CN101400801A - Oligonucleotide microarray for identifying pathogens - Google Patents

Abstract

A method for detecting a target nucleic acid of a pathogen in a test sample, the method comprising: preparing a target nucleic acid detecting reagent and contacting the target nucleic acid detecting reagent with the oligonucleotide microarray. A kit for detecting a target nucleic acid of a pathogen in a test sample is also described. The kit comprises at least one pair of primers and an oligonucleotide microarray containing at least one probe.

Description

Translated fromChinese

用于鉴定病原体的寡核苷酸微阵列Oligonucleotide microarrays for identification of pathogens

本申请要求于2005年12月30日提交的美国临时专利申请60/755,504的权益。This application claims the benefit of USProvisional Patent Application 60/755,504, filed December 30, 2005.

发明背景Background of the invention

本发明涉及用于检测检验样品中病原体存在情况的检验试剂盒和方法。检验样品可以获自患者或者来自可能含有病原体的食物或饮料的来源。The present invention relates to test kits and methods for detecting the presence of pathogens in test samples. A test sample may be obtained from a patient or from a source of food or drink that may contain the pathogen.

诸如脑炎、脑膜炎、脑(脊)膜炎、肺炎、肠胃炎、心内膜炎、尿道感染的疾病由病原体感染引起。病原体包括不同的细菌、病毒、真菌和原生动物。不同的致病性感染往往具有类似的症状，这致使难以提供感染或疾病的起因的确诊。例如，脑炎和脑(脊)膜炎患者一般都会出现发烧、头痛和惊厥。快速而准确地鉴定引起这些症状的病原体对于确定开出什么药方至关重要。当前用于鉴定病原体的诊断方法包括：镜检、微生物培养、血清免疫测试和聚合酶链式反应(PCR)。这些方法需要经过严格训练的人员而且非常费时。Diseases such as encephalitis, meningitis, meningitis, pneumonia, gastroenteritis, endocarditis, urinary tract infection are caused by pathogen infection. Pathogens include different bacteria, viruses, fungi and protozoa. Different causative infections often have similar symptoms, making it difficult to provide a definitive diagnosis of the cause of the infection or disease. For example, patients with encephalitis and meningitis typically present with fever, headache, and seizures. Rapid and accurate identification of the pathogen causing these symptoms is critical to determining what medication to prescribe. Current diagnostic methods used to identify pathogens include: microscopy, microbial culture, serum immune tests, and polymerase chain reaction (PCR). These methods require highly trained personnel and are very time consuming.

按照常规的PCR方法，将含有起模板作用的基因的检验样品与设计成用于扩增具有特定长度的至少一种基因的至少一对PCR引物混合。通过使所扩增的PCR产物进行琼脂糖凝胶电泳并使其染色，就可使PCR产物显现。按照PCR方法，根据所显现的PCR产物长度来鉴定检验样品中所含的基因。在同一样品中可以扩增多对引物以便更有效地筛选多种基因。According to a conventional PCR method, a test sample containing a gene serving as a template is mixed with at least one pair of PCR primers designed to amplify at least one gene having a specific length. PCR products can be visualized by subjecting the amplified PCR products to agarose gel electrophoresis and staining them. According to the PCR method, the genes contained in the test sample are identified based on the length of the PCR product revealed. Multiple pairs of primers can be amplified in the same sample for more efficient screening of multiple genes.

然而，随着引物试剂中所含PCR引物的数量增多，在电泳凝胶上的“噪声”也随之增多。当由于非特异性退火产生PCR产物时，“噪声”就会出现。出于此原因，当使用PCR反应时，难以确定不只是仅少数基因存在。However, as the number of PCR primers contained in the primer reagent increases, so does the "noise" on the electrophoresis gel. "Noise" occurs when PCR products are generated due to non-specific annealing. For this reason, when using PCR reactions, it is difficult to be sure that more than just a few genes are present.

此外，有必要设计好引物，以使经PCR反应扩增出的PCR产物在分辨率有限的电泳凝胶上可以辨别。要设计出可产生适当的可分辨PCR产物且噪声低的引物并不容易并且耗时。因此，严格的基于PCR/电泳的方法对于筛选大量基因表达是低效的。In addition, it is necessary to design primers so that the PCR products amplified by the PCR reaction can be distinguished on electrophoretic gels with limited resolution. It is not easy and time consuming to design primers that produce adequately resolvable PCR products with low noise. Therefore, stringent PCR/electrophoresis-based methods are inefficient for screening large amounts of gene expression.

发明概述Summary of the invention

本发明涉及用于检测生物样品或检验样品中的目标核酸的方法和检验试剂盒。使用寡核苷酸微阵列快速地高通量筛选病原体的存在情况。寡核苷酸微阵列含有已知病原体的各种聚核苷酸(探针)，并且以一个杂交测定来鉴别病原体。The present invention relates to methods and test kits for detecting target nucleic acids in biological samples or test samples. Rapid high-throughput screening for the presence of pathogens using oligonucleotide microarrays. Oligonucleotide microarrays contain various polynucleotides (probes) of known pathogens, and a hybridization assay is used to identify pathogens.

一方面，本发明涉及一种简易而快速地确定生物样品或检验样品中是否存在含有目标核酸的多核苷酸的方法。In one aspect, the present invention relates to a simple and rapid method for determining the presence or absence of a polynucleotide containing a target nucleic acid in a biological sample or test sample.

另一方面，本发明涉及一种用于检测生物样品或检验样品中病原体的目标核酸的方法，所述方法包括：利用与不止一种病原体中的保守区结合的一对或更多对引物来扩增样品中的目标核酸；使扩增的目标核酸与寡核苷酸微阵列接触；且检测目标核酸与探针的结合情况，其中与特定病原体探针结合则表明样品中存在该病原体。所述微阵列包括含与不同病原体互补的多核苷酸序列的两种或以上的探针或者探针组。In another aspect, the present invention relates to a method for detecting a target nucleic acid of a pathogen in a biological sample or a test sample, the method comprising: using one or more pairs of primers that bind to a conserved region in more than one pathogen to detect amplifying target nucleic acid in the sample; contacting the amplified target nucleic acid with an oligonucleotide microarray; and detecting binding of the target nucleic acid to the probe, wherein binding to a specific pathogen probe indicates the presence of the pathogen in the sample. The microarray includes two or more probes or probe groups containing polynucleotide sequences complementary to different pathogens.

又一方面，本发明涉及一种辨别样品中的大肠杆菌(E.coli)和沙门氏菌的方法。所述方法包括：利用与大肠杆菌和沙门氏菌中的保守区结合的一对或更多对引物扩增样品中的核酸；使扩增的目标核酸与寡核苷酸微阵列接触；且检测核酸与作为指示样品中存在大肠杆菌和/或沙门氏菌的探针的结合情况。所述微阵列包括含与大肠杆菌和沙门氏菌中的可变区互补的多核苷酸序列的两种或以上探针。In yet another aspect, the present invention relates to a method of distinguishing Escherichia coli (E. coli) from Salmonella in a sample. The method includes: amplifying nucleic acid in a sample using one or more pairs of primers that bind to conserved regions in Escherichia coli and Salmonella; contacting the amplified target nucleic acid with an oligonucleotide microarray; Binding as a probe indicating the presence of E. coli and/or Salmonella in the sample. The microarray includes two or more probes comprising polynucleotide sequences complementary to variable regions in E. coli and Salmonella.

在另一方面，本发明涉及一种用于检测在生物样品或检验样品中病原体的目标核酸的试剂盒，所述试剂盒包括：至少一对引物和寡核苷酸微阵列，所述微阵列包含固定化在固相支持体上的至少一种探针。In another aspect, the present invention relates to a kit for detecting a target nucleic acid of a pathogen in a biological sample or a test sample, said kit comprising: at least one pair of primers and an oligonucleotide microarray, said microarray Contains at least one probe immobilized on a solid support.

根据以下的说明且结合附图，并通过例示阐明本发明原理，本发明的其他方面和优点将显而易见。Other aspects and advantages of the invention will become apparent from the following description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the invention.

附图简述Brief description of the drawings

图1A和图1B显示了16S rDNA的细菌DNA序列的保守引物区和可变探针区。Figure 1A and Figure 1B show the conserved primer region and variable probe region of the bacterial DNA sequence of 16S rDNA.

图2A和图2B显示了18S rDNA的真菌DNA序列的保守引物区和可变探针区。Figure 2A and Figure 2B show the conserved primer region and variable probe region of the fungal DNA sequence of 18S rDNA.

图3是本发明实施方案的图式概述。Figure 3 is a schematic overview of an embodiment of the invention.

发明详述Detailed description of the invention

1.病原体1. Pathogens

术语“病原体”指的是疾病因子或致病因子。术语病原体最通常是指感染性生物体。它们包括但不限于：细菌、病毒、真菌和原生动物。本发明的方法和试剂盒可用于鉴定已经引起传染病或食物中毒的病原体和微生物。病原体的实例包括但不限于：立克次氏体、衣原体、支原体、螺旋体、链球菌、沙门氏菌(almonella)、葡萄球菌、支原体、单核细胞增生李斯特氏菌(L.monocytogenes)、脑膜炎奈瑟氏菌(N.meningtides)、大肠杆菌、流感嗜血杆菌(H.influenzae)、布氏疏螺旋体(B.burgdorferi)、钩端螺旋体、变形菌、厌氧菌(anaecrobacter)、结核分支杆菌(M.tuberculosis)、肠球菌、脊髓灰质炎病毒1、肠道病毒71、肠道病毒70、艾柯病毒2、艾柯病毒4、艾柯病毒6、艾柯病毒9、艾柯病毒11、艾柯病毒12、艾柯病毒26、柯萨奇病毒A13、柯萨奇病毒A15、柯萨奇病毒A18、柯萨奇病毒A20、柯萨奇病毒A21、柯萨奇病毒B3-A、柯萨奇病毒B3-C、HSV-1和HSV-2。The term "pathogen" refers to a disease agent or causative agent. The term pathogen most commonly refers to an infectious organism. They include, but are not limited to: bacteria, viruses, fungi and protozoa. The methods and kits of the invention can be used to identify pathogens and microorganisms that have caused infectious disease or food poisoning. Examples of pathogens include, but are not limited to: Rickettsia, Chlamydia, Mycoplasma, Spirochetes, Streptococcus, Salmonella, Staphylococcus, Mycoplasma, Listeria monocytogenes (L. monocytogenes), meningitidis N.meningtides, Escherichia coli, H.influenzae, B.burgdorferi, Leptospira, Proteus, anaerobic bacteria, Mycobacterium tuberculosis ( M.tuberculosis), Enterococcus, Poliovirus 1, Enterovirus 71, Enterovirus 70, Ecovirus 2, Ecovirus 4, Ecovirus 6, Ecovirus 9, Ecovirus 11, Artovirus Coxsackievirus 12, Ecovirus 26, Coxsackievirus A13, Coxsackievirus A15, Coxsackievirus A18, Coxsackievirus A20, Coxsackievirus A21, Coxsackievirus B3-A, Coxsackievirus Viruses B3-C, HSV-1 and HSV-2.

