CN112708690A

CN112708690A - 念珠菌检测及分型的方法和试剂盒

Info

Publication number: CN112708690A
Application number: CN202110040671.0A
Authority: CN
Inventors: 李立威; 岑博顿; 韩斌; 华绍炳
Original assignee: Hangzhou Dalton Biosciences Ltd
Current assignee: Hangzhou Dalton Biosciences Ltd
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-04-27
Anticipated expiration: 2041-01-13
Also published as: CN112708690B

Abstract

本发明提供了一种念珠菌检测及分型的方法和试剂盒。一方面，本发明揭示了一种优化的基于DNA‑RNA杂合体的检测方法，包括在检测体系中添加RNase A，以其降解非特异性杂交的杂合体，提高检测的灵敏度和准确性。另一方面，本发明揭示了对于多种念珠菌具有特异性检测/分型功能的试剂盒。结合核酸杂交化学发光免疫技术，本发明人开发了可实现对多种念珠菌进行检测和分型的技术。

Description

念珠菌检测及分型的方法和试剂盒

技术领域

本发明属于生物技术领域，更具体地，本发明涉及一种念珠菌检测及分型的方法和试剂盒。

背景技术

念珠菌广泛存在于自然界中，土壤、植物、某些水果、奶制品等均可分离出念珠菌。念珠菌也是人体正常携带的真菌，在免疫力低下时致病，不是由别处传播而来。正常人皮肤、口腔、肠道、肛门、阴道中都可分离出本菌。

念珠菌是一种真菌，当全身及阴道局部免疫能力下降，尤其是局部细胞免疫力下降，念珠菌大量繁殖，引发阴道炎症状，即霉菌性阴道炎(VVC)。其典型症状是外阴瘙痒，且瘙痒症状时轻时重，严重时坐卧不宁、寝食难安，炎症较重时还可能出现排尿痛、性交痛等。引发霉菌性阴道炎的主要念珠菌种类为白色念珠菌，约占所有病例的80％～90％。其余常见的其它种类有热带念珠菌、光滑念珠菌、近平滑念珠菌、乳酒念珠菌、克柔念珠菌等。除白色念珠菌外，其它种类的念珠菌对某些抗真菌药物(如氟康唑)有一定的天然耐药性，故随着抗真菌药物的广泛应用，感染白色念珠菌以外种类的念珠菌的患者逐渐增加。此类患者需要更换使用其它药物，否则极易复发。

目前对于霉菌性阴道炎临床诊断方法主要为镜检或样本真菌培养。镜检或荧光染色的镜检灵敏度低，对镜检人员有一定的经验要求，且不能对念珠菌的种类进行区分。由于体外念珠菌生长缓慢，培养法鉴别不同念珠菌种类需要3-5天，耗时很长。另外也有核酸扩增法对念珠菌进行检测，其步骤复杂，场地设备条件高，对人员要求也很高。

综上，鉴于念珠菌在大自然中的广泛存在，以及其感染人类并具有致病性，本领域亟待开发有效地进行念珠菌分型以及检测的方法和试剂，以及时快速地测出念珠菌。

发明内容

本发明的目的在于提供一种念珠菌检测及分型的方法和试剂盒。

在本发明的第一方面，提供一种检测念珠菌的方法，所述方法包括：(1)将待测的念珠菌DNA样本解链处理，得到单链DNA样本；(2)将步骤(1)获得的单链DNA样本、特异性识别目标DNA的探针以及检测抗体、RNase A共同加样到包被有捕获抗体的固相载体上；所述捕获抗体与检测抗体为特异性抗DNA-RNA杂合体的抗体，所述检测抗体携带可检测信号；当DNA样本存在目标DNA时，在固相载体上形成捕获抗体-双链杂交体-检测抗体三元复合物；(3)根据检测抗体的可检测信号，得知目标DNA的存在与否或存在量。

在一个优选例中，所述的捕获抗体与所述的检测抗体基于相同的抗体或不同的抗体(即检测抗体在不携带可检测信号的情况下可以是与捕获抗体相同的或不同的)。优选的，所述的捕获抗体与所述的检测抗体基于不同的抗体。

在另一优选例中，所述的RNase A在混合体系中的终浓度为0.02～0.5mg/ml；较佳地0.05～0.3mg/ml；例如0.06mg/ml，0.08mg/ml，0.1mg/ml，0.15mg/ml，0.2mg/ml，0.25mg/ml，0.3mg/ml或0.4mg/ml。

在另一优选例中，所述的念珠菌包括：白色念珠菌，热带念珠菌，近平滑念珠菌，乳酒念珠菌，光滑念珠菌，克柔念珠菌。

在另一优选例中，所述特异性识别目标DNA的探针包括：(a)针对白色念珠菌的SEQID NO:1，SEQ ID NO:2探针；(b)针对近平滑念珠菌的SEQ ID NO:3探针；(c)针对乳酒念珠菌的SEQ ID NO:4探针；(d)针对光滑念珠菌的SEQ ID NO:5探针；(e)针对热带念珠菌的SEQID NO:6探针；(f)针对克柔念珠菌的SEQ ID NO:7探针。

在另一优选例中，通过分组法进行分型检测，包括：将(a)的探针作为一组，将(b)～(f)的探针作为一组，两组分别在独立的体系中进行反应，从而检测后将念珠菌分型为：白色念珠菌或其它念珠菌；该其它念珠菌为热带念珠菌、近平滑念珠菌、乳酒念珠菌、光滑念珠菌、克柔念珠菌或其组合。

在另一优选例中，所述的探针上，还设置荧光基团，针对不同型的念珠菌的探针其荧光基团不同，从而进行念珠菌的分型。

在另一优选例中，所述的可检测信号包括(但不限于)：碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶。

在另一优选例中，(3)中，所述检测抗体的可检测信号的检测包括：加入识别所述可检测信号的底物，测定底物的显色情况；较佳地，采用化学发光法测定底物的显色情况。

在另一优选例中，所述的方法是非疾病诊断性的方法，也即为不以获得疾病诊断结果为直接目的的方法。例如，将所述方法用于离体的或来自于自然界(如土壤、植物、某些水果、奶制品)的念珠菌的检测。

在本发明的另一方面，提供一种用于检测念珠菌的检测试剂盒，其中含有：

(1)特异性识别念珠菌DNA中目标DNA的探针，所述的探针能够与目标DNA结合形成双链杂交体；

(2)检测抗体，其是抗所述双链杂交体的抗体，其携带有可检测信号；

(3)固相载体及捕获抗体，其是抗所述双链杂交体的抗体；所述捕获抗体与所述固相载体是分离的或所述捕获抗体包被于所述固相载体；和

(4)RNase A。

在一个优选例中，所述的念珠菌包括：白色念珠菌，热带念珠菌，近平滑念珠菌，乳酒念珠菌，光滑念珠菌，克柔念珠菌。

在另一优选例中，所述的所述的探针被分组保存，其中，(a)的探针作为一组，(b)～(f)的探针作为一组，两组分别在独立的体系中保存。

在另一优选例中，所述的检测试剂盒中还含有核酸解链试剂，如碱处理试剂。

在另一优选例中，所述的检测试剂盒中还含有洗涤试剂。

在另一优选例中，所述的检测试剂盒中还含有识别所述可检测信号的底物。

在另一优选例中，所述的检测试剂盒中还含有显色剂。

本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。

附图说明

图1、本发明的方法的检测原理示意图。

图2、RNase A对于非特异性杂交的杂合体的特异性降解作用的示意图。

具体实施方式

本发明人经过深入的研究，提供了一种念珠菌检测及分型方法。一方面，本发明揭示了一种优化的基于DNA-RNA杂合体的检测方法，包括在检测体系中添加RNase A，以其降解非特异性杂交的杂合体，提高检测的灵敏度和准确性。另一方面，本发明揭示了对于多种念珠菌具有特异性检测/分型功能的试剂盒。结合核酸杂交化学发光免疫技术，本发明人开发了可实现对多种念珠菌进行检测和分型的技术。

术语

如本发明所用，“待测样本”或“待测核酸(DNA)样本”是指待检测的核酸样本，其中含有一种核酸或多种核酸，需要了解其中是否存在与念珠菌相关的目标核酸。

如本发明所用，“目标核酸(DNA)”是指感兴趣的核酸，例如其是一种与念珠菌相关的核酸。

如本发明所用，“DNA-RNA杂合体”是指一种核酸，其含有两条链，其中一条链是DNA链，另一条链是RNA链，所述的DNA链和RNA链的核苷酸序列是互补的。

如本发明所用，“探针”是指一种具有已知核苷酸序列的单链核酸(本发明中优选RNA)，其具有与目标核酸基本上互补的核苷酸序列结构，可以与“目标核酸”形成双链。所述的“探针”可以携带荧光基团或不携带荧光基团。例如，荧光基团可以连接在探针的5’末端或3’末端。

如本发明所用，“捕获抗体”是指可被包被在固相载体上的，特异性地识别和结合所述的DNA-RNA杂合体的抗体，其不结合单链的核酸(包括DNA或RNA等)。所述的“捕获抗体”通过识别双链杂交体的双螺旋结构而实现与双链杂交体的结合，而并非是碱基序列特异性的。将抗体包被在固相载体上是本领域技术人员熟知的技术。术语“捕获抗体”可以与“包被抗体”互换使用。

如本发明所用，“检测抗体”或“酶偶联抗体”是指特异性地识别和结合所述的DNA-RNA杂合体的抗体，其不结合单链的核酸(包括DNA或RNA等)。所述的“检测抗体”通过识别双链杂交体的双螺旋结构而实现与双链杂交体的结合，而并非是碱基序列特异性的。所述的“检测抗体”携带有可检测信号，用于报告双链杂交体的捕获情况。

试剂盒

念珠菌具有多种多样的菌株种类，不同的种类致病性或有害程度有显著性的不同。因此，鉴定待测样本中是否存在念珠菌以及念珠菌是否是高危类型，对于自然界中念珠菌的检测、分类，或临床检测、预后、指导用药方案是非常关键的。但是，鉴于念珠菌的种类较多，且不同种类的菌株之间基因组序列的同源性也非常高，如何准确地、便捷地(最好实时地)判断出特定类型的念珠菌一直是本领域高度关注的课题，目前临床所用方法实际上存在较高比例的假阳性和假阴性现象。

本发明人经过深入的研究后，制备了可针对多种念珠菌进行检测的检测试剂盒，所述试剂盒中，包含可特异性识别多种念珠菌的RNA探针。所述的探针是在经过本发明人广泛筛选后优选的，长度合适且易于人工合成，特异性良好，不会与本发明的目标念珠菌以外的其它念珠菌种类发生交叉反应。

作为本发明的优选方式，所述试剂盒中，所述的探针被混合，形成两个探针组：针对白色念珠菌的SEQ ID NO:1，SEQ ID NO:2探针，针对近平滑念珠菌的SEQ ID NO:3探针、针对乳酒念珠菌的SEQ ID NO:4探针、针对光滑念珠菌的SEQ ID NO:5探针、针对热带念珠菌的SEQ ID NO:6探针、针对克柔念珠菌的SEQ ID NO:7探针。从而，可实现将念珠菌分为白念珠菌，或其它念珠菌；该其它念珠菌为热带念珠菌、近平滑念珠菌、乳酒念珠菌、光滑念珠菌、克柔念珠菌或其组合。如此设置，可实现对耐药性较高的菌株与其它念珠菌的准确区分，这对于临床应用是非常有用的。

尽管上述的分组模式是本发明所优选的，作为本发明的其它实施方式，可采取其它念珠菌分组检测形式，本发明也不限于其它一些根据检测所需的分组检测形式。

作为本发明的实施方式，所述的探针试剂中除了含有特异性RNA探针之外还包括一定浓度的盐离子组份，如柠檬酸三钠，磷酸二氢钠等，保证特异性RNA探针和单链DNA在含有一定盐离子浓度下根据碱基互补配对进行杂交。

作为可选择的方式，探针试剂中的念珠菌RNA探针也可以是携带标记物的，如生物素，地高辛，他克莫司；相应的捕获微孔板上包被的为亲和素，地高辛抗体，他克莫司结合蛋白；或者相应检测试剂中为亲和素标记的酶，地高辛抗体标记的酶，他克莫司结合蛋白标记的酶。

作为可选择的方式，探针试剂中的念珠菌RNA探针也可以连接荧光基团，从而便于在需要时进行进一步的分型。

作为本发明的优选方式，所述的试剂盒中，还包括捕获抗体和检测抗体。

所述的捕获抗体与所述的检测抗体可以应用相同的抗体或不同的抗体来制备，即检测抗体在不携带可检测信号的情况下可以是与捕获抗体相同的或不同的。以DNA-RNA杂合体作为抗原与以蛋白质作为抗原不同，抗DNA-RNA杂合体所产生的抗体无特定的序列或抗原决定簇的要求，其特异性识别双链杂交体所特有的双螺旋结构。抗DNA-RNA抗体(无论是单克隆或多克隆抗体)能与任何DNA-RNA双链杂交体相结合。利用特定的双链杂交体来制备抗所述双链杂交体的抗体的方法是本领域已知的技术，例如可以按照Kitagawa&Stollar的方法(Kitagawa Y,Stollar BD,Mol Immunol 1982，19：413-420)来制备多克隆抗体；或可以按照Fliss等的方法(Fliss I,Laurent M,Emond E,et al.,Appl EnvironMicrobiol,1993,59(8):2698-2705)来制备单克隆抗体。

作为本发明的优选方式，检测抗体的溶液中还含有一定浓度的NaCl、MgCl₂、ZnCl₂和Tris-HCl，保证检测抗体在含有一定盐离子的溶液中和DNA-RNA杂合体结合时使检测抗体的非特异性吸附降到最低。

本发明对所采用的固相载体没有特别的限制，只要其能够与包被抗体相结合(偶联、连接)即可。例如，所述的固相载体选自：微量滴定板(如96孔板)、载玻片、试纸或微球。将抗体包被到固相载体上的技术也是本领域技术人员所熟知的。

所述的可检测信号是连接或偶联于检测抗体上的、用于报告检测抗体的结合情况的报告分子。优选的，所述的可检测信号选自：碱性磷酸酶(AP)，辣根过氧化物酶(HRP)，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶。这些可检测信号具有特定的底物，在与底物接触后可以发生显色反应或其它可被检测到或可见的反应，从而报告检测抗体的结合情况。所述的底物比如：用于碱性磷酸酯酶的对硝基苯磷酸酯(p-nitrophenyl phosphate，p-NPP)、CDP-Star；用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS；等等。

所述探针包括RNA探针和单链DNA探针。

所述的固相载体包括但不限于：多孔板、微球、磁珠、试纸、玻片。

所述的各探针可分离地存在，或可按照检测需要进行混合。将针对白色念珠菌的DNA互补RNA序列设为探针A；将针对近平滑念珠菌的DNA互补RNA序列、针对热带念珠菌的DNA互补RNA序列、针对乳酒念珠菌的DNA互补RNA序列、针对光滑念珠菌的DNA互补RNA序列、针对克柔念珠菌的DNA互补RNA序列混合设为探针B。

所述抗体偶联物包括但不限于：碱性磷酸酶、辣根过氧化物酶、生物素、亲和素、地高辛、量子点、荧光基团(FAM、HEX、CY3、ROX、CY5等)。

作为本发明的实施方式，所述试剂盒中，还可包含阴性质控品(NC)、念珠菌质控品。尤其可还包括念珠菌低值质控品。

为了便于操作，所述的试剂盒中还可含有进行核酸解链处理、洗涤、显色等操作所需要的试剂。用于解链处理的如变性试剂(如碱处理试剂)，用于显色处理的如指示剂染料。

作为本发明的优选方式，所述试剂盒中，还包括样本保存液，所述的样本保存液主要成份为含有一定浓度盐的TE缓冲液，使核酸稳定地保存在TE缓冲液中。

作为本发明的优选方式，洗涤液为一定pH值的缓冲液，具有减少非特异性吸附的作用。

本发明的检测试剂盒特异性高、灵敏度高，成本低廉，无需DNA扩增，无需特殊试验条件，操作简单易培训，使实验污染的降到最低，可实现对多种念珠菌进行分型检测。

此外，所述的试剂盒中还可包括使用说明书等。

检测方法

本发明的方法基于杂交捕获技术，但抗体捕获的对象是特殊的，即由DNA与RNA互补形成的DNA-RNA杂合体，而非常规的蛋白质。当使用RNA探针时，首先将样本中的念珠菌双链DNA变性分解成单链，单链DNA与特异性RNA探针结合为DNA-RNA杂合体，DNA-RNA杂合体与固相载体微孔板上的特异性抗DNA-RNA抗体结合被固定在固相微孔板上，同时与偶联有可检测信号的抗DNA-RNA杂合体的特异性抗体结合，通过检测该可检测信号，定性检测样本中的念珠菌核酸。同时，通过使用不同的探针，可以对白色念珠菌和其他多种念珠菌进行区分。本发明的原理的一种实施方式示例于图1中。

常规的杂交捕获的技术方法中，变性、杂交、捕获、检测、底物发光等每一步按顺序进行。本发明中，本发明人对捕获和检测两个步骤进行了合并，在灵敏度不变的前提下减少了操作步骤和耗费的时间。

因此，作为本发明的优选方式，单链DNA样本、特异性识别目标DNA的探针以及检测抗体、RNase A共同加样到包被有捕获抗体的固相载体，进行振荡反应，包被在捕获微孔板上的抗DNA-RNA杂合体的特异性高亲合力单克隆抗体对杂交液中的配对的DNA-RNA杂合体进行捕获。

本发明人意外地发现，RNase A的添加可对非特异性杂交产生的DNA-RNA杂合体上的单链RNA部分进行切割，减少抗体对其的结合。其工作原理如图2所示：当DNA-RNA杂合体正常杂交时，其不对此类杂合体进行降低；而当DNA-RNA杂合体为非特异性杂交的情形时，RNase A可对其进行降解。也即，RNase A的运用大大提高了检测的准确性，以及提高了检测的灵敏度。

在本发明的具体实施方式中，本发人在使用现有的杂交捕获技术时，A组探针对高浓度的热带念珠菌Candida tropicalis、B组探针对高浓度的白色念珠菌Candidaalbicans存在一定程度的交叉反应。而使用添加RNase A的改进的方法时，这些交叉反应信号消失了。

本领域人员已知，抗DNA-RNA杂合体抗体不识别单链或双链RNA、一般认为此类体系中无需添加RNaseA来消除多余的RNA。然而，本发明人却发现，RNaseA的添加可有效地对非特异杂交的DNA-RNA杂合体进行消化切割。在捕获、检测两步分离时，固定的捕获抗体已经结合了非特异杂交的DNA-RNA杂合体，此时再对其进行消化切割没有意义；而捕获、检测同步进行时尚处于游离状态的非特异杂交的DNA-RNA杂合体被RNaseA先一步切割，从而减少了捕获抗体对其结合的能力，进而减少了非特异性信号的产生。

作为本发明的一种优选实施方式，所述的RNA探针中，本发明人将针对白念珠菌的两种探针混合为A组，用于检测白色念珠菌Candida albicans；将针对另四株念珠菌的探针混合为B组，用于检测近平滑念珠菌Candida parapsilosis、乳酒念珠菌Candida kefyr、光滑念珠菌Candida glabrata、热带念珠菌Candida tropicalis、克柔念珠菌Candidakrusei。两组探针分别对同一样本(两份)进行检测，根据各自的阴阳性结果可以判断样本中含有的念珠菌类型：

A组阳性、B组阴性：判断为含白色念珠菌；

A组阴性、B组阳性：判断为含近平滑念珠菌、乳酒念珠菌、光滑念珠菌、热带念珠菌、克柔念珠菌中的一种或多种；

A组阳性、B组阳性：判断含有白色念珠菌的同时，也含有近平滑念珠菌、乳酒念珠菌、光滑念珠菌、热带念珠菌、克柔念珠菌中的一种或多种；

A组阴性、B组阴性：判断不含有上述6种念珠菌，或含量低于检测限。

本发明的方法可无需采用对实验室环境要求高的PCR技术，克服了PCR技术中实验污染不易控制、假阴性高、存在的竞争抑制以及受样本保存及DNA提取过程等影响的缺点。

与一般镜检法相比，本发明灵敏度更高，结果客观，且可以区分白色念珠菌和其他种类念珠菌。与培养法相比，本发明时间短，仅需4小时左右即可得到结果。与核酸扩增法相比，本发明无需核酸提取，无需核酸扩增实验室，对操作人员要求低。

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。

材料和方法

DNA-RNA杂合体(用于制备抗DNA-RNA杂合体抗体)的制备

Poly(A)和Poly(dT)购自美国Sigma公司。在0.15M NaCl 0.015M柠檬酸钠pH7.0的溶液中混合Poly(A)和Poly(dT)，两者浓度均为10μM。65℃孵育60分钟，即得DNA-RNA杂合体。

抗体的制备

前述制备的DNA-RNA杂合体与甲基化的牛血清白蛋白(methylated bovine serumalbumin,即mBSA)混合后再加Freund氏佐剂(Complete Freund’s Adjuvant)注射到山羊体内。第二次开始使用Incomplete Freund氏佐剂。山羊抗体用常规免疫纯化方法进行纯化制备。

本发明以下实施例中所用的鼠抗DNA-RNA杂合体单克隆抗体购自美国Z-BioMed,Inc.公司。该抗体的细胞株(S9.6)来自美国ATCC(#HB-8730)。

将鼠抗DNA-RNA杂合体单克隆抗体与碱性磷酸酶(AP，即Alkaline Phosphatase)偶联，获得抗DNA-RNA杂合体抗体-碱性磷酸酶偶联体(AP偶联抗体)。

利用上述方法，可获得鼠抗DNA-RNA杂合体单克隆抗体；鼠抗DNA-RNA杂合体单克隆抗体与碱性磷酸酶(AP)的偶联体。

山羊抗DNA-RNA杂合体抗体的包被

首先将山羊抗DNA-RNA杂合体抗体包被于96-孔板(Costar High Binding WhitePlate高结合白色板)上。每孔用100μl PBS(10mM磷酸缓冲液，pH 7.4，150mM氯化钠)含有0.5μg/100μl山羊抗DNA-RNA杂合体抗体，4℃，包被16小时。包被后的96-孔板用300μl PBS清洗3次后用含有3％BSA的300μl PBS进行封闭处理。

实施例1、RNA探针序列设计

本发明人设计了一系列适用于进行白念珠菌、近平滑念珠菌、乳酒念珠菌、光滑念珠菌的检测探针，具体如下：

1.1Ca-RNL探针(SEQ ID NO:1；针对白色念珠菌Candida albicans)

AGACAAAGUAGAAAGGAGUAACCGAUCUAAGCUAGAAAUAGUUAAAUAAUAAGGAUAUGAAUUGAAACAUCUUAGUAAUAUCUAAUAGAACCAACAGGGAUAAUAAAAAUACCGGUGAGGGAAAUUAUUAUUAAGUAUAAAAGUAAUACCAUGAGGUAUGAAGAUAGUAGUGCUACUACUAAUACGAAUAAGAGAUAUUAAGUUAUAAGUACCGUGAGGGAAAGUGGUAAGGGAGUUGUUAUUGACCGCCCAAAGGUAUUUAAUACCUUAGGGCGGUGAGUAUAUAAUAUACCCUGUAUAAACACUGAUAUAAGGAGCAGCUAUAGUAUAGCGUACCUCUUGUAUAAUGGGUCAGUGAGUCAUAUUGAAUAGCGAAAUAAAUAAUUAUAUAUAAUAAGUAUAAGUUAUUGGAUAUGGACCCGAAAGUAUAUGAUCUAUACAUGGUCAGGUUAAAUAGUAGAGAGACCCGCCCUUAGGGGCGGGUGCUAGAUUAUUUAGACCGAACAGGUGGGUGUAGCAAUACCUUCUGAAGAACUGUGUAUCGUUGUGAAAGGCUAAUCUCAUAUACAGAUAGCUGGUUAUCCGUGAAAUAUAUAUUAGUAUAGCCUUUAUUAAUUAUUUACUUGGUAAAUCACUAGAUAUAUGUUGGGAGUAAAUAUAGAACUUAUCAACUGUAUCUAAUAUCUGAACAAGUUAACAUCUCACCUAUAAGAUGAGUGUAUAAUUAAAUAAAGAGUCAGUCUUUAUGCGAUAAGGUAUAAGGACCAAAAGGGAAUAGCCUAGACUGUGAAAUGAAGGUCCCUAAUUAUAGUUAAGUGGAUAUAAUCCCAUGGUAUUAACCAUACUCUAUUUAAGCAAUUAGUCAGUUGAUAUGAUAGUAAUCAUCUGUUAAUGAACGUGUAACAACACACUAAUCGAAUAUAAAUGAAUAGAGGAUGGGAGUAGAUAAUCGGGCCUAUAACUAUAGACCAAAUUACAGAUCCACCCGACCGGAGGUCGGGUGGAUGGUAACGGAUCAUUCUAUGUAUACAAUAGAACAGAUAAUGCUGACUUGAGUAACGUUAGAUAGUAUGAAACUAUCCCCAUAAUGCAUAAGGUAUUACUACUAAUUAACGGCCUCUAAUAUAUAAUAUGAAAAUAUUAUAUAGAUGAGUAAUAUAACUAGAAAUUAAUAUAACCGUACUGUAGUCCGACACUGGUGUGCAAUAUAAUUAAUAUAUAUAUAAUAAUAUAUAAUUAUAUAAUGGGUAUGAGAUAAUCCCUGUGAAUGAACUCGGCAAUUAAGGGUAUAACCUUUAUAAUUUAGUGCGACUGUUUACCAAAAACAUAGCCUACUGCUAACAUGGAAUGUAAGUAUAGUAGGUGAUACCUGCCCGGUGCUAGUAAGAUAAUUAAUAAUAUACUGGUUAACGGCGGUCUUAUCAUGAGGAUCCGAAGGUAGCGAAAUUCCUUGACGUAUAAUUGGCGUCCUGCAUGAAUGAUUACACGAUGCUAUAGAUGUAUCCACAGGGAGCUCAGUGAAAUAGUACUUGCAGUGUAGAUACUGUAUGUAGCAACAGGUUGACGAGAAGACCCUAUGCAGCUUUACUAUAAGACUAUAUUGUUACAAUAUUAUAAUUUAAGUAUAUAAAAUAAUUUAGCUUAUAUAUAUAAUAUAUUUAACUAACUAUACCUUCAGGUAUAGGACAUUAUAGUCUGGUUAGUUUCGAUGGGGCGUCGGCAUCACACAAAAUAACUGAUGUGUCUAACUGAUAAUUAAUAUAAACAUUCUAUUCAUUAUAUAAGAAUAUGAAUAUAAUAGUAUGACCAAGUACAGUAAGACUAGAUUGUAACUAGUACAUAAUAAUAUGUAUAUAACAUAUAUUAAAUGAUAUGGAUAAUAACCUAACUGUGCUAUGAUAAGGAGACUUACAAGUCGACUUAGUACGUAAGUAUAGUAUAAUGAACAGGUGAUUUCUUCAGCAUAGAGUGCCGAUAACGGAUCAAAGUUACGCUAGGGAUAAACUGUUAGUCCCUUUAUAUAGAGAUAUAUAGAAUAACAAUCGGGUGAAUUGCAAGAAUACCACCCACCUCCUUUGGAGGUGGGUGACAAUUUGCAGCGAAGUAUAUAUUAGCUUAAUAAUAAAAUAUUAUUUAGAUAUAUAAACGUUCAACGACUAGAAGGUGAGUAAGCUAACAAUAACCCUUCCACGAGCGCCCGACAUCUUAAUGAACCAACAAUUACAAUUAAUUAAAGGUCAUUAUGAUGAUGACAUAGUCUGAACUAAAUAGUGAUAUUUAGAUAUAAUAUUUAAAUGAUAUUAUGAUAACAAAAUUGAACAGGGUAAUACCGCUAGAGAGUUGAUAUCGUCAGCGGUGUUUGCCACCUCGAUUAGAUAUAGUCGAGUAUAAAUGGGGUGAAUUGCAAGAAAGUCUAAGGGUCAUAACCACCCGACCUUCGUGGAAGGUCGGGUGGUAUUAUGGAAGGACCUAUGAUGACUUGCAGCGAAGUAUUAUUUAUACAUAUAACUCAACUCCUUUGGAGUUGGGUUAUAAAGAAUAAUAAACGUUCAACGACUAGAGAGUGAGUAGUAUAACAAUAUCUCUCCACGAGCGCUUCAGAAUAUAUAAGUAUAUAUAUUUAUGACAUAGUCUGAACUAUAAUGAAAAUUAUAGAUAUUAGUAUAAAGAGGCUAAUGAUAACAAUAUUGGUCGUCUAGGCUCAACCUCACGGUCGCAGCAACUGUGAAGGGUAGGACUGUUCGUCCUGUAAUGAGCUACUUGAGAUGGGUUAAUUACGGCGUGAGCCAGUAAUGAUUCUAUCAUCUGUUGCAUGUGGCUCUGCUCUAUUAUACGUGUACGAGAGGAUGGUAUAAUAUAGUCCUCUGGUGUAUAACUAGUAAUAUAAGUUAUAAGCUAAGACUAAUAAGGAUACGUAUUGACUGCAUAUCAAAUACUAAACCUAGAGUACCGCCACAUAAACAUUGAA

1.2Ca-Com探针(SEQ ID NO:2；针对白色念珠菌Candida albicans)

GUCCAUAGUUAAGAGCACCCUGGUAACCCCGUUUGCUAAUAGCACAACCAAUUGAAGCUGGUAUUUGGUGGCUCUGGUGUCAAUUUAUAGCCAACAAUAAACAUUUUCAAAUCCGUCUAAACCGGUCAAAAGAAGAGUUGAGCUUCCAUCUCUGGGUCAAAAAAGGCCGUUUUGGCCAUAGUUAAGACCACCCCCUUUCUGUAGCACAACCAAUUGAAGUUGGUAUUUGGUGGCUCUAGUGCCGAUUUGUAGAGUCAAGUUAUAGUGUUUGCAUCCGAGAGUUUUGAUUUAUUCAGUGUUGUUUUCAUUGGUUGAGGGCAAAAAAUUCGCAUCGAGCAGAAAAGGUCGUGCCCGGGGCAUAGUGGAUAGACAACGAUUACUGGUGAACCACAUGUGCUACAAAGACCAAACUAGGGCCGUUUUGAAGCUACAAUCAUGUAGAGUAUUGGGUGUGAAUUAGGCAUGAUUCGGAUCAAAAUUGGUGAAGCUAUUGAAGAAAACGUUUUCUCCGUGGAAAUGUGAAAUUAACUCCGCUAAGGCUGACACAGUCAGUUUCGAUGCUAGAAAGACCCAACUAGUGCCAAUUCAUAGCCAUAAGAUGUAAGUACUAUGACUGUAAGAGCUGUUAGAAACAAGGUUCAACUGCUUUCUGUAGAACAAAAAAGGCCGUUUUUGCCAUAGUUAAGGAAUUCGCGGUGAUGUCCGUUGAAGACUGCGCGAUGAAAAAUAACGCUACAAAAAUCAAACUAGUGCCGAUUUAUACCUUUGGAUUAUGAGUUCUAUCCCUGCAAGAACUGUUAGAAACGAAAUACAACUGUUAUCUGUGGAACAAAAAAGGCCGUUUUGGCCAUAGUUAAGGGAGCCGCAGCUAUGUCUGAUCACAACUACGCGACCAAAUUCAACGCUACAAAAAUCAAACUAGUGCCGAUUUAUACCUUUGGAUUAUGAGUUCUAUCCCUGCAAGAACUGUUAGAAACGAAAUACAACUGUUAUCUGUGGAACAAAAAAGGCCGUUUUGGCCAUAGUUAAGGGAGCCGCAGCUAUGUCUGAUCACAACUACGCGACCAAAUUCAACGCUACAAAAAUCAAACUAGUGCCGAUUUAUACCUUUGGAUUAUGAGUUCUAUCCCUGCAAGAACUGUUAGAAACGAGAUUUAACUGCAUCCUGUGGAACAAAAAAGGCCGUUUCGUCCAUAGUUAAGACGGAAAAAUUAUGUAUAUUGUUGACAGAAGAUCGAAUUUGAAUGAGUUAAUGACAAGGCUAGUAUCGAUUUGGAACCACAAAAUGUGAGUGUCAAAGCCGUGGGAUACUGUUAGAAAAGAGAUACAGCUGCAUACCGUGGGACAAAAAAGGCCGUUUUGGCCAUAGUUAAGAAUCACUCGUGUUUUUAUUUUGACUACUGCUCGACCAAUAGAGGCGUUACAAAGACCAAACUAAUACCGUUUUGAAGCCAAGACAAAUGUUUAUUAUACGCACAGAUGGUGAUUGAGGUAGGCGAACGAUUGGAGAUGCUACAACAAAAAAGGCCGUUUUUUUAUCCCUUUUUUCCUAACAAAUUUUCGACGAAAAACACCAAGAUGCCUUGCCACCUAAAUCCGUUGGACAGAAAAACAUCAAUUUUGAUGCCAGCAAGCUCGUUCAAAGAUCAAUUUGAAGCUACAUGUUUGGGGAAGAAACCACUAGAUUUUGAAAACAAUCGAUGGUAUAUUCGAAGCUCCAUUGCGAUUAAGGUGGAUAUUUACAUAGUGAAAAUUGGGGGUUGUCUGUACAGCAACUUUUUGUUAGACCAACUAUGGCUUUUUUGGUAUCAAUCUGUAGCCACCGUUUUUUAGGUAUAUGCCCAGUAAUAUGGUGAAUGUAGAUGAUGUAUUGAAUGAACACGCCAUGUUUUUAGUUUUGGUCAAUCGUUUUUGAAAAAUCGCUACCAAAUGGGCCAGAAUUGUUAGGAAAAGUCUAAAAUCGUUAAUAACUCUCCCAAUAUAGGUGCUAGACUUCCCAAAGUCGGGUCACUUUAAAGGCGCAUUUUGUAAGCAACUGUCAAUCUAAGUUUGGUCGAAGGAAGUCCAAUAUUGGUGGAGAUAUUGUCAAAAAACCAAUAUUUUCAAGUUGCAUCCAAACUACAAUAGUUAAGAAGGAAAGUUGAAGUGUGGGAGGUAGAGGAGGGCUACAAGAGCCAAUCAGGUGCCGAUUUGUGGCCUACAGUUGUGGUUUUAAAACCCGAGAGAAUCGUUAAUAGCGGCAAUUAGUUGGGUGCUG

1.3Cp探针(SEQ ID NO:3；针对近平滑念珠菌Candida parapsilosis)

GUAUCGGUGAGAGAAUCUGUUGUUGUAUACAAGUAAUACUUAGAAGUAUGAAUAAAGUAGUAUCUACUACUAAUACAUAAACUAUAACAUAAAGUUAAAAGUACUGUAAAGGAAAGAGUUAAAAAAUCAAAUUAUAUAAUAAAAAUAUAUAUAAGAGAUAAAAAGAAAUAUAUUAAUAAAGAGCAACUAUAGUAUAGUGUACCUCUUGUAUAAUGGGUCAGUGAGUUAUUUUUAUAAGCGAAAUAAGAAUUAUAUAUAAUAAGAUAAAGUUUAUAGGAAUAGACCCGAAAGCACAUGAUCUAUAUAUGUAUAGAUAUAUAUUUAAAAUAAAUAUAUGAUCAAAUAGGUAGAUGUAGCAAUAUCUUCUAAAGAUAUGUGUAUAGUAGUGAAAGGCAAAACUGAUGUGCAGAUAGCUGGUUAUCUACGAAAUAUAUGUUAGUAUAGCCCUUAUAAUAGCGUAAACCCUGGUAAAGCACUUAAUACCAUAUGGGAAUAAAUUUAUUUACUAUAUGUAUUAAACCUCUGAACAGGAGUGAUAUGACAUAUAAGGAGUCAGACUUAUUGCGAUAAGGUAAUAAGUCGAAAAGGGAAUAACCUAGACUAUUAAAUGAAGGUCCCAAAUCCUCAUUAAGUGAAUACAAUCCUAUGAGUAGAUAAAUACCUCAAUAUAUUCCAUAAAGGAAUAAUUUUGAUCAGACAAUUAGUCAGUGGGUUUGAUAAUAAUCACCUGUUAAAGAACAUGUAAAAAUGCACUAAUAGAAUUUAUAUCAAAAAAAGAUAUAUAAAUCAAAAGAUAGGAACAAAUAUACGGGUCUAAAUGUAGGAACCGAAUAAUAGAUAUAAUAAAAAAUAUUAUAUAGUAGUAGAUUAUAUAGUGAUAAUAACUAUAAAGACAAUGCUGACUUGAGUAACGAUAGGUAAUUAAGGAUUACCCCUACAAAACACAAGGUUUAACUAUAAAUUAACGGUAUCUAAGUAUAUAUAUGAAGAUAUAUAUAUGAUGAUAGAUAGUACAGGAAAAUGUAGUAACCGUACCGCAGUCCGACCCAGGUGUGUAAAUGAUAAAAAAUAAUCAUAAUAUAAAUCUUAUAACUAAUAAGAAAGAUAUUGAGUAGGAAUGGGUGAAGUAUCAUAAAUGAACUCGGCAAAUAAGGCAAGGCCUGAAUAGUACGACUGUUUACCAAAAACACAGCUUACUGCAAAUAGGAAACUAUCGGUAUAGUAAGUGAUAUCUGUCCGGUGUAAGUUAGAUAAAUAAAUAAUUGACUUAUAAACGACGGUCUUAUCAUGAGGAUCUGAAAGUAGCAAAAUUCAUUGGCAUAUAAUUGGUGUCCCGCAUGAAUGAUUAUACGAUACUAUAGCUGUAUCUAUGGUAUACCCAGUGAAAUAGUAAUCGCAGUGAAGAUACUGUGUAGUCAUAGAUUGACAAAAAGACCCUAUGCAGCUUUACUAUAUUCUUAUACUGUUAUAUAUAUAUAUAUUUAAAUUAAAUAUUUUUAUUUAUAUGUUUAUUUCUUUAUAUAUAUAACUAACCUUUAUUAAGGUACCAUAUAAGAUGGUUAGUUUCGAUGGGGCGUCGGCAUCACAAAACAUAACUGAUGUGUCUAAAUGAUAUUUUAUAUUCCCUUAUAUUUUAUUACUUUUUAGUUUUAUUUAUAAGGCCAUCAUCUUUUUAAGAUAAUAAUAGAAUAAAUAAGUACAGAUACACUAGUUGGCAACUAGUAUUAAUUAAUAUAAUAUUAUUUAUUAUAUUAUUCCUCAUUUUUGAUGUGGAAUCAUAUAUGUAAUACAUAUAUAAUUAAUUUUAUUUAUAAGGAUAAUAACAUAACUGUGCUAUGAUAAUGACACAUACUUGUGGAAUUAGUACUUAAGUAUAGUAUAUUGAACAGGCAAUAUCAUUAGAAUUGAGUGUCGAUUACGGAUUAAAGUUACGCUAGGGAUAUUAAGUUGUCCCCGUAUAUCGUGAGAUAUACUUAAUUAAUAGGGUUAAUUACAAAGAUAUCUUAUUUUCAUAAGACAAUUUGUAGCGAAGUAUAUAUUAGUAUUUAUAUAUAUAUAAACGUUCAACGACUAAAAGAUGAGUUUACUAACAAUAAUUCUUUCAUGAAAGCCCUACACCUAAUUUAAUUUUAGGUGAUGACAUAGUCUAAUCUAUAUAGUGAUAUAUAGAUAUAUAUAUUAAAUAAUAUAUAUGUUAAUAAUAAAUGAACAGGGUAAUAUCGCUAGAGAGUUGAUAUCGUCAGCGAUGUUUGCCACCUCGAUUAAAGAUAGUCGAGUAUAAAUAGGGUGAAUUGCAAGAAAAUCUAAGUAUAUAUAUAUAUAUAUGAUAAUUUGCAGCGAAGUUAUGUUAAAAACAUAAAACGUUCAACGACUAGAAAGAAUAAUAUCUUUCCAAGAGAGCCCUAAAUUUACAGUAUAUAAAUAUAUAAAUAUAUAUAUAUUAUAAGAAUUGUAAAUUAUGACAUAGUCUAAACUAUAAUGAAAAUUAUAGAAAUAUAUUUUAAUAAAAUAUAUGUUAAUAAAAUUGGUCGUCUAGACUUAUCCUAUUGGUCGUAGAAGCUAAUAAGGGUACGACUGUUCGUCGUUUAAAAAGUUACUUGAGAUGGGUUAAUUACGACGUGAGUCAGUAAUGAUUUUAU

1.4Ck探针(SEQ ID NO:4；针对乳酒念珠菌Candida kefyr)

CAGAGAUAUUAUGAGUAUUGGUGAGAGAAAAUAAUAUAGUCAAAUUUAUAUAUUUAUAAUAUAUAUAAAUAAAUAAGUAUUAUGUAAAUAAAUUAUGUAAGAAUAUAGGACAAACUAGUUCUAAGACUAAAUACUAUUAAUAAACAAAAUAAGUACCGUAAGGGAAAAUAAGUAAUUGGUUAUUUUAUAAGCAGUUAUAAAUAUUUUAUAUAAAUAUUAAUAACGUACCUUUUGUAUAAUGGGUCAGCAAGUAAUUAUUAUUAGCAAAAUUAUUUUAAUAUAAUUAUAAAUAAUUUGAAUAUUAAACAUUGUAUAAUAUAAUAAUAUAUAUUUAAUAUAUAUUAUAGUUAGUUAAUGAUAAUUGACCCGAAAGCGGACGAUCUUAAUAUGACCAGACGGUUAAAAACGGUCGAACAGGUUGAUGUUGCAAUAUCAUCUGAUGAGUCGUGUUAAGCGGUGAAAGACAAAUCUGGUUCGCAGAUAGCUGGUUUUCCACGAAAUAUAUGUUAGUAUAGUCUCUAUUAAAUAAUUAAUAUUAUUAUUAUAUUUUUUAUAUAAUAAUAAAGAAUGGUACAGCUAUAAAUAUUAUUAUUUGGGGAAUAAUUGCUAUUUUAUCAAAGAUAUUUAAUCUCGAAAUGUUUAUUAAUAUUAAUAGAGAAUCAGACUGUGCGCGAUAAGGUGAACAGUCUAAAGGGAAACAGCCCAGAAUAAGAUAUAAAGUUCUUAAUUAGAUAAUAAGUGAAAUAAAUAUUUAAAUAUUAUAAUACAAUCAGUUAAUGGGUUUGACAAUAACCAUUUUUUAAUGAACAUGUAACAAUGCACUGAUCUAUUUUUUUAUAAAUAAUAUUUAAAACCAUAAUUAUUUUUAAUAAUUAUGUAAAUAAAUAAUAUAACGGAUCUUUUUUAAUUAUAAUUAUCUAACUGAAUAUCUAUAUAAUAUUAUAUAUAAAUAAAUGUAAUAUUAUGGUAGUGGAACAUUUAAUGAAUAAUAAGUAUAUUAAUAUUUACGGAUUAAUAUAUGAUAUAUAAAUUAAAUAGAGAAUGCUGACAUGAGUAACGAAAAAAAGGUUUAAUCCUUUUCACCUAAAGCAUAAGGUUUAACAAUAAAAGUACGGCCCUUAAGUAAAAUAUGAAUAAAAUUAUAUUGUACGAUAGGUAAAAUCUAAAUAUAUAUAUUAUAUCUAUAUUAUAUAUAUUAUAUUAUAAUAUAUAUAAACUAAUAUAGAUCAAGAAAUAGAUAUAUAUACAAAUACCGUAAUGUAUACCGACUCUGGUAUGUUAGUAGAGAAUAUGAAGGUGAAUUAGAUAAUUAAAGAGAAGGAACUCGGCAAAAAUAGCUCUAACGUUAGUCAAAAAAGAGUAAAAAAAACAAGGUUGUCCAACUGUUUACUAAAAACACCGCACUUUGCAAAAACGUUAAGUUAUAGUAUAAGGUGUGAACUCUGCUCCAUGCUUAAUUAAUAAAUAUGAAUAAUUAUUAUGAUUAUUAUAUUAAAUUUAAGUAAAUAGCAGCCUUAUUAUGAGGGUUCUAAGGUAGCGAAAUUCCUAGGCCAAUAAUUGUGGUCCUGCAUGAAUGACGUAAUGAUACAACAACUGUCUCCUCUUUAAGCUAAGUGAAAUUGAAUUCGCAGUGAAGAUGCUGUGUACCUAUAGUAAGACGAAAAGACCCUUUGCAGCUUUACUGUAAUUAGAUAGAUCGAAUUAUUAUGUAUUUUGUUCAGUAUAUUAAGUAAUAGAUUAUAUAUAAAUGAAAUACUUAUCUAAAUAUCUUAAAUAUUAUUUUUAAUAAUAUUUUAUAAUAAUUCUUAUCUUUAUAAUAUAUUUAAUUGAUAUAUAUUCUUAAAUAUAUAUUAUAUUAUAUAAUAUUUAUAUUGUAUAAUAUAAUUAAAGAGAAAAUCUCUAAUUGGUAGUUUUGAUGGGGCGUCAUUUUCAGCAAAAGUAUCUGAAUAAGUCCAUAUAUAAAUUAUAAUGUUAUUGAUUAACAAAUUAUAUAUUAUAUAUAAUAAUUAUUCACAUCUUUAUUUUAUAAUAUGAUUUUAAUUAUAAUAUAAAAUAAAGUAAAUAAUAGAAAAGAUUAUGUACAGUAAAAUUGUCAUGAAAACAAUAAUAUUAAAAAAUAUUAAAUUAAUAUAAUGGUAUAACUGUGCAACAAUUGUAACACUCACAAGUGAAACAAGUACGUAAGUAUGGCAUAAUGAACAAAUAACACUGAUUGUAAUGGUUAUUGAUAACGAAUAAAAGUUACGCUAGGGAUAAUUUUUUGUCCCAUUAUAUAUAUAUAUAUAUAUAAAAAGAUAAUAUAUGUAUAUAUAAUUAUCCCUUUUUUACCUUUUUCAAGGAAAAAAAAGGGAGUAAAUUGGGUGAAUUGCAAGGAACUCCUAUUAUUAUUUAAUAAUAAUUAAAAAAUAAUAAAAUAAAUAAUAAGGACAAUUUGCAGCGAAGCUUAUAAUAACCUAUUUAUUAUAAUAUUAUAAUAUUAUAAUAAAGAUUUUAUAAGAACGUUCAACGACUAGAUAAUGAGUUAGGUUAACAAUAAUUUAUCCACGAGUGCCCAAUAUUAAAUAUUAUUAUAAAUAUUUAAUAAUGACAUAGUCUGAACAAUAUAGUAAUAUAUUGAAAUAAUUAAUUAAAAAUAAUUAUAUAAACAAAAUUGAACAGGGUAAUAUAACGAAAGAGUAGAUAUUGUAAGUUAUGUUUGCCACCUCGAUGUCGACUCUACCUAU

1.5Cg探针(SEQ ID NO:5；针对光滑念珠菌Candida glabrata)

AUAAGACUAAGUGAACUGAAACAUCUUAGUAACUUAAGGAAUAAAAAUCAACAGAGAUAUUAUGAGUAUUGGUGAGAGAAAAUAAUAAUAGGUCGUAAAAAGUAUUAUGUAUUAAUUAAUAUGUAAGAAUAUAGGAUUAACAAUUUCUAAGACUAAAUACUAUUAAUAAGUUAAGAAAGUACCGUGAGGGAAAGUAUGUAAAUGGUUAUUUUAUAAGCAAUUAUAGAUAUUAUAUAUAAUAUCAAUAAUGUACCUUUUGUAUAAUGGGUCAGCAAGUAAUUAAUAUUAGCAAAAUUAAAUAAUUAUAUAAAAAUAGAAAUAAAUAUAUAUAUAAAAAUAUAUUAAGAGUUAAUAUUAAUUGACCCGAAAGCAUAUGAUCUUACUAUGACAAGAUGGUUAAAAACGAUCGAACAGGUUAAUGUUGCAAAAUUAUCUGAUUAGUUGUGGUAAGGUAUGAAAGAUAAAUCUGAUAUGCAGAUAGCUGGUUUUCUAUGAAAUAUAUGUUAGUAUAGCCCUUAUAAUUAAAUAAUUAAUAAUAAUAUAAUAAUUUAUUAUAUUAUUAUUGAAUGGUACAGCAAUUAAUAUAUAUUGGGGAAUUAUUAUAAUUUUAUCAAUAAUAUUUAAUCUCGAAAUAUUUAAUUAUAUAUAAUUAAUUUAAUUAAUUAUAUAAUUUAUAAGGAGUCAGAUUAUGUGCGAUAAGGUAAAUAAUCUAAAGGGAAACAGCCCAGAUUAAUAUAUAAAGUUCCAAAUUUUAUAAAAUAUAUAUAAAAAAAAUAUAUUUAAUUUAUAAAUUUAAUUUAUAAAUAAUAAUAAGUGAAAAUAAUAUUAAAAUAUUAUUAAAUAAUCAGUUAAUGGGUUUGACAAUAACCAUUUUUUAAUGAACAUGUAACAAUGCACUGAUCAAUAAUAUUAUAUAAUAUUUCAAAUUAAAUUAAUUUUUAAUUAAUUUAUUAAUAAACAAUAUACGGAUCUUAUUAAUUAUAAAAAUUAUUAAACUGAAUAUUUAAAUAUUAUUAAUUAUUAUUAAAAUAAUUAAUAAUAUGGUAAUAGAACAUCUAAUGAAAGAGUAUAUUAAUUAAAUAUUAAUAUAUUAAUAAAUAAUUAGAUAGAGAAUGCUGACAUGAGUAACGAUAAAAAGGUUUAAACCUUUUCACCUAAAACAUAAGGUUUUACUAUAAAAGUACGGCACCUAAUAUAAAUAUGAAUAUAAAUAUAUUAUAGAUGGUAAAAAUCUAAUUUAAUAAUAAAAAUCUUUAUAGAUAUAUAUUAUAUAUAUAUAUAAAUUAAUAAAGAUCAGGAAAUAAAUUAUUAAAUGUAUACCGUAAUGUAUACCGACUCAGGUAUGUUAGUAGAGAAUAUGAAGGUGUAUUAGAUAAUUAAGGAUAAGGAACUCGGCAAAAAUAGCUCUAACGUUAGUCAAUAAAGAGAUAUAAAAACAAGGUUGUACAACUGUUUACUAAAAACACCGCACUUUGCAAAAACUAUUUAAGUUAUAGUAUAAGGUGUGAACUCUGCUCAAUGCUUAAUAUAUAAAUUAUAUAUUUAUUAAAGAAUAUAUAAAUAAAUUUAAGUAAAUAGCAGCCUUAUUAUGAGGGUUAUAAUGUAGCGAAAUUCCUUGGCUGAUAAUUACAGUCCCGCAUGAAUGACGUAAUGAUACAACAACUGUCUCAUCCUUAAACUAAGUGAAAUUGAAUUUGCAGUGAAGAUGCUGUAUAUUUGUAGUUAGACAAGAAGACCCUAUGCAGCUUUACUAUAAUUAGAUAGAUCGAAUUAUUAUUUAUUAUAUUCAGCAUAUUAAGUAAUUUAUAUAUAUAUAUAUAUAUAUAAUAAUCGUUUAGAUAUAAAUGAGAUACUUAUUAUAAUAAUAUAAUAAUUCUAAUCUUUAAUAAAUAUAUAUAUAUUUAAUAGAAAAUUAAUAUAUUCUUAAAUAUAUUUUAUAAUAUUAUUAUUAUUAAUAAUAUUAUAUAUUAAGAGACAAUCUCUAAUUGGUAGUUUUGAUGGGGCGUCAUUUUCAGCAAAAGUAUCUGAAUAAGUCCAUAUAUAAAUUAUUAUAAAAUUUUAUUUAUAAUAUAAAGUAUAUAUUAUAUAUACUAAUUAUAAAAUAUAUUAUAUAUUUUUAUAUAUAUAAUAUAAUAAUAGAAAAAUUAUAUACAGUAAAAUUGUCUGGAAAACAAUAAUAUUUAUUUAUUUUUAAUAAAUAUUAAAUUAAUAUAAUAGUAUAACUGUGUAAAGAUUAUGACACAAACAAGUGGAAUAAGUACGUAAGUAUGGUAUAAUGAACAAAUAAUACUGAUUGUAAAGGUUAUUGAUAACGAAUAAAAGUUACGCUAGGGAUAACAGGGUAAUAUAACGAAAGAGUAGAAAUUGUAAGUUAUGUUUGCCACCUCGAUGUCGACUCUACCUAUCCUAUUGGUUGUAAAAGCUAAUAAGGGUUCGACUGUUCGUCGAUUAAAAGGUAACGUGAGUUGGGUUAAAUACGACGUGAGUCAGUAUGGUUUUUAUCUACUACAAAUAAUAUUAUCUUAUUAAAUCUCAUUAUUAGUACGCAAGGACCAUAAUGAAUU

1.6Ct探针(SEQ ID NO:6；针对热带念珠菌Candida tropicalis)

UAGAGUGAGGGGAGUAAGGGAGAGGGAUCCUAAUGGGAGUAGGAGAGGUAAUUGAAAGACUACAUUUAAUAACAAUGUCUAAGUAUGGACGUAAAGACUAUGAUUAACCUAGACAAAGGAAAGGGUCCUUAAUGGGACCGUUCUAAGCUAGAAAUAGUGACGAUAUUGGAGGCAUCUGAAUUGAAACAUCUUAGUAAGAUGCUAGUAAACCAAUAGGGAGCACUUCAGUACCGGUGAGGGAAAAAGUGCUAGUAUAUAAGUAAUAUCAUGUGAUAUGAAUAUAGUAGUGCUACUACUAAUACUAAUGCCUAAUCAUAAGUAACAAGUACCGUGAGGGAAAUACAUACAUUACUGCUUAAUUAAUCGGAAUAUAAUAUUAAGGUUAUAUAAAGACAGUAAACAAAUACUUAGAUAAGGAGCAGCUAUAGAAUAGCGUACCUCUUGUAUAAUGGGUCAGUGAGUUAGCUUCAUUAGCGAAAGAAACAUUGGAUUUAAUCGGAUGAUAUAGUUAAUGGGGCUAGACCCGAAAGUAUAUGAUCUAUACAUGACCAGGUUAUGUAGUUAACACUUUAUUGACUAUGUAGACCUAACAGGUGGGUGUUGCAAUACCCUCUGGAGAGUUGUGUAUCGAUGUGAAAGGCUACUCUCAUAUACAGAUAGCUGGUUGUCUGUGAAAUAUAUCUUAGUAUAGCGAUUAAAUAUGCAUUACUAGGUAAAUCACUAGAUUUGAACAUAUCUCUUAUGUCAUGGACAUUAAUUGAUAUUUAUGGGAGCUAUUAGAGCUUAUCAUACGAAUCUAACAUCUGAACUAGUUAAGUAGCAUUAAUUUGAUCAGUCAAUCUAGGUGCGAUAAGGUACCUAGAUGAGAAGGGAAUAACCUAGAUUACUCGGUGUAAGUCCUUAAUAACAACUAAGUGGUAUUAAUCCUAUCCCUUAAGCAUUAUGUUUAAGAAUUUCUAAUAAGACAGUUAAUCAGUGGAUAUGAUAGUAAUCAUCUGCUAGAGAACGUGUAACAACGCAUUAACUGAUACAUAGAUAGUAGGAGUAGAUAUACGGGCCUAAGUUGUUGACUGAACAGUAAAUAGUUACUCUAAUAGUAACUAUGGUAACAGACCACACCAUACUAUUAAUGGUGCAGAAUAUGCUGACUUGAGUAACGUAAGGAGGGAUAAGCCCUUCCCCUAUAUGUACAAGGUUUUACUACAAAUUAACGGCCUCUAAUCGUAUCUAUGAAGAUAGAUAGGAGAUGAGUAAUAUGACUAGAAAUAGUGCUAACCGUACUGUAGACCGACACUGGUGUACUAUAUCUUGAUUAUAUAAGAUAAAAUAGGGUAUGAGAUAAGCCAUAUGAAUGAACUCGGCAAUAAGAGUAUAACCUCUUGAAUAUAGUGCGACUGUUUACCAAAAACAUAGCCUAUUGCAGUCCCAUAAGGGAUGGUAUAAUAGGUGCUACCUGCCCGGUGCUAGAGAGAUAACACAUAAUGCUUUAGUUAACGGCGGUCUUAUCAUGAGGAUCCGAAGGUAGCGAAAUUCCUAGACGUUUAAUUGGCGUCCUGCAUGAAUGAGUACACGAUGCUAUAGAUGUAUCCAUAUGGUUCUCAGUGAAAUAGUAAUUGCAGUGAAGAUACUGUAUCAGCAAUAGAUUGACGAGAAGACCCUAUACAGCUUUACUAUAGAGUUACCUUGCUAUUCACUACCCUUUAUAAGCCUAUGUACUCGUACAGCUUAGUAAGCGUAGUCCGUAGCUAAUCUCCAUAGAAAUAUGCUUACUUAAUