0.056980 | A1 G20 C15 T10 |
14208.3386 | 0.034440 | A1 G23 C20 T2 |
14208.3074 | 0.065560 | A6 G11 C3 T26 |
14208.3300 | 0.043020 | A6 G14 C8 T18 |
14208.3525 | 0.020480 | A6 G17 C13 T10 |
14208.3751 | 0.002060 | A6 G20 C18 T2 |
14208.3439 | 0.029060 | A11 G8 C1 T26 |
14208.3665 | 0.006520 | A11 G11 C6 T18 |
14208.3890 | 0.016020 | A11 G14 C11 T10 |
14208.4116 | 0.038560 | A11 G17 C16 T2 |
14208.4030 | 0.029980 | A16 G8 C4 T18 |
14208.4255 | 0.052520 | A16 G11 C9 T10 |
14208.4481 | 0.075060 | A16 G14 C14 T2 |
14208.4395 | 0.066480 | A21 G5 C2 T18 |
14208.4620 | 0.089020 | A21 G8 C7 T10 |
14079.2624 | 0.080600 | A0 G14 C13 T19 |
14079.2849 | 0.058060 | A0 G17 C18 T11 |
14079.3075 | 0.035520 | A0 G20 C23 T3 |
14079.2538 | 0.089180 | A5 G5 C1 T35 |
14079.2764 | 0.066640 | A5 G8 C6 T27 |
14079.2989 | 0.044100 | A5 G11 C11 T19 |
14079.3214 | 0.021560 | A5 G14 C16 T11 |
14079.3440 | 0.000980 | A5 G17 C21 T3 |
14079.3129 | 0.030140 | A10 G5 C4 T27 |
14079.3354 | 0.007600 | A10 G8 C9 T19 |
14079.3579 | 0.014940 | A10 G11 C14 T11 |
14079.3805 | 0.037480 | A10 G14 C19 T3 |
14079.3494 | 0.006360 | A15 G2 C2 T27 |
14079.3719 | 0.028900 | A15 G5 C7 T1914 --> |
14079.3944 | 0.051440 | A15 G8 C12 T11 |
14079.4170 | 0.073980 | A15 G11 C17 T3 |
14079.4084 | 0.065400 | A20 G2 C5 T19 |
14079.4309 | 0.087940 | A20 G5 C10 T13 |
在计算的正向链16种组成和反向链18种组成中,只有一对(以粗体表示)是互补的,对应于炭疽杆菌PCR产物的实际碱基组成。
实施例4
炭疽芽胞杆菌和蜡样芽胞杆菌区域的BCS
合成了炭疽芽胞杆菌(A14G9C14T9)和蜡样芽胞杆菌(A15G9C13T9)的含有C到A碱基变化的保守性杆菌区域并接受ESI-TOFMS。结果显示于图7,其中这两个区域用本发明的方法清楚地区分(MW=14072.26对14096.29)。
实施例5
其它生物体的鉴定
在本发明的另一个实施例中,病原体霍乱弧菌可以ΔM>600Da与副溶血性弧菌相区分,采用了表2中所示的三套16S引物(16S_971、16S_1228或16S_1294),如表4所示。列出的两种支原体(出殖道支原体和肺炎支原体)也可相互区分,如三种分枝杆菌可区分一样。扩增产物的直接质量测定可鉴定和区分大量的生物体,而碱基组成签名的测定提供了对紧密相关生物体显著提高的分辨能力。在仅仅根据质量差异无法彼此有效区分的炭疽芽胞杆菌和蜡样芽胞杆菌的情况下,片段化模式或组成分析可用于分辨这种差异。这两种生物体之间的单个碱基差异产生了不同的片段化模式,尽管存在炭疽杆菌位置20存在不明确/未鉴定的碱基N,但这两种生物体是可鉴定的。
表4a-b显示表1引物对的例子,其区分了病原体和背景。
表4a
生物体名字 | 23S_855 | 16S_1337 | 23S_1021 |
炭疽杆菌 | 42650.98 | 28447.65 | 30294.98 |
金黄色葡萄球菌 | 42654.97 | 28443.67 | 30297.96 |
表4b
生物体名字 | 16S_971 | 16S_1294 | 16S_1228 |
霍乱弧菌 | 55625.09 | 35856.87 | 52535.59 |
副溶血性弧菌 | 54384.91 | 34620.67 | 50064.19 |
表4显示禽分枝杆菌和链霉菌sp中16S_1100-1188区域的预计分子量和碱基组成。
表5
区域 | 生物体名字 | 长度 | 分子量 | 碱基组成 |
16S_1100-1188 | 分枝杆菌avium | 82 | 25624.1728 | A16G32C18T16 |
16S_1100-1188 | 链霉菌sp | 96 | 29904.871 | A17G38C27T14 |
表5显示不同种类细菌16S_1100-1188引物扩增反应的碱基组成(单链)结果。表中重复的菌种(如肉毒梭菌)是在16S_1100-1188区域含有不同碱基组成的不同菌株。
表6
含有不同碱基组成的相同生物体是不同的菌株。粗体或斜体表示的生物体群在扩增区域有相同碱基组成。这些生物体中的一些可用多个引物相区分。例如,炭疽杆菌可采用引物16S_971-1062与蜡样芽胞杆菌和苏去金芽胞杆菌相区分(表6)。产生独特碱基组成签名的其它引物对见表6(粗体)。含有非常类似的威胁生物体和普遍存在的无威胁生物体(如炭疽群),用聚合的引物对系列以高分辨率相区分。表6中已知的生物战物质是炭疽杆菌、耶尔森鼠疫杆菌土拉热弗朗西丝菌和普氏立克次体。
表7
炭芽胞杆菌和蜡芽胞杆菌在16S_971区域中的序列如下所示。粗体表示两个菌种之间的单个碱基差异,可用本发明的方法检测。炭疽杆菌在位置20有不确定的碱基。
炭疽芽胞杆菌_16S_971
GCGAAGAACCUUACCAGGUNUUGACAUCCUCUGACAACCCUAGAGAUAGGGC
UUCUCCUUCGGGAGCAGAGUGACAGGUGGUGCAUGGUU(SEQIDNO:1)
蜡样芽胞杆菌_16S_971
GCGAAGAACCUUACCAGGUCUUGACAUCCUCUGAAAACCCUAGAGAUAGGGC
UUCUCCUUCGGGAGCAGAGUGACAGGUGGUGCAUGGUU(SEQIDNO:2)
实施例6
sspE56聚体的ESI-TOFMS加校准
在ESI-MS研究PCR产物中可采用内部质量标准品获得的质量测定准确性见图8。此质量标准品是加到溶液中的20聚体硫代磷酸寡核苷酸,该溶液含炭疽杆菌孢子外膜蛋白sspE产生的56聚体PCR产物。预计的PCR产物的质量可将炭疽杆菌和其它种类的杆菌如苏去金芽胞杆菌和蜡样芽胞杆菌区分开来。
实施例7
炭疽杆菌的ESI-TOF合成性16S_1228双链体
ESI-TOFMS谱获自含5μM的炭疽杆菌16SrRNA基因核苷酸1228区域的预期正向和反向PCR产物的各个合成类似物的水溶液。结果(图9)显示正向和反向链的分子量可精确地测定并容易地区分两条链。显示[M-21H+]21-和[M-20H+]20-的带电状态。
实施例8
合成的炭疽杆菌16S_133746碱基对双链体的ESI-FTICRMS
ESI-FTICR-MS谱获自含5μM的炭疽杆菌16SrRNA基因核苷酸1337区域的预期正向和反向PCR产物的各个合成类似物的水溶液。结果(图10)显示二链的分子量可用此方法相区分。显示[M-16H+]16-到[M-10H+]10-的带电状态。此插入强调了可在FTICR-MS仪器上辩认的分辨率,使得离子的带电状态可通过峰之间的质量差异来测定,峰差异的不同在于单一13C替换。
实施例9
炭疽杆菌saspB基因的56聚体寡核苷酸的ESI-TOFMS与内部质量标准的
ESI-TOFMS谱获自含内部质量标准的炭疽杆菌saspB基因的合成56聚体寡核苷酸(5μM),1.7μL/分钟的ESI作为样品消耗的函数。结果(图11)噪音显示比信号随着更多的扫描而改进并作了小结,仅100次扫描后标准品和产物就已可见。
实施例10
用三丁铵(TBA)-三氟醋酸(TFA)缓冲液的内部质量标准的ESI-TOFMS
20聚体硫代磷酸质量标准的ESI-TOFMS谱在5mMTBA-TFA缓冲液加入到该溶液后获得。此缓冲液带从寡核苷酸获得电荷并使最大丰度的带电状态从[M-8H+]8-转变到[M-3H+]3-(图12)。
Claims (18)
1.一种鉴定病毒、真菌或孢子生物体的方法,其特征在于,所述方法包括:
a)使所述生物体的核酸接触至少一对寡核苷酸引物,此引物对于具体病毒种类、具体真菌种类或具体孢子种类没有特异性且能与所述核酸的序列杂交,所述序列在所述病毒生物体、所述真菌生物体或所述孢子生物体之间是保守的,其中所述序列与在所述病毒生物体、所述真菌生物体或所述孢子生物体之间可变的可变核酸序列侧接;
b)扩增所述可变核酸序列产生扩增产物;
c)测定所述扩增产物的碱基组成,其中所述碱基组成包括G残基、A残基、T残基和C残基以及其类似物和其质量标志的残基的数量,所述类似物选自2,6-二氨基嘌呤、丙炔T、丙炔G、吩嗪和G-夹子;和
d)将所述碱基组成与在多种已知生物体上进行步骤a)-c)获得的一种或多种扩增产物的已知碱基组成相比较,其中一种匹配可鉴定所述病毒、真菌或孢子生物体。
2.如权利要求1所述的方法,其特征在于,与所述至少一对寡核苷酸引物杂交的所述序列是保守的。
3.如权利要求1所述的方法,其特征在于,所述扩增步骤包括聚合酶链式反应。
4.如权利要求1所述的方法,其特征在于,所述扩增步骤包括连接酶链式反应或链置换扩增。
5.如权利要求1所述的方法,其特征在于,所述核酸是核糖体RNA。
6.如权利要求1所述的方法,其特征在于,所述核酸编码RNAseP或RNA依赖的RNA聚合酶。
7.如权利要求1所述的方法,其特征在于,所述扩增产物在碱基组成测定前离子化。
8.如权利要求1所述的方法,其特征在于,该方法还包括在使所述核酸与所述至少一对寡核苷酸引物接触之前,从所述生物体中分离核酸的步骤。
9.如权利要求1所述的方法,其特征在于,该方法还包括采用不同寡核苷酸引物对进行步骤a)-d),并将结果与在步骤d)的不同的多种已知生物体上进行步骤a)-c)获得的一种或多种扩增产物的碱基组成相比较。
10.如权利要求1所述的方法,其特征在于,所述一种或多种已知碱基组成包含在碱基组成数据库中。
11.如权利要求1所述的方法,其特征在于,所述扩增产物通过电喷射离子化、基质辅助的激光解吸或快速原子轰击来离子化。
12.如权利要求1所述的方法,其特征在于,所述碱基组成用质谱测定。
13.如权利要求12所述的方法,其特征在于,所述质谱选自傅立叶变换离子回旋共振质谱法、离子陷阱、四极、磁扇面、飞行时间、Q-TOF或三重四极。
14.如权利要求1所述的方法,其特征在于,该方法还包括在存在与腺苷、胸苷、鸟苷或胞苷有不同的分子量的腺嘌呤、胸苷、鸟苷或胞苷类似物时进行步骤b)。
15.如权利要求1所述的方法,其特征在于,所述寡核苷酸引物在所述引物中各个三联体位置1和2上含有碱基类似物,其中所述碱基类似物能以比天然碱基更高的亲和力结合其互补物。
16.如权利要求15所述的方法,其特征在于,所述引物在所述引物中各个三联体位置3上含有通用碱基。
17.如权利要求16所述的方法,其特征在于,所述通用碱基选自肌苷、胍尿苷、5-硝基吲哚、3-硝基吡咯、dP、dK和1-(2-脱氧-β-D-呋喃核糖)-咪唑-4-酰胺。
18.一种鉴定细胞生物体的方法,其特征在于,所述方法包括:
a)使所述生物体的核酸接触至少一对寡核苷酸引物,此引物对于具体细胞种类没有特异性且能与所述核酸的序列杂交,所述序列在所述细胞生物体之间是保守的,其中所述序列与在所述细胞生物体之间可变的可变核酸序列侧接;
b)扩增所述可变核酸序列产生扩增产物;
c)测定所述扩增产物的碱基组成,其中所述碱基组成包括G残基、A残基、T残基和C残基以及其类似物和其质量标志的残基的数量,所述类似物选自2,6-二氨基嘌呤、丙炔T、丙炔G、吩嗪和G-夹子;和
d)将所述碱基组成与在多种已知生物体上进行步骤a)-c)获得的一种或多种扩增产物的已知碱基组成相比较,其中一种匹配可鉴定所述细胞生物体。
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