2.生物样品和检验样品2. Biological samples and test samples

文中所用的术语“生物样品”是指取自生物体或生物体的组分(例如细胞)的样品。在一个实施方案中，生物体是哺乳动物。在另一个实施方案中，生物体是人类。样品可以是任何生物组织或生物液体。通常生物样品取自患者。这些样品包括但不限于：组织、细胞、血液、血清、脑脊液、尿、细胞裂解物、血浆、粪便、唾液、血细胞、细针活检样品、腹膜液和胸膜液，或来自以上样品的细胞。生物样品同样包括组织切片，例如用于组织学用途的冰冻切片。The term "biological sample" as used herein refers to a sample taken from an organism or a component (eg, cells) of an organism. In one embodiment, the organism is a mammal. In another embodiment, the organism is a human. The sample can be any biological tissue or biological fluid. Typically a biological sample is taken from a patient. These samples include, but are not limited to: tissue, cells, blood, serum, cerebrospinal fluid, urine, cell lysates, plasma, feces, saliva, blood cells, fine needle biopsy samples, peritoneal and pleural fluid, or cells derived from the above. Biological samples also include tissue sections, such as frozen sections for histological purposes.

在某些实施方案中，本发明涉及一种方法，其中生物样品选自组织、细胞、血液、血清、脑脊液、尿、细胞裂解物、血浆、粪便、唾液、血细胞、细针活检样品、腹膜液和胸膜液，或来自以上样品的细胞。In certain embodiments, the present invention relates to a method wherein the biological sample is selected from the group consisting of tissue, cells, blood, serum, cerebrospinal fluid, urine, cell lysate, plasma, feces, saliva, blood cells, fine needle biopsy, peritoneal fluid and pleural fluid, or cells from the above samples.

本文所用的术语“检验样品”是指取自非活体来源的样品。检验样品的来源包括但不限于：食物、饮料、土壤、地下水、海水和湖底沼泽水。在这些检验样品中通常含有病原体和被病原体感染的细胞。As used herein, the term "test sample" refers to a sample taken from a non-living source. Sources of test samples include, but are not limited to: food, beverages, soil, groundwater, seawater, and lake bottom marsh water. Pathogens and pathogen-infected cells are usually contained in these test samples.

根据是否存在特定病原体的目标核酸来鉴定生物样品和检验样品的病原体。在某些实施方案中，目标核酸包括：病原体的基因组材料、线粒体DNA、rRNA、tRNA、mRNA、病毒RNA、质粒DNA以及它们的片段。Biological samples and test samples are identified for pathogens based on the presence or absence of target nucleic acids for a particular pathogen. In certain embodiments, target nucleic acids include: genomic material of pathogens, mitochondrial DNA, rRNA, tRNA, mRNA, viral RNA, plasmid DNA, and fragments thereof.

根据症状和感染途径，病原体被分类成众多组别。为了对应多个组别，制备出多种目标核酸检测试剂和寡核苷酸微阵列。使用目标核酸检测试剂和寡核苷酸微阵列，它们根据症状而选择。因此，可以在根据症状和感染途径所预测的病原体之中高度准确地鉴定出真正的病原体。Pathogens are classified into numerous groups according to symptoms and route of infection. In order to correspond to multiple groups, various target nucleic acid detection reagents and oligonucleotide microarrays were prepared. Using target nucleic acid detection reagents and oligonucleotide microarrays, they are selected according to the symptoms. Therefore, the true pathogen can be identified with high accuracy among the pathogens predicted from the symptoms and the route of infection.

3.目标核酸3. Target nucleic acid

目标核酸是待检验的病原体所固有的多核苷酸。此多核苷酸是遗传材料，其包括：基因组DNA/RNA、线粒体DNA、rRNA、tRNA、mRNA、病毒RNA和质粒DNA。通过检测病原体特有的目标核酸的存在情况，可以推断病原体自身的存在情况。类似地，存在对病原体的属特异性的目标核酸则表明了存在该属成员。The target nucleic acid is a polynucleotide inherent to the pathogen to be tested. Such polynucleotides are genetic material which includes: genomic DNA/RNA, mitochondrial DNA, rRNA, tRNA, mRNA, viral RNA and plasmid DNA. By detecting the presence of a target nucleic acid specific to the pathogen, the presence of the pathogen itself can be inferred. Similarly, the presence of a target nucleic acid specific for a genus of a pathogen indicates the presence of a member of that genus.

术语“核苷酸序列”或“核酸”或“多核苷酸”可交换使用并且指的是核苷酸的杂聚物或这些核苷酸的序列。“寡核苷酸”指的是约50个或以下核苷酸的多核苷酸。这些术语同样指的是DNA或RNA，它们可以是单链或双链的，并且可以相对于cDNA而言代表正义链或负义链。本文的序列中，A是腺嘌呤，C是胞嘧啶，T是胸腺嘧啶，G是鸟嘌呤，而N是A、C、G或T(U)。可以预期，若多核苷酸是RNA，那么本文所提供的序列中的T(胸腺嘧啶)以U(尿嘧啶)来代替。The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" are used interchangeably and refer to a heteropolymer of nucleotides or a sequence of such nucleotides. "Oligonucleotide" refers to a polynucleotide of about 50 nucleotides or fewer. These terms also refer to DNA or RNA, which may be single- or double-stranded, and may represent, with respect to cDNA, the sense or the negative strand. In the sequences herein, A is adenine, C is cytosine, T is thymine, G is guanine, and N is A, C, G or T(U). It is contemplated that where the polynucleotide is RNA, T (thymine) is replaced by U (uracil) in the sequences provided herein.

术语“互补的”或“互补性”指的是通过碱基配对多核苷酸的自然结合。例如，序列5’-AGT-3’与互补的序列3’-TCA-5’结合。两条单链分子之间的互补性可以是“部分的”，致使只有某些核酸结合；或者可以是“完全的”，致使在单链分子之间存在完全互补性。核酸链之间的互补程度显著地影响核酸链之间杂交的效率和强度。The term "complementary" or "complementarity" refers to the natural association of polynucleotides through base pairing. For example, the sequence 5'-AGT-3' is combined with the complementary sequence 3'-TCA-5'. Complementarity between two single-stranded molecules can be "partial", such that only some nucleic acids bind, or "complete", such that complete complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands.

本文所用的术语“纯化的”或“基本上纯化的”指的是：所指示的核酸或多肽以基本上不存在其他生物大分子(诸如多核苷酸、蛋白质等)的情况下存在。The term "purified" or "substantially purified" as used herein means that the indicated nucleic acid or polypeptide is present in the substantial absence of other biomacromolecules (such as polynucleotides, proteins, etc.).

本文所用的术语“分离的”指的是：核酸与在该核酸天然来源中与其一起存在的至少一种其他组分分离。在一个实施方案中，所述核酸仅在(如果有的话)溶剂、缓冲剂、离子或正常地存在于核酸溶液中的其他组分存在的情况下存在。术语“分离的”和“纯化的”不包括存在于它们的天然来源中的核酸。The term "isolated" as used herein means that a nucleic acid is separated from at least one other component with which it is present in its natural source. In one embodiment, the nucleic acid is present only in the presence, if any, of solvents, buffers, ions, or other components normally present in nucleic acid solutions. The terms "isolated" and "purified" exclude nucleic acids as they exist in their natural source.

本文所使用的“基本上等同的”或“基本上类似的”指的是：由于一个或更多个置换、缺失或添加而不同于参比序列的核苷酸序列，而所述置换、缺失或插入的实际结果并不导致参比序列与被试序列之间不利的功能相异。通常此基本上等同的序列有不超过约35％的不同(即与相应的参比序列相比，基本上等同序列中个别核苷酸的置换、添加和/或缺失的数量除以基本上等同序列中核苷酸的总数量约等于0.35或更少)。这种序列被认为与所示序列具有65％的序列同一性。在一个实施方案中，本发明的基本上等同的序列与参比序列有不超过30％不同(70％序列同一性)；在此实施方案的变体中，有不超过25％不同(75％序列同一性)；在此实施方案的又一个变体中，有不超过20％不同(80％序列同一性)；在此实施方案的又一个变体中，有不超过10％不同(90％序列同一性)；在此实施方案的又一个变体中，有不超过5％不同(95％序列同一性)。在一个实施方案中，所述核苷酸序列具有至少约65％的同一性。在其它实施方案中，所述核苷酸序列具有至少约75％、80％、85％、90％、95％、98％或99％的同一性。As used herein, "substantially identical" or "substantially similar" refers to: a nucleotide sequence that differs from a reference sequence due to one or more substitutions, deletions or additions, and the substitutions, deletions Or the actual result of the insertion does not result in an unfavorable functional divergence between the reference sequence and the test sequence. Typically such substantially identical sequences differ by no more than about 35% (i.e., the number of individual nucleotide substitutions, additions and/or deletions in the substantially identical sequence divided by the substantially identical The total number of nucleotides in the sequence is approximately equal to 0.35 or less). This sequence is believed to have 65% sequence identity to the sequence shown. In one embodiment, substantially identical sequences of the invention differ from a reference sequence by no more than 30% (70% sequence identity); in variants of this embodiment, they differ by no more than 25% (75% sequence identity). sequence identity); in yet another variant of this embodiment, no more than 20% differ (80% sequence identity); in yet another variant of this embodiment, no more than 10% differ (90% sequence identity); in yet another variant of this embodiment, no more than 5% differ (95% sequence identity). In one embodiment, the nucleotide sequences are at least about 65% identical. In other embodiments, the nucleotide sequences are at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical.

在某些方面，本发明涉及制备“目标核酸检测试剂”。目标核酸检测试剂可以或是目标核酸自身或是扩增的目标核酸。In certain aspects, the invention relates to the preparation of "target nucleic acid detection reagents". The target nucleic acid detection reagent can be either the target nucleic acid itself or an amplified target nucleic acid.

使用常规的技术将目标核酸序列从生物样品和检验样品中分离。根据要分离的是什么类型的多核苷酸(DNA还是RNA)来确定所用的技术。分离技术也可以根据待调查的病原体的类型和生物/检验样品的量来作出修改。Target nucleic acid sequences are isolated from biological samples and test samples using conventional techniques. The technique used will depend on what type of polynucleotide (DNA or RNA) is being isolated. Isolation techniques can also be modified depending on the type of pathogen being investigated and the amount of organism/test sample.

4.检测试剂4. Detection reagent

在某些实施方案中，在与寡核苷酸微阵列杂交之前，目标核酸先被扩增以产生“目标核酸检测试剂”。在其他实施方案中，在与寡核苷酸微阵列杂交之前，目标核酸不被扩增，这时“目标核酸检测试剂”和目标核酸是等同的。在一个实施方案中，用一种或更多种标记分子标记目标核酸或所扩增的目标核酸。标记分子的附着有利于在与寡核苷酸微阵列杂交时检测目标核酸或所扩增的目标核酸。可以通过本领域技术人员所知熟的许多方法中的任一种掺入标记分子。在一个实施方案中，可以在制备样品的目标核酸中的扩增步骤时，同时掺入标记分子。因此，例如使用包含标记分子的引物或核苷酸的聚合酶链式反应(PCR)将提供含有所述标记分子的扩增产物。在某些实施方案中，标记分子包括：生物素、磁珠、荧光染料、放射性标记、酶、比色标记、有色玻璃或塑料珠。在一个实施方案中，如前文所述，使用含有标记分子的核苷酸(荧光素标记的UTP和/或CTP)进行转录扩增，致使标记掺入到所转录的核酸中。In certain embodiments, target nucleic acids are amplified prior to hybridization to an oligonucleotide microarray to produce "target nucleic acid detection reagents." In other embodiments, the target nucleic acid is not amplified prior to hybridization to the oligonucleotide microarray, in which case "target nucleic acid detection reagent" and target nucleic acid are synonymous. In one embodiment, the target nucleic acid or amplified target nucleic acid is labeled with one or more labeling molecules. Attachment of label molecules facilitates detection of target nucleic acids or amplified target nucleic acids upon hybridization to the oligonucleotide microarray. Labeling molecules can be incorporated by any of a number of methods known to those skilled in the art. In one embodiment, the labeling molecule can be incorporated simultaneously during the amplification step in the preparation of the target nucleic acid of the sample. Thus, for example, polymerase chain reaction (PCR) using primers or nucleotides comprising a marker molecule will provide an amplification product comprising said marker molecule. In certain embodiments, labeling molecules include: biotin, magnetic beads, fluorescent dyes, radioactive labels, enzymes, colorimetric labels, colored glass or plastic beads. In one embodiment, transcriptional amplification is performed using nucleotides containing label molecules (fluorescein-labeled UTP and/or CTP), as described previously, resulting in incorporation of the label into the transcribed nucleic acid.

作为选择，标记分子可以直接加到原始目标核酸(例如：mRNA、polyA、mRNA、cDNA等)中。使标记分子附着核酸的方法为本领域的技术人员所熟知，这些方法包括：例如通过经由激酶反应使目标核酸磷酸化来进行切口平移或末端标记，随后通过核酸接头使样品核酸与标记分子连接。Alternatively, marker molecules can be added directly to the original target nucleic acid (eg, mRNA, polyA, mRNA, cDNA, etc.). Methods for attaching marker molecules to nucleic acids are well known to those skilled in the art and include, for example, nick translation or end-labeling by phosphorylating target nucleic acid via a kinase reaction, followed by ligation of sample nucleic acid to marker molecules via nucleic acid adapters.

适用于本发明的可检测标记分子包括：通过波谱学、光化学、生物化学、免疫化学、电学、光学或化学的方法可检测的任何组合物。本发明的有用标记分子包括：生物素，用标记的链霉抗生物素缀合物染色；磁珠(例如Dynabeads^TM)；荧光染料(例如荧光素、德克萨斯红、罗丹明、绿色荧光蛋白等)；放射性标记(例如³H、¹²⁵I、³⁵S、¹⁴C或³²P)；酶(例如辣根过氧化物酶、碱性磷酸酶和在ELISA中通常使用的其他酶)；以及比色标记，诸如胶体金或有色玻璃或塑料(例如聚苯乙烯、聚丙烯、橡胶等)珠。教导使用这些标记的专利包括：美国专利号3,817,837、3,850,752、3,939,350、3,996,345、4,277,437、4,275,149和4,366,241。在一个实施方案中，标记分子是含掺入所扩增目标核酸中的标记分子的核苷酸。Detectable label molecules suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labeling molecules of the invention include: biotin, stained with a labeled streptavidin conjugate; magnetic beads (eg, Dynabeads^™ ); fluorescent dyes (eg, fluorescein, Texas Red, rhodamine, green fluorescent proteins, etc.); radioactive labels (such as³ H,¹²⁵ I,³⁵ S,¹⁴ C, or³² P); enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA); and Colorimetric markers such as colloidal gold or colored glass or plastic (eg polystyrene, polypropylene, rubber, etc.) beads. Patents that teach the use of these labels include: US Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. In one embodiment, the marker molecule is a nucleotide containing marker molecule incorporated into the amplified target nucleic acid.

含标记分子的核苷酸可以是用荧光物质(Cy3、Cy5等)或生物素标记的核苷酸(单体)，因为它具有高检测灵敏度而且易于操作。含标记分子的核苷酸被带入通过PCR反应扩增的多核苷酸中，并对扩增的多核苷酸进行标记。在用生物素标记核苷酸的情况下，被标记的多核苷酸通过酶联免疫吸附测定(ELISA)显现。Nucleotides containing labeled molecules may be nucleotides (monomers) labeled with fluorescent substances (Cy3, Cy5, etc.) or biotin because it has high detection sensitivity and is easy to handle. Nucleotides containing marker molecules are incorporated into polynucleotides amplified by PCR reactions, and the amplified polynucleotides are labeled. In the case of labeling of nucleotides with biotin, the labeled polynucleotides are visualized by enzyme-linked immunosorbent assay (ELISA).

在某些实施方案中，本发明包括：通过PCR扩增目标核酸的扩增步骤，以及检测在PCR反应扩增产物中是否存在含目标核酸的多核苷酸的基因检测步骤。在基因检测步骤中，例如使用寡核苷酸微阵列来检测多核苷酸的存在情况。In certain embodiments, the present invention comprises: an amplification step of amplifying a target nucleic acid by PCR, and a genetic detection step of detecting the presence or absence of a polynucleotide containing the target nucleic acid in the amplified product of the PCR reaction. In the genetic detection step, for example, an oligonucleotide microarray is used to detect the presence of polynucleotides.

5.目标核酸扩增步骤5. Target nucleic acid amplification step

在某些实施方案中，目标核酸检测试剂通过扩增目标核酸来提供。在一个实施方案中，使用PCR扩增目标核酸检测试剂。在目标核酸扩增步骤中，使用样品提取物、用于扩增含目标核酸的多核苷酸的基因检测引物试剂以及标记性核苷酸进行PCR反应。样品提取物是如下获得的提取物：按照用于提取并纯化DNA或RNA的已知的多核苷酸提取方法来提取并纯化检验样品中所含的多核苷酸。样品提取物在PCR反应中作为模板使用。In certain embodiments, target nucleic acid detection reagents are provided by amplification of the target nucleic acid. In one embodiment, the target nucleic acid detection reagent is amplified using PCR. In the target nucleic acid amplification step, a PCR reaction is performed using a sample extract, a gene detection primer reagent for amplifying a polynucleotide containing the target nucleic acid, and a labeled nucleotide. The sample extract is an extract obtained by extracting and purifying a polynucleotide contained in a test sample according to a known polynucleotide extraction method for extracting and purifying DNA or RNA. Sample extracts are used as templates in PCR reactions.

如果一种核酸与第二种核酸两者在严格条件下与同一探针核酸杂交，则此核酸与第二种核酸基本上相同。基本上相同的核酸之间的同源性可以为80％、90％、95％或以上。A nucleic acid is substantially identical to a second nucleic acid if the nucleic acid and the second nucleic acid both hybridize under stringent conditions to the same probe nucleic acid. The homology between substantially identical nucleic acids may be 80%, 90%, 95% or more.

为了进行基因扩增步骤，让样品提取物、病原体鉴定引物试剂、被标记的核苷酸以及PCR反应所必需的其他试剂和酶于一个反应管中进行PCR反应。通过RCR反应扩增多核苷酸。To perform the gene amplification step, a PCR reaction is performed in one reaction tube with sample extracts, pathogen identification primer reagents, labeled nucleotides, and other reagents and enzymes necessary for the PCR reaction. Polynucleotides are amplified by RCR reactions.

通常，理想的是在与寡核苷酸微阵列杂交之前扩增核酸样品。合适的扩增方法包括但不限于：聚合酶链式反应(PCR)(Innis等，PCRProtocols.A guide to Methods and Application.Academic Press，Inc.SanDiego，(1990))、连接酶链式反应(LCR)(参见Wu和Wallace，Genomics，4：560(1989)；Landegren等，Science，241：1077(1988)；和Barringer等，Gene，89：117(1990))、转录扩增(Kwoh等，Proc.Natl.Acad.Sci.USA，86：1173(1989))、自动维持序列扩增(Guatelli等，Proc.Nat.Acad.Sci.USA，87：1874(1990))。在一个实施方案中，使用PCR扩增目标核酸。在另一个实施方案中，使用反转录酶(RT)-PCR扩增目标核酸。在又一实施方案中，使用Tth DNA聚合酶(可得自例如Promega(Cat.No.M210A))进行PT-PCR。Tth DNA聚合酶是取自嗜热真细菌嗜热栖热菌(Thermus thermophilus)HB-8的酶的重组体形式，是在74℃下复制DNA的热稳定酶，并且在95℃下表现出20分钟的半衰期。Tth DNA聚合酶在镁存在下催化核苷酸沿5’→3’的方向聚合成双链DNA，而在锰存在下使用RNA模板沿5’→3’的方向使核苷酸聚合成DNA。TthDNA聚合酶经常用于PCR、RT-PCR、反转录以及在升高温度下的引物延伸反应。Often, it is desirable to amplify the nucleic acid sample prior to hybridization to the oligonucleotide microarray. Suitable amplification methods include, but are not limited to: polymerase chain reaction (PCR) (Innis et al., PCR Protocols. A guide to Methods and Application. Academic Press, Inc. San Diego, (1990)), ligase chain reaction (LCR ) (see Wu and Wallace, Genomics, 4:560 (1989); Landegren et al., Science, 241:1077 (1988); and Barringer et al., Gene, 89:117 (1990)), transcription amplification (Kwoh et al., Proc. USA, 86: 1173 (1989)), Self-sustaining sequence amplification (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87: 1874 (1990)). In one embodiment, PCR is used to amplify the target nucleic acid. In another embodiment, the target nucleic acid is amplified using reverse transcriptase (RT)-PCR. In yet another embodiment, PT-PCR is performed using Tth DNA polymerase (available, eg, from Promega (Cat. No. M210A)). Tth DNA polymerase is a recombinant form of the enzyme from the thermophilic eubacterium Thermus thermophilus (Thermus thermophilus) HB-8. It is a thermostable enzyme that replicates DNA at 74°C and exhibits 20 minute half-life. Tth DNA polymerase catalyzes the polymerization of nucleotides into double-stranded DNA in the 5'→3' direction in the presence of magnesium, and uses RNA templates to polymerize nucleotides into DNA in the 5'→3' direction in the presence of manganese. Tth DNA polymerase is frequently used in PCR, RT-PCR, reverse transcription, and primer extension reactions at elevated temperatures.

本发明的扩增方法利用了以下酶活性：DNA聚合酶和依赖DNA的RNA聚合酶。用于本发明方法和组合物的DNA聚合酶能够按照本发明的方法实施引物延伸。因此，所选聚合酶是能够使核酸引物沿着目标核酸模板延伸的聚合酶，所述目标核酸模板至少主要由脱氧核糖核苷酸组成。聚合酶能够从多核苷酸置换核酸链，其中所述多核苷酸是所述置换链与之结合的多核苷酸。The amplification method of the present invention utilizes the following enzymatic activities: DNA polymerase and DNA-dependent RNA polymerase. DNA polymerases useful in the methods and compositions of the invention are capable of performing primer extension according to the methods of the invention. Thus, the polymerase of choice is one that is capable of extending a nucleic acid primer along a target nucleic acid template consisting at least essentially of deoxyribonucleotides. A polymerase is capable of displacing a strand of nucleic acid from a polynucleotide to which the displacing strand binds.

任何反转录酶皆可用于本发明的实施中，包括但不限于：Superscript RTII、“regular”MMLV-RT、AMV RT或者它们的组合。Any reverse transcriptase can be used in the practice of the present invention, including but not limited to: Superscript RTII, "regular" MMLV-RT, AMV RT, or combinations thereof.

当样品提取物中不存在预测的病原体时，以及当样品提取物中不存在多核苷酸时，则在PCR产物中不存在扩增的多核苷酸，并且在随后的检测基因步骤中检测不出多核苷酸。When the predicted pathogen is absent from the sample extract, and when the polynucleotide is absent from the sample extract, then the amplified polynucleotide is absent from the PCR product and cannot be detected in subsequent detection gene steps polynucleotide.

当样品提取物中存在预测的病原体时，来源于所述病原体的多核苷酸则在PCR反应中充当模板。通过病原体鉴定引物试剂中的相应扩增引物与模板结合，PCR反应得以进行，由此扩增出含病原体所固有的目标核酸的多核苷酸。标记性核苷酸被带入所扩增的多核苷酸(DNA)中，即被带入PCR反应产物中。由此PCR反应产物被标记。When the predicted pathogen is present in the sample extract, the polynucleotide derived from said pathogen serves as a template in a PCR reaction. By binding the corresponding amplification primers in the pathogen identification primer reagent to the template, the PCR reaction is carried out, thereby amplifying the polynucleotide containing the target nucleic acid inherent in the pathogen. The labeled nucleotides are incorporated into the amplified polynucleotide (DNA), ie into the PCR reaction product. The PCR reaction product is thus labeled.

用于扩增目标核酸的引物根据从选自以下的病原体中分离的多核苷酸来设计：细菌、病毒、真菌和原生动物。在其他实施方案中，用于扩增目标核酸的引物根据从选自以下的病原体中分离的多核苷酸来设计：立克次氏体、衣原体、支原体、螺旋体、链球菌、沙门氏菌、葡萄球菌、支原体、单核细胞增生李斯特氏菌(L.monocytogenes)、脑膜炎奈瑟氏菌(N.meningtides)、大肠杆菌、流感嗜血杆菌(H.influenzαe)、布氏疏螺旋体(B.burgdorferi)、钩端螺旋体、变形菌、厌氧菌、结核分支杆菌(M.tuberculosis)、肠球菌、脊髓灰质炎病毒1、肠道病毒71、肠道病毒70、艾柯病毒2、艾柯病毒4、艾柯病毒6、艾柯病毒9、艾柯病毒11、艾柯病毒12、艾柯病毒26、柯萨奇病毒A13、柯萨奇病毒A15、柯萨奇病毒A18、柯萨奇病毒A20、柯萨奇病毒A21、柯萨奇病毒B3-A、柯萨奇病毒B3-C、HSV-1和HSV-2。在一些实施方案中，PCR利用了含从以上所列病原体的至少一种中分离的多核苷酸序列的引物。Primers for amplifying target nucleic acids are designed based on polynucleotides isolated from pathogens selected from the group consisting of bacteria, viruses, fungi and protozoa. In other embodiments, primers for amplifying target nucleic acids are designed based on polynucleotides isolated from pathogens selected from the group consisting of Rickettsia, Chlamydia, Mycoplasma, Spirochetes, Streptococcus, Salmonella, Staphylococcus, Mycoplasma, Listeria monocytogenes (L.monocytogenes), Neisseria meningitidis (N.meningtides), Escherichia coli, Haemophilus influenzae (H.influenzαe), Borrelia brucei (B.burgdorferi) , Leptospira, Proteus, Anaerobic bacteria, Mycobacterium tuberculosis (M.tuberculosis), Enterococcus, Poliovirus 1, Enterovirus 71,Enterovirus 70, Ecovirus 2, Ecovirus 4, Ecovirus 6, Ecovirus 9, Ecovirus 11, Ecovirus 12, Ecovirus 26, Coxsackievirus A13, Coxsackievirus A15, Coxsackievirus A18, Coxsackievirus A20, Coxsackievirus A20 Saatchievirus A21, Coxsackievirus B3-A, Coxsackievirus B3-C, HSV-1 and HSV-2. In some embodiments, PCR utilizes primers comprising a polynucleotide sequence isolated from at least one of the pathogens listed above.

在某些实施方案中，特定的引物对能够与不止一种病原体的多核苷酸退火。此引物对可退火至某些病原体中保守的区域。然而，引物对邻接的核苷酸序列是可变区。使用此引物对使多种病原体的核酸序列能够被扩增，而且还允许这些病原体在与寡核苷酸微阵列杂交时能够得以辨别。当使用来自许多细菌和真菌的16S/18S rDNA基因的两个相同区作为引物时，这两个区通过PCR扩增提供了197个碱基对(细菌)或248个碱基对(真菌)的DNA片段。在引物区之间的DNA片段中发现许多低保守区，这些低保守区提供了探针以根据其特定序列通过杂交来辨别病原体。参见出示了16S rDNA的细菌DNA序列的图1A和1B。出示了这两个保守的引物区以及这两个保守区之间的可变的探针区。图2A和2B出示了真菌DNA序列的所述18S rDNA片段中的引物区和探针区。In certain embodiments, a particular primer pair is capable of annealing to polynucleotides of more than one pathogen. This primer pair anneals to regions conserved in some pathogens. However, the nucleotide sequence adjacent to the primer pair is the variable region. Use of this primer pair enables the nucleic acid sequences of a variety of pathogens to be amplified and also allows the pathogens to be distinguished when hybridized to an oligonucleotide microarray. When the two identical regions of the 16S/18S rDNA gene from many bacteria and fungi were used as primers, the two regions were amplified by PCR to provide 197 base pairs (bacteria) or 248 base pairs (fungus) DNA fragments. Many less conserved regions are found in the DNA fragments between the primer regions, and these less conserved regions provide probes to identify pathogens by hybridization based on their specific sequences. See Figures 1A and 1B showing the bacterial DNA sequence of the 16S rDNA. The two conserved primer regions and the variable probe region between the two conserved regions are shown. Figures 2A and 2B show the primer and probe regions in the 18S rDNA fragment of the fungal DNA sequence.

在一个实施方案中，PCR利用含选自SEQ ID NO：1-12(表1)的多核苷酸的引物。引物对的一些组合包括：SEQ ID NO：1和2；SEQ IDNO：1和4；SEQ ID NO：1和6；SEQ ID NO：3和2；SEQ ID NO：3和4；SEQ ID NO：3和6；SEQ ID NO：5和2；SEQ ID NO：5和4；SEQ ID NO：5和6；SEQ ID NO：5和6；SEQ ID NO：7和8；SEQ ID NO：9和10和/或SEQ ID NO：11和12。In one embodiment, PCR utilizes primers comprising a polynucleotide selected from SEQ ID NO: 1-12 (Table 1). Some combinations of primer pairs include: SEQ ID NO: 1 and 2; SEQ ID NO: 1 and 4; SEQ ID NO: 1 and 6; SEQ ID NO: 3 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 3 and 6; SEQ ID NO: 5 and 2; SEQ ID NO: 5 and 4; SEQ ID NO: 5 and 6; SEQ ID NO: 5 and 6; SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10 and/or SEQ ID NO: 11 and 12.

表1Table 1

 引物来源序列(5’→3’)SEQ ID NO细菌+actcctacgggaggcagcag1细菌-attaccgcggctgctggcac2细菌+ccagactcctacgggaggcagcag(353)3细菌-gattaccgcggctgctggcac(553)4细菌+ccatactcctacgggaggcagcag(353)5细菌-tattaccgcggctgctggcac(553)6肠道病毒：ctccggcccctgaatgcgg(372)7肠道病毒：acccaaagtagtcggttccg(478)8HSVggaactcctccaccgccca(74869)9HSVgtaccagggcgtcctgggc(75086)10真菌ggccgttcttagttggtggagt11真菌atgctctatccccagcacgac12 Primer source Sequence (5'→3') SEQ ID NO bacteria+ actcctacgggaggcagcag 1 bacteria- attaccgcggctgctggcac 2 bacteria+ ccagactcctacgggaggcagcag(353) 3 bacteria- gattaccgcggctgctggcac(553) 4 bacteria+ ccatactcctacgggaggcagcag(353) 5 bacteria- tattaccgcggctgctggcac(553) 6 Enteroviruses: ctccggcccctgaatgcgg(372) 7 Enteroviruses: acccaaagtagtcggttccg(478) 8 HSV ggaactcctccaccgccca(74869) 9 HSV gtaccagggcgtcctgggc(75086) 10 fungus ggccgttcttagttggtggagt 11 fungus atgctctatccccagcacgac 12

序列后面的数字指的是在各种模板上引物退火的核苷酸位置。Numbers following the sequence refer to the nucleotide position at which the primer anneals on the various templates.

6.微阵列6. Microarray

“微阵列”是在固相支持体表面上形成的离散区的线性或者二维微点阵，每一个离散区都具有确定的区域。使用下文描述的展示策略中的一种，将与目标核酸序列或其亚序列互补的寡核苷酸探针微阵列固定在固相支持体上。本发明的方法使用含展现与一种或更多种目标核酸序列互补性的探针的寡核苷酸微阵列。通常这些探针是DNA，并且以高密度的微阵列(即“基因芯片”)的方式固定在固体表面上。基本上任何可能的基片(substrate)都可为本发明所利用。基片可以是以颗粒、链、沉淀物、凝胶、薄片、管、球体、容器、毛细管、垫、切片、薄膜、平皿、载玻片等存在的生物基片、非生物基片、有机基片、无机基片或它们的任何组合。基片可以具有任何适宜的形状，例如圆盘、方形、球形、环形等。基片通常是平的，但可以呈现多种可供选择的表面结构。例如，基片可以含有于其上进行合成的凸起区或凹陷区。基片及其表面可以形成在上面进行本文所述反应的刚性支持体。同样选择基片及其表面来提供合适的光吸收特征。例如，基片可以是聚合的朗缪尔-布罗杰特膜、功能化玻璃(functionalized glass)、Si、Ge、GaAs、GaP、SiO₂、SiN₄、改性硅或者众多种类的凝胶或聚合物中的任一种，诸如(聚)四氟乙烯、(聚)偏二氟乙烯、聚苯乙烯、聚碳酸酯或他们的组合。其他基片材料将在本领域技术人员查阅此公开内容时显而易见。在一个实施方案中，基片是平片玻璃。A "microarray" is a linear or two-dimensional microarray of discrete regions, each having a defined area, formed on the surface of a solid support. Using one of the display strategies described below, a microarray of oligonucleotide probes complementary to a target nucleic acid sequence or a subsequence thereof is immobilized on a solid support. The methods of the invention use oligonucleotide microarrays containing probes that exhibit complementarity to one or more target nucleic acid sequences. Typically these probes are DNA and are immobilized on a solid surface in a high density microarray (ie "gene chip"). Essentially any possible substrate can be utilized with the present invention. Substrates can be biological substrates, non-biological substrates, organic-based sheets, inorganic substrates, or any combination thereof. The substrate may have any suitable shape, such as disc, square, spherical, circular, and the like. The substrate is usually flat, but can exhibit a variety of alternative surface structures. For example, the substrate may contain raised or recessed regions upon which synthesis is performed. The substrate and its surface can form a rigid support upon which the reactions described herein are carried out. The substrate and its surface are also selected to provide suitable light absorbing characteristics. For example, the substrate can be a polymeric Langmuir-Blodgett film, functionalized glass, Si, Ge, GaAs, GaP,_SiO2 ,_SiN4 , modified silicon, or numerous types of gels or Any of polymers such as (poly)tetrafluoroethylene, (poly)vinylidene fluoride, polystyrene, polycarbonate or combinations thereof. Other substrate materials will be apparent to those skilled in the art upon reviewing this disclosure. In one embodiment, the substrate is a flat sheet of glass.

各种策略可用于使寡核苷酸探针微阵列定制和展现在基片上，藉此使杂交图谱和可推断出的关于目标核酸的序列信息最大化。例示性展现和定制策略在PCT专利公开说明书WO 94/12305中描述，该文献在此引作参考。Various strategies are available for customizing and displaying microarrays of oligonucleotide probes on substrates, thereby maximizing hybridization patterns and inferred sequence information about target nucleic acids. Exemplary presentation and customization strategies are described in PCT Patent Publication WO 94/12305, which is hereby incorporated by reference.

探针可以是寡脱氧核糖核苷酸或寡核糖核苷酸，或者是能够通过互补碱基配对与目标核酸序列杂交的以上核苷酸聚合物的修饰形式。修饰形式包括2’-O-甲基寡核糖核苷酸和所谓的PNA，在PNA中寡脱氧核糖核苷酸通过肽键而非通过磷酸二酯键连接。探针可以通过任何键与支持体连接(例如3’、5’或通过碱基)。3’连接更常用，因为这样定位与用于寡核苷酸固相合成的化学法相一致。众多的单个探针被固定在基片上的离散位置中，以便辨别目标核酸检测试剂与每种类型探针的杂交。众多离散的单一探针中的每一个被称为“点”。点的尺寸可以在直径0.7mm至1mm左右，在每个点中有0.35μM至40μMDNA。例如，用于检验链球菌和沙门氏菌两种病原体的多核苷酸存在情况的DNA微阵列可具有两个点：一个由链球菌探针组成，另一个由沙门氏菌探针组成。在某些实施方案中，有6、12、24、36、56、64、96、108或384个不同的探针点固定在一个基片上。在一个实施方案中，微阵列可以是1.5mm²。Probes may be oligodeoxyribonucleotides or oligoribonucleotides, or modified versions of the above nucleotide polymers capable of hybridizing to a target nucleic acid sequence through complementary base pairing. Modified forms include 2'-O-methyl oligoribonucleotides and so-called PNAs in which the oligodeoxyribonucleotides are linked by peptide bonds rather than by phosphodiester bonds. Probes can be attached to the support via any linkage (eg 3', 5' or via a base). The 3' ligation is more commonly used because this orientation is consistent with the chemistry used for solid phase synthesis of oligonucleotides. A multitude of individual probes are immobilized in discrete locations on the substrate to allow discrimination of hybridization of target nucleic acid detection reagents to each type of probe. Each of the many discrete single probes is called a "point". The spot size can be around 0.7 mm to 1 mm in diameter, with 0.35 μΜ to 40 μΜ DNA in each spot. For example, a DNA microarray testing for the presence of polynucleotides for both Streptococcus and Salmonella pathogens may have two spots: one consisting of Streptococcus probes and the other consisting of Salmonella probes. In certain embodiments, there are 6, 12, 24, 36, 56, 64, 96, 108 or 384 different probe spots immobilized on a substrate. In one embodiment, the microarray may be 1.5 mm² .

在一些微阵列中，所有探针的长度都相同。其他微阵列使用不同长度的探针。探针的长度可以变化以优化特定的杂交条件。优化杂交条件以减少“噪声”。通过改变杂交反应的pH、温度和离子条件来修改杂交条件。术语“严格”用来指明通常在本领域中理解为核酸序列之间需要高度互补性以便杂交得以发生的条件。严格条件可以包括高度严格条件，例如于65℃下，在0.5M NaHPO₄、7％十二烷基硫酸钠(SDS)、1mM EDTA中进行杂交。在寡核苷酸杂交的情况下，其他例示性的严格杂交条件包括于37℃下，在6×SSC/0.05％焦磷酸钠中洗涤。更高温度例如48℃、55℃、60℃和64℃可用于更长的寡核苷酸。In some microarrays, all probes are the same length. Other microarrays use probes of different lengths. Probe lengths can be varied to optimize specific hybridization conditions. Optimize hybridization conditions to reduce "noise". Hybridization conditions are modified by changing the pH, temperature and ionic conditions of the hybridization reaction. The term "stringent" is used to designate conditions generally understood in the art as requiring a high degree of complementarity between nucleic acid sequences in order for hybridization to occur. Stringent conditions can include highly stringent conditions, eg, hybridization is performed at 65°C in 0.5M_NaHPO4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA. In the case of oligonucleotide hybridization, other exemplary stringent hybridization conditions include washing in 6 x SSC/0.05% sodium pyrophosphate at 37°C. Higher temperatures such as 48°C, 55°C, 60°C and 64°C can be used for longer oligonucleotides.

在另一个实施方案中，本发明涉及进一步包括洗涤寡核苷酸微阵列和检测标记分子存在情况的方法。例如于68℃下在0.1×SSC/0.1％SDS中进行洗涤，和在适度的严格条件下进行洗涤(即于42℃下在0.2×SSC/0.1％SDS中进行洗涤)。In another embodiment, the present invention relates to a method further comprising washing the oligonucleotide microarray and detecting the presence of marker molecules. For example, washes are performed at 68°C in 0.1×SSC/0.1% SDS, and under moderately stringent conditions (ie, at 42°C in 0.2×SSC/0.1% SDS).

在一些实施方案中，探针被设计成与从病原体中分离的多核苷酸互补，所述病原体选自：细菌、病毒、真菌、原生动物。在其他实施方案中，探针被设计成与从选自以下的病原体中分离的多核苷酸互补：立克次氏体、衣原体、支原体、螺旋体、链球菌、沙门氏菌、葡萄球菌、支原体、单核细胞增生李斯特氏菌(L.monocytogenes)、脑膜炎奈瑟氏菌(N.meningtides)、大肠杆菌、流感嗜血杆菌(H.influenzae)、布氏疏螺旋体(B.burgdorferi)、钩端螺旋体、变形菌、厌氧菌、结核分支杆菌(M.tuberculosis)、肠球菌、脊髓灰质炎病毒1、肠道病毒71、肠道病毒70、艾柯病毒2、艾柯病毒4、艾柯病毒6、艾柯病毒9、艾柯病毒11、艾柯病毒12、艾柯病毒26、柯萨奇病毒A13、柯萨奇病毒A15、柯萨奇病毒A18、柯萨奇病毒A20、柯萨奇病毒A21、柯萨奇病毒B3-A、柯萨奇病毒B3-C、HSV-1和HSV-2。在另一个实施方案中，探针选自SEQ ID NO：13-52(表2)。In some embodiments, the probe is designed to be complementary to a polynucleotide isolated from a pathogen selected from the group consisting of: bacteria, viruses, fungi, protozoa. In other embodiments, the probe is designed to be complementary to a polynucleotide isolated from a pathogen selected from the group consisting of Rickettsia, Chlamydia, Mycoplasma, Spirochetes, Streptococcus, Salmonella, Staphylococcus, Mycoplasma, L.monocytogenes, N.meningtides, Escherichia coli, H.influenzae, B.burgdorferi, Leptospira , Proteus, Anaerobic bacteria, Mycobacterium tuberculosis (M.tuberculosis), Enterococcus, Poliovirus 1, Enterovirus 71,Enterovirus 70, Ecovirus 2, Ecovirus 4, Ecovirus 6 , Ecovirus 9, Ecovirus 11, Ecovirus 12, Ecovirus 26, Coxsackievirus A13, Coxsackievirus A15, Coxsackievirus A18, Coxsackievirus A20, Coxsackievirus A21 , Coxsackievirus B3-A, Coxsackievirus B3-C, HSV-1 and HSV-2. In another embodiment, the probe is selected from SEQ ID NO: 13-52 (Table 2).

表2Table 2

 探针来源序列(5’→3’)SEQ ID NO链球菌ccgagcaacgccgcgtgagtgaagaaggttttcggatc13沙门氏菌tgcagccatgccgcgtgtatgaagaaggccttcgggtt14沙门氏菌tttttccccggggaggaaggtgttgtggttaata15沙门氏菌tttttccccgtgttgtggttaataaccgcagcaa16沙门氏菌cccccgtactttcagcggggaggaaggtgttgtggttaat17葡萄球菌cggagcaacgccgcgtgagtgatgaaggtcttcggatc18支原体tgaagcaatgccgcgtgagtgatgacggccttcgggtt19单核细胞增生李斯特氏菌          cggagcaacgccgcgtgtatgaagaaggttttcggatc20脑膜炎奈瑟氏菌tccagccatgccgcgtgtctgaagaaggccttcgggtt21大肠杆菌tgcagccatgccgcgtgtatgaagaaggccttcgggtt22大肠杆菌gaagggagtaaagttaatacctttgctcattgacg23大肠杆菌tttttcccccggggaggaagggagtaaagttaata24大肠杆菌cgtcaatgagcaaaggtattaactttactccctt25大肠杆菌ccccccgtcaatgagcaaaggtattaactttactccctt26流感嗜血杆菌cgcagccatgccgcgtgaatgaagaaggccttcgggtt27布氏疏螺旋体cggagcgacactgcgtgaatgaagaaggtcgaaagatt28钩端螺旋体agcagcgacgccgcgtgaacgatgaaggtcttcggatt29变形菌tgcagccatgccgcgtgtatgaagaaggccttagggtt30厌氧菌tgcagcaacgccgcgtgagtgataaggcttcgggttgt31结核分支杆菌tgcagcgacgccgcgtgggggatgacggccttcgggtt32肠球菌ccgagcaacgccgcgtgagtgaagaaggttttcggatc33脊髓灰质炎病毒1ccacggagcaagtgccctcaatccagagggtggctt34肠道病毒71ctgcggagcacatgctcacaaaccagtgggtggtgt35肠道病毒70ccatggagcaaatgctcacaatccagtgagtggttt36艾柯病毒2ctgcggagcaggtacccacgagccagtgggcagcct37艾柯病毒4ctgcggagcacacgctcacaagccagtgagtggtgt38艾柯病毒6ctgcggagcaggtgctcacaatccagtgggtggcct39艾柯病毒9ctgtggagcacatgcccctaatccaaggggtagtgt40 probe source Sequence (5'→3') SEQ ID NO Streptococcus ccgagcaacgccgcgtgagtgaagaaggttttcggatc 13 salmonella tgcagccatgccgcgtgtatgaagaaggccttcgggtt 14 salmonella tttttccccggggaggaaggtgttgtggttaata 15 salmonella tttttccccgtgttgtggttaataaccgcagcaa 16 salmonella cccccgtactttcagcggggaggaaggtgttgtggttaat 17 staphylococcus cggagcaacgccgcgtgagtgatgaaggtcttcggatc 18 Mycoplasma tgaagcaatgccgcgtgagtgatgacggccttcgggtt 19 Listeria monocytogenes cggagcaacgccgcgtgtatgaagaaggttttcggatc 20 Neisseria meningitidis tccagccatgccgcgtgtctgaagaaggccttcgggtt twenty one Escherichia coli tgcagccatgccgcgtgtatgaagaaggccttcgggtt twenty two Escherichia coli gaagggagtaaagttaatacctttgctcattgacg twenty three Escherichia coli tttttcccccggggaggaagggagtaaagttaata twenty four Escherichia coli cgtcaatgagcaaaggtattaactttactccctt 25 Escherichia coli ccccccgtcaatgagcaaaggtattaactttactccctt 26 Haemophilus influenzae cgcagccatgccgcgtgaatgaagaaggccttcgggtt 27 Borrelia brucei cggagcgacactgcgtgaatgaagaaggtcgaaagatt 28 Leptospira agcagcgacgccgcgtgaacgatgaaggtcttcggatt 29Proteus tgcagccatgccgcgtgtatgaagaaggccttagggtt 30 anaerobic bacteria tgcagcaacgccgcgtgagtgataaggcttcgggttgt 31 Mycobacterium tuberculosis tgcagcgacgccgcgtgggggatgacggccttcgggtt 32 Enterococcus ccgagcaacgccgcgtgagtgaagaaggttttcggatc 33 Poliovirus 1 ccacggagcaagtgccctcaatccagagggtggctt 34 Enterovirus 71 ctgcggagcacatgctcacaaaccagtgggtggtgt 35Enterovirus 70 ccatggagcaaatgctcacaatccagtgagtggttt 36 Ecovirus 2 ctgcggagcaggtacccacgagccagtgggcagcct 37 Ecovirus 4 ctgcggagcacacgctcacaagccagtgagtggtgt 38 Ecovirus 6 ctgcggagcaggtgctcacaatccagtgggtggcct 39 Ecovirus 9ctgtggagcacatgcccctaatccaaggggtaggtgt 40

 艾柯病毒11ctgcggagcacatacccctaatccaaggggcagtgt41艾柯病毒12ctgtggagcaagtgcccacaacccagtgggtggctt42艾柯病毒26ctgcggagcaggcacccacaagccagtgggcagcct43柯萨奇病毒A13ccatggagcaagtgatcacaatccagtgatattctt44柯萨奇病毒A15ccacggagcaggtgacttcaagccagaagttggcct45柯萨奇病毒A18ccacggagcaagtgctcacgaaccagtgagtggctt46柯萨奇病毒A20ccatggagcaggcggtcacagaccagtgactagctt47柯萨奇病毒A21ccacggagcaaccgctcacaacccagtgagtaggtt48柯萨奇病毒B3-Actgtggatcatgcgccctcaaaccagagggaagcgt49柯萨奇病毒B3-Cctgcggagcatgcacccacaagccagtgggtagcgt50HSV-1gttgggccacgcgccccccgagctggtggacggccccgg51HSV-2gcttggtgacgcgcgcgcccagctcctccacggcctccg52 Ecovirus 11 ctgcggagcacatacccctaatccaaggggcagtgt 41 Ecovirus 12 ctgtggagcaagtgcccacaacccagtgggtggctt 42 Ecovirus 26 ctgcggagcaggcacccacaagccagtgggcagcct 43 Coxsackievirus A13 ccatggagcaagtgatcacaatccagtgatattctt 44 Coxsackievirus A15 ccacggagcaggtgacttcaagccagaagttggcct 45 Coxsackievirus A18 ccacggagcaagtgctcacgaaccagtgagtggctt 46 Coxsackie virus A20 ccatggagcaggcggtcacagaccagtgactagctt 47 Coxsackievirus A21 ccacggagcaaccgctcacaacccagtgagtaggtt 48 Coxsackievirus B3-A ctgtggatcatgcgccctcaaaccagagggaagcgt 49 Coxsackievirus B3-C ctgcggagcatgcacccacaagccagtggggtagcgt 50 HSV-1 gttgggccacgcgccccccgagctggtggacggccccgg 51 HSV-2 gcttggtgacgcgcgcgcccagctcctccacggcctccg 52

业已开发出若干技术来设计、合成、杂交和解释上文所述类型的高密度寡核苷酸微阵列。Several techniques have been developed to design, synthesize, hybridize, and interpret high-density oligonucleotide microarrays of the type described above.

可以使用光引导合成(light directed synthesis)以在基片表面建立寡核苷酸探针(Fodor等，Science，251：767-73(1991))。光引导合成使基于半导体的光刻法与固相化学合成相联合。该方法起始于当用光化学可移除的保护基团修饰的接头与固体基片即基片表面连接的时候。已经合成了带光不稳定的保护基的接头与亚磷酰胺，并由Pease等，PNAS，91：11241-11245(1994)描述。光通过光刻掩膜被引导到合成表面的特定区域，并激活这些区域以便进行随后的化学偶联。将具有光不稳定保护基的一系列核苷酸中的第一个与基片一起温育，化学偶联发生在在前述步骤中已被阐明的那些位置上。然后光通过掩膜的不同部分被定向到下一个合成位置，构建出化学步骤，即规定合集的聚核苷酸探针，而每一种探针在基片的表面上都具有它自己唯一的地址。寡核苷酸微阵列与扩增产生的荧光标记DNA或RNA杂交，杂交通过落射荧光共焦显微镜(epi-fluorescence confocal microscopy)进行检测(Fodor等，1993)。Light directed synthesis can be used to create oligonucleotide probes on the substrate surface (Fodor et al., Science, 251:767-73 (1991)). Light-guided synthesis combines semiconductor-based photolithography with solid-phase chemical synthesis. The method begins when a linker modified with a photochemically removable protecting group is attached to a solid substrate, ie, the surface of the substrate. Linkers and phosphoramidites with photolabile protecting groups have been synthesized and described by Pease et al., PNAS, 91:11241-11245 (1994). Light is directed through a photolithographic mask to specific regions of the synthetic surface and activates these regions for subsequent chemical coupling. The first of a series of nucleotides bearing a photolabile protecting group is incubated with the substrate and chemical coupling occurs at those positions that have been elucidated in the preceding steps. The light is then directed through different parts of the mask to the next synthesis site, building the chemical step, a defined collection of polynucleotide probes, each with its own unique DNA on the surface of the substrate. address. Oligonucleotide microarrays are hybridized to fluorescently labeled DNA or RNA produced by amplification, and the hybridization is detected by epi-fluorescence confocal microscopy (Fodor et al., 1993).

在一个实施方案中，寡核苷酸微阵列具有含被测的每一种病原体所固有的全部探针的点。In one embodiment, the oligonucleotide microarray has spots containing all probes inherent to each pathogen being tested.

因此，本发明涉及用于检测生物样品或检验样品中的病原体的目标核酸的方法。所述方法包括：制备目标核酸检测试剂；并使目标核酸检测试剂与寡核苷酸微阵列接触。在一些实施方案中，病原体选自链球菌、沙门氏菌、葡萄球菌、支原体、单核细胞增生李斯特氏菌(L.monocytogenes)、脑膜炎奈瑟氏菌(N.meningtides)、大肠杆菌、流感嗜血杆菌(H.influenzae)、布氏疏螺旋体(B.burgdorferi)、钩端螺旋体、变形菌、厌氧菌、结核分支杆菌(M.tuberculosis)、肠球菌、脊髓灰质炎病毒1、肠道病毒71、肠道病毒70、艾柯病毒2、艾柯病毒4、艾柯病毒6、艾柯病毒9、艾柯病毒11、艾柯病毒12、艾柯病毒26、柯萨奇病毒A13、柯萨奇病毒A15、柯萨奇病毒A18、柯萨奇病毒A20、柯萨奇病毒A21、柯萨奇病毒B3-A、柯萨奇病毒B3-C、HSV-1和HSV-2。在一些实施方案中，寡核苷酸微阵列包含探针，所述探针包含从以上列举的病原体的至少一种中分离的多核苷酸序列。在另一个实施方案中，探针包含选自SEQ ID NO：13-52的多核苷酸序列。Accordingly, the present invention relates to methods for detecting target nucleic acids of pathogens in biological samples or test samples. The method includes: preparing a target nucleic acid detection reagent; and contacting the target nucleic acid detection reagent with an oligonucleotide microarray. In some embodiments, the pathogen is selected from Streptococcus, Salmonella, Staphylococcus, Mycoplasma, Listeria monocytogenes (L.monocytogenes), Neisseria meningitidis (N.meningtides), Escherichia coli, influenzae H.influenzae, B.burgdorferi, Leptospira, Proteus, Anaerobic bacteria, M.tuberculosis, Enterococcus, Poliovirus 1, Enterovirus 71.Enterovirus 70, Ecovirus 2, Ecovirus 4, Ecovirus 6, Ecovirus 9, Ecovirus 11, Ecovirus 12, Ecovirus 26, Coxsackievirus A13, Coxsackie Chiavirus A15, Coxsackievirus A18, Coxsackievirus A20, Coxsackievirus A21, Coxsackievirus B3-A, Coxsackievirus B3-C, HSV-1 and HSV-2. In some embodiments, the oligonucleotide microarray comprises probes comprising polynucleotide sequences isolated from at least one of the above-listed pathogens. In another embodiment, the probe comprises a polynucleotide sequence selected from SEQ ID NO: 13-52.

7.试剂盒7. Kit

在某些方面，本发明还涉及用于检测检验样品中病原体的目标核酸的试剂盒。所述试剂盒包括至少一对引物，和包含至少一种探针的寡核苷酸微阵列。在一个实施方案中，所述引物对包含选自ID NO：1-12的引物。在另一个实施方案中，探针包含选自SEQ ID NO：13-52的多核苷酸序列。In certain aspects, the invention also relates to kits for detecting target nucleic acids of pathogens in a test sample. The kit includes at least one pair of primers, and an oligonucleotide microarray comprising at least one probe. In one embodiment, the primer pair comprises primers selected from ID NO: 1-12. In another embodiment, the probe comprises a polynucleotide sequence selected from SEQ ID NO: 13-52.

本发明将通过以下实施例进行更详细的解释。本发明的实施例和实施方案应该被认作是阐述性而非限制性的，本发明并非旨在受限于文中所给出的细节，而是可以在所附权利要求书的范围和等同范围内作出修改。The present invention will be explained in more detail by the following examples. The examples and embodiments of the invention should be considered as illustrative and not restrictive, and the invention is not intended to be limited to the details given herein, but may be construed within the scope and equivalents of the appended claims Make changes within.

8.实施例8. Example

实施例1Example 1

DNA微阵列制备方法DNA microarray preparation method

1.玻璃表面洗涤1. Glass surface cleaning

将洁净的载玻片浸入H₂SO₄/H₂O₂2:1中30分钟。用H₂O洗涤3次，然后用甲醇洗涤2次。在加压空气流中吹干，然后在80℃下烘10分钟。Immerse clean slides in H₂ SO₄ /H₂ O₂ 2:1 for 30 minutes. Wash 3 times with_H2O , then 2 times with methanol. Blow dry in a stream of pressurized air, then bake at 80°C for 10 minutes.

2.玻璃的硅烷化2. Silanization of glass

在2％N-(2-氨乙基)-3-氨丙基三甲氧基硅烷(EDA)的1mM醋酸溶液中于室温(RT)下超声处理30分钟。用H₂O洗涤3次，然后用甲醇洗涤2次。在N₂气流中吹干，然后在110℃下烘15分钟。Sonicate in 2% N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (EDA) in 1 mM acetic acid for 30 minutes at room temperature (RT). Wash 3 times with_H2O , then 2 times with methanol. Blow dry in N₂ flow, and then bake at 110°C for 15 minutes.

3.用异双官能交联剂修饰硅烷化载玻片3. Modification of Silanized Glass Slides with Heterobifunctional Crosslinkers

于室温下浸入交联剂溶液(含384mg PDITC(2mmol)的80ml含10％无水吡啶的DMF)2小时。用DMF冲洗3次，然后用二氯乙烷冲洗2次。在N₂中吹干。Immerse in a crosslinker solution (384 mg PDITC (2 mmol) in 80 ml DMF containing 10% anhydrous pyridine) for 2 hours at room temperature. Rinse 3 times with DMF and then 2 times with dichloroethane. Blow dry under_N2 .

4.微阵列印迹和DNA固定化4. Microarray Blotting and DNA Immobilization

于60％湿度下在活化的载玻片上点上含40μM NH₂-寡脱氧核苷酸(ODN)的0.1M碳酸盐缓冲液(pH 9.0)。于37℃饱和湿度下反应1小时以上。用水冲洗并浸没在含6-氨基己醇(100mM)的水溶液中。用100℃的水洗涤2-3分钟，然后在加压空气下吹干。0.1 M carbonate buffer (pH 9.0) containing 40 μM NH₂ -oligodeoxynucleotide (ODN) was spotted on the activated glass slide at 60% humidity. The reaction was carried out at 37° C. under saturated humidity for more than 1 hour. Rinse with water and immerse in an aqueous solution containing 6-aminohexanol (100 mM). Wash with 100°C water for 2-3 minutes, then blow dry under pressurized air.

DNA固定化结构DNA Immobilization Structure

实施例2Example 2

DNA微阵列杂交方法DNA microarray hybridization method

1.PCR扩增和标记1. PCR amplification and labeling

将1μl DNA样品加入到100μl PCR反应缓冲液中，所述反应缓冲液含有10mM Tris-HCl(pH 9.0)、50mM KCl、3.75mM MgCl2、0.1％Triton X-100、0.1mM dNTP(每种)和0.2μM Cy5标记的引物(每种)。然后加入5u Taq DNA聚合酶，按以下温度条件进行PCR扩增：1 μl of DNA sample was added to 100 μl of PCR reaction buffer containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 3.75 mM MgCl, 0.1% Triton X-100, 0.1 mM dNTP (each) and 0.2 μM Cy5-labeled primers (each). Then add 5u Taq DNA polymerase, and carry out PCR amplification according to the following temperature conditions:

i).96℃      2分钟i).96℃ for 2 minutes

ii).95℃     20秒ii).95℃ for 20 seconds

iii).60℃    20秒iii).60℃ for 20 seconds

iv).72℃     30秒iv).72℃ for 30 seconds

回到步骤ii)，28个循环Back to step ii), 28 cycles

v).72℃      2分钟v).72℃ for 2 minutes

总反应时间约1小时20分钟。The total reaction time is about 1 hour and 20 minutes.

2.DNA微阵列杂交2. DNA microarray hybridization

用5倍体积的含0.75M NaCl、75mM柠檬酸钠和0.1％SDS pH 7.2的杂交缓冲液稀释PCR溶液。于61℃下杂交1小时。Dilute the PCR solution with 5 volumes of hybridization buffer containing 0.75M NaCl, 75mM sodium citrate and 0.1% SDS pH 7.2. Hybridization was performed at 61°C for 1 hour.

3.DNA微阵列洗涤3. DNA Microarray Washing

将杂交后的载玻片用0.3M NaCl、30mM柠檬酸钠和0.1％SDS在50℃下洗涤15分钟，共洗涤3次，然后用0.3M NaCl、30mM柠檬酸钠在50℃下洗涤15分钟。用水冲洗然后在加压空气中干燥。The hybridized slides were washed with 0.3M NaCl, 30mM sodium citrate and 0.1% SDS at 50°C for 15 minutes for a total of 3 times, and then washed with 0.3M NaCl and 30mM sodium citrate for 15 minutes at 50°C . Rinse with water and dry in pressurized air.

4.微阵列读取4. Microarray Readout

DNA微阵列杂交结果通过FuJiFilm^TM荧光扫描仪FLA3000读取，用HeNe激光(633nm)激发Cy5。用打包软件ArrayGuage^TM分析信号密度。DNA microarray hybridization results were read by FuJiFilm^TM fluorescence scanner FLA3000, and Cy5 was excited by HeNe laser (633nm). Signal density was analyzed with the packaged software ArrayGuage^™ .

实施例3Example 3

从由患者分离的组织切片中直接收集细胞。使用任何已知的方案从这些细胞中提取DNA。使用两对引物SEQ ID NO：1-2和SEQ ID NO：7-8通过PCR扩增约2-10ng的DNA。将[³²P]-dCTP包括在PCR反应中，以标记扩增的DNA，从而产生带标记分子的目标核酸检测试剂。Cells are collected directly from tissue sections isolated from patients. DNA is extracted from these cells using any known protocol. About 2-10 ng of DNA was amplified by PCR using two pairs of primers SEQ ID NO: 1-2 and SEQ ID NO: 7-8. [³² P]-dCTP is included in the PCR reaction to label the amplified DNA, thereby producing a target nucleic acid detection reagent with the labeled molecule.

带标记分子的目标核酸检测试剂经由离心柱(spin column)纯化以除去多余的[³²P]-dCTP，在95℃下温育变性，随后直接在冰上冷却。然后在高严格条件下，使变性的带标记分子的目标核酸检测试剂在含探针的寡核苷酸微阵列存在下温育，所述探针含SEQ ID NO：13-52。杂交在0.5M NaHPO₄、7％十二烷基硫酸钠(SDS)、1mM EDTA中于65℃下进行2小时，随后在0.1×SSC/0.1％ SDS中于68℃下洗涤3次。此微阵列曝光于X射线胶片并显影出放射自显影图。Target nucleic acid detection reagents with labeled molecules were purified by spin column to remove excess [³² P]-dCTP, denatured by incubation at 95° C., and then directly cooled on ice. Denatured target nucleic acid detection reagents with labeled molecules are then incubated under high stringency conditions in the presence of oligonucleotide microarrays containing probes comprising SEQ ID NOs: 13-52. Hybridization was performed in 0.5M_NaHPO4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C for 2 hours, followed by 3 washes in 0.1 x SSC/0.1% SDS at 68°C. The microarray was exposed to X-ray film and an autoradiogram was developed.

实施例4Example 4

从任何一般的饮用水源(例如来自贮水池或来自水)中分离饮用水样品。将样品浓缩和/或过滤以分离任何病原体。使用任何已知的方案从这些细胞中提取DNA。使用两对引物SEQ ID NO：1-2和SEQ ID NO：7-8通过PCR扩增约2-10ng的DNA。将[³²P]-dCTP包括于PCR反应中，以标记扩增DNA，从而产生带标记分子的目标核酸检测试剂。Drinking water samples are isolated from any common source of drinking water, such as from a reservoir or from water. Samples are concentrated and/or filtered to isolate any pathogens. DNA is extracted from these cells using any known protocol. About 2-10 ng of DNA was amplified by PCR using two pairs of primers SEQ ID NO: 1-2 and SEQ ID NO: 7-8. [³² P]-dCTP is included in the PCR reaction to label the amplified DNA, thereby producing a target nucleic acid detection reagent with labeled molecules.

带标记分子的目标核酸检测试剂经过离心柱纯化以除去多余的[³²P]-dCTP，在95℃下温育变性，随后直接在冰上冷却。然后在高严格条件下，使变性的带标记分子的目标核酸检测试剂在含探针的寡核苷酸微阵列存在下温育，所述探针含有SEQ ID NO：13-52。杂交在0.5M NaHPO₄、7％十二烷基硫酸钠(SDS)、1mM EDTA中于65℃下进行2小时，随后在0.1×SSC/0.1％ SDS中于68℃下洗涤3次。此微阵列曝光于X射线胶片并显影放射自显影图。Target nucleic acid detection reagents with labeled molecules are purified by spin columns to remove excess [³² P]-dCTP, denatured by incubating at 95°C, and then directly cooled on ice. Denatured target nucleic acid detection reagents with labeled molecules are then incubated under high stringency conditions in the presence of oligonucleotide microarrays containing probes comprising SEQ ID NOs: 13-52. Hybridization was performed in 0.5M_NaHPO4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C for 2 hours, followed by 3 washes in 0.1 x SSC/0.1% SDS at 68°C. The microarray was exposed to X-ray film and the autoradiogram was developed.

尽管已用了至少一个说明性实施例来描述本发明，但在阅读本说明书时，许多变更和修改对于本领域的技术人员将是显而易见的。因此，意图在于：所附权利要求书应考虑现有技术尽可能宽泛地被解释为包括所有的这些变更和修改。While the invention has been described using at least one illustrative embodiment thereof, upon reading the specification, many alterations and modifications will become apparent to those skilled in the art. It is therefore intended that the appended claims be interpreted as broadly as possible in view of the prior art to include all such changes and modifications.

序列表sequence listing

<110>霍尼韦尔国际公司<110> Honeywell International Corporation

     Gu，YuandongGu, Yuandong

     Xu，LeonXu, Leon

<120>用于鉴定病原体的寡核苷酸微阵列<120> Oligonucleotide microarray for identification of pathogens

<130>1100.1430101<130>1100.1430101

<150>60/755,504<150>60/755,504

<151>2005-12-30<151>2005-12-30

<160>52<160>52

<170>PatentIn version 3.4<170>PatentIn version 3.4

<210>1<210>1

<211>20<211>20

<212>DNA<212>DNA

<213>细菌<213> bacteria

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<212>DNA<212>DNA

<213>细菌<213> bacteria

<400>2<400>2

<210>3<210>3

<211>24<211>24

<212>DNA<212>DNA

<213>细菌<213> bacteria

<400>3<400>3

<210>4<210>4

<211>21<211>21

<212>DNA<212>DNA

<213>细菌<213> bacteria

<400>4<400>4

<210>5<210>5

<211>24<211>24

<212>DNA<212>DNA

<213>细菌<213> bacteria

<400>5<400>5

<210>6<210>6

<211>21<211>21

<212>DNA<212>DNA

<213>细菌<213> bacteria

<400>6<400>6

<210>7<210>7

<211>19<211>19

<212>DNA<212>DNA

<213>病毒<213> virus

<400>7<400>7

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<212>DNA<212>DNA

<213>病毒<213> virus

<400>8<400>8

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<211>19<211>19

<212>DNA<212>DNA

<213>病毒<213> virus

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<210>10<210>10

<211>19<211>19

<212>DNA<212>DNA

<213>病毒<213> virus

<400>10<400>10

<210>11<210>11

<211>22<211>22

<212>DNA<212>DNA

<213>真菌<213> fungus

<400>11<400>11

<210>12<210>12

<211>21<211>21

<212>DNA<212>DNA

<213>真菌<213> fungus

<400>12<400>12

<210>13<210>13

<211>38<211>38

<212>DNA<212>DNA

<213>Streptococcus<213> Streptococcus

<400>13<400>13

<210>14<210>14

<211>38<211>38

<212>DNA<212>DNA

<213>Salmonella typhimurium<213>Salmonella typhimurium

<400>14<400>14

<210>15<210>15

<211>34<211>34

<212>DNA<212>DNA

<213>Salmonella typhimurium<213>Salmonella typhimurium

<400>15<400>15

<210>16<210>16

<211>34<211>34

<212>DNA<212>DNA

<213>Salmonella typhimurium<213>Salmonella typhimurium

<400>16<400>16

<210>17<210>17

<211>40<211>40

<212>DNA<212>DNA

<213>Salmonella typhimurium<213>Salmonella typhimurium

<400>17<400>17

<210>18<210>18

<211>38<211>38

<212>DNA<212>DNA

<213>Staphylococcus<213>Staphylococcus

<400>18<400>18

<210>19<210>19

<211>38<211>38

<212>DNA<212>DNA

<213>Mycoplasma<213> Mycoplasma

<400>19<400>19

<210>20<210>20

<211>38<211>38

<212>DNA<212>DNA

<213>Listeria monocytogenes<213>Listeria monocytogenes

<400>20<400>20

<210>21<210>21

<211>38<211>38

<212>DNA<212>DNA

<213>N.meningitides<213> N. meningitides

<400>21<400>21

<210>22<210>22

<211>38<211>38

<212>DNA<212>DNA

<213>E.Coli<213> E. Coli

<400>22<400>22

<210>23<210>23

<211>35<211>35

<212>DNA<212>DNA

<213>Enterococcus Coli<213>Enterococcus Coli

<400>23<400>23

<210>24<210>24

<211>35<211>35

<212>DNA<212>DNA

<213>E.Coli<213> E. Coli

<400>24<400>24

<210>25<210>25

<211>34<211>34

<212>DNA<212>DNA

<213>E.Coli<213> E. Coli

<400>25<400>25

<210>26<210>26

<211>39<211>39

<212>DNA<212>DNA

<213>E.Coli<213> E. Coli

<400>26<400>26

<210>27<210>27

<211>38<211>38

<212>DNA<212>DNA

<213>H.influenzae<213>H.influenzae

<400>27<400>27

<210>28<210>28

<211>38<211>38

<212>DNA<212>DNA

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<400>28<400>28

<210>29<210>29

<211>38<211>38

<212>DNA<212>DNA

<213>Leptospira<213> Leptospira

<400>29<400>29

<210>30<210>30

<211>38<211>38

<212>DNA<212>DNA

<213>Proteus mirabilis<213>Proteus mirabilis

<400>30<400>30

<210>31<210>31

<211>38<211>38

<212>DNA<212>DNA

<213>Anaerobacter<213> Anaerobacter

<400>31<400>31

<210>32<210>32

<211>38<211>38

<212>DNA<212>DNA

<213>M.tuberculosis<213>M. tuberculosis

<400>32<400>32

<210>33<210>33

<211>38<211>38

<212>DNA<212>DNA

<213>Enterococcus Coli<213>Enterococcus Coli

<400>33<400>33

<210>34<210>34

<211>36<211>36

<212>DNA<212>DNA

<213>脊髓灰质炎病毒1<213> Poliovirus 1

<400>34<400>34

<210>35<210>35

<211>36<211>36

<212>DNA<212>DNA

<213>肠道病毒71<213> Enterovirus 71

<400>35<400>35

<210>36<210>36

<211>36<211>36

<212>DNA<212>DNA

<213>肠道病毒70<213>Enterovirus 70

<400>36<400>36

<210>37<210>37

<211>36<211>36

<212>DNA<212>DNA

<213>艾柯病毒2<213> Ecovirus 2

<400>37<400>37

<210>38<210>38

<211>36<211>36

<212>DNA<212>DNA

<213>艾柯病毒4<213> Ecovirus 4

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<210>39<210>39

<211>36<211>36

<212>DNA<212>DNA

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<400>39<400>39

<210>40<210>40

<211>36<211>36

<212>DNA<212>DNA

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<211>36<211>36

<212>DNA<212>DNA

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<210>42<210>42

<211>36<211>36

<212>DNA<212>DNA

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<400>42<400>42

<210>43<210>43

<211>36<211>36

<212>DNA<212>DNA

<213>艾柯病毒26<213> Ecovirus 26

<400>43<400>43

<210>44<210>44

<211>36<211>36

<212>DNA<212>DNA

<213>柯萨奇病毒A13<213> Coxsackievirus A13

<400>44<400>44

<210>45<210>45

<211>36<211>36

<212>DNA<212>DNA

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<400>45<400>45

<210>46<210>46

<211>36<211>36

<212>DNA<212>DNA

<213>柯萨奇病毒A18<213> Coxsackievirus A18

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<210>47<210>47

<211>36<211>36

<212>DNA<212>DNA

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<400>47<400>47

<210>48<210>48

<211>36<211>36

<212>DNA<212>DNA

<213>柯萨奇病毒A21<213> Coxsackievirus A21

<400>48<400>48

<210>49<210>49

<211>36<211>36

<212>DNA<212>DNA

<213>柯萨奇病毒B3-A<213> Coxsackievirus B3-A

<400>49<400>49

<210>50<210>50

<211>36<211>36

<212>DNA<212>DNA

<213>柯萨奇病毒B3-C<213> Coxsackievirus B3-C

<400>50<400>50

<210>51<210>51

<211>39<211>39

<212>DNA<212>DNA

<213>人疱疹病毒1<213> Human herpesvirus 1

<400>51<400>51

<210>52<210>52

<211>39<211>39

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<400>52<400>52

Claims (20)

1. one kind is used for detection of biological sample or check sample one or more plants the method for the target nucleic acid of pathogenic agent, and described method comprises:

Utilization is a pair of or more to increase target nucleic acid in the sample of primer in conjunction with the conserved regions of more than a kind of pathogenic agent;

The target nucleic acid of amplification is contacted with oligonucleotide microarray, and described microarray comprises two kinds or above probe or the two or more probe groups that contains with different pathogens complementary polynucleotide sequence;

Detect the situation that combines of target nucleic acid and probe, wherein combine and then mean this pathogenic agent of existence in sample with the special pathogen probe.

2. the process of claim 1 wherein that described pathogenic agent is selected from: suis, Salmonellas, staphylococcus, mycoplasma, monocyte hyperplasia listeria spp (L.monocytogenes), Neisseria meningitidis (N.meningtides), intestinal bacteria, hemophilus influenzae (H.influenzae), B. burgdorferi (B.burgdorferi), Leptospira, mycetozoan, anerobe, Mycobacterium tuberculosis (M.tuberculosis), faecalis, poliovirus 1, Enterovirus 71, Enterovirus 70, ECHO virus 2, ECHO virus 4, ECHO virus 6, ECHO virus 9, ECHO virus 11, ECHO virus 12, ECHO virus 26, CA 13, CA 15, CA 18, CA 20, CA 21, Coxsackie B virus 3-A, Coxsackie B virus 3-C, HSV-1 and HSV-2.

3. the method for claim 2, wherein said probe is selected from SEQ ID NO:13-52.

4. the method for claim 3, wherein said target nucleic acid is intestinal bacteria and Salmonellas, and described probe is selected from SEQ ID NO:14-17 and 22-26.

5. the process of claim 1 wherein that described amplification step comprises utilizing is selected from a pair of of SEQ IDNO:1-12 or more primer is carried out polymerase chain reaction (PCR).

6. the method for claim 5, it is right that wherein said primer is selected from following primer: SEQ IDNO:1 and 2; SEQ ID NO:1 and 4; SEQ ID NO:1 and 6; SEQ ID NO:3 and 2; SEQ ID NO:3 and 4; SEQ ID NO:3 and 6; SEQ ID NO:5 and 2; SEQ ID NO:5 and 4; SEQ ID NO:5 and 6; SEQ ID NO:5 and 6; SEQ ID NO:7 and 8; SEQ ID NO:9 and 10; And SEQ ID NO:11 and 12.

7. the method for claim 6, wherein said primer is to being SEQ ID NO:1-2 and SEQID NO:7-8.

8. the method for claim 5, wherein said probe comprises the polynucleotide sequence that is selected from SEQ ID NO:13-52.

9. the method for claim 8, wherein microarray comprises 2 kinds or above probe from least a pathogenic agent.

10. the method for claim 1, wherein said biological sample is selected from tissue, cell, blood, serum, cerebrospinal fluid, urine, cell lysate, blood plasma, ight soil, saliva, hemocyte, fine needle biopsy samples, peritoneal fluid and Pleural fluid, or from the cell of above sample.

11. the process of claim 1 wherein that described microarray comprises the probe of the pathogenic agent relevant with common symptoms exhibited.

12. the process of claim 1 wherein that described microarray comprises the probe of the pathogenic agent relevant with common route of infection.

13. a method of distinguishing intestinal bacteria in the sample and Salmonellas, described method comprises:

Utilization is a pair of or more to the nucleic acid in the primer amplification sample in conjunction with the conserved regions in intestinal bacteria and the Salmonellas;

The target nucleic acid of amplification is contacted with oligonucleotide microarray, described microarray comprise contain with intestinal bacteria and Salmonellas in two kinds or above probe of variable region complementary polynucleotide sequence;

Detect the nucleic acid and the situation that combines as the probe of indicating intestinal bacteria and Salmonellas in sample, to exist.

14. the method for claim 13, wherein said microarray comprises: the two or more probe groups of colibacillary two or more probe groups and Salmonellas, wherein each probe groups comprises and two kinds of described pathogenic agent complementary or above different probe.

15. the method for claim 13, wherein said primer is to being selected from SEQ ID NO:1-6.

16. the method for claim 15, wherein said probe is selected from SEQ ID NO:14-17 and 22-26.

17. test kit that is used for detecting the target nucleic acid of at least a pathogenic agent of check sample, described test kit comprises: at least one pair of primer that is selected from SEQ ID NO:1-12, and the oligonucleotide microarray that comprises at least a probe, described probe contains the polynucleotide sequence that is selected from SEQ ID NO:13-52, and described probe is fixed on the solid support.

18. the test kit of claim 17, wherein said primer is right to being selected from following primer: SEQ ID NO:1 and 2; SEQ ID NO:1 and 4; SEQ ID NO:1 and 6; SEQ ID NO:3 and 2; SEQ ID NO:3 and 4; SEQ ID NO:3 and 6; SEQ ID NO:5 and 2; SEQ ID NO:5 and 4; SEQ ID NO:5 and 6; SEQ ID NO:5 and 6; SEQ ID NO:7 and 8; SEQ ID NO:9 and 10; And SEQ ID NO:11 and 12.

19. the test kit of claim 18, wherein said probe is selected from SEQ ID NO:14-17 and 22-26.

20. the test kit of claim 17, wherein said at least a probe comprise two kinds or above probe from two kinds or above pathogenic agent.

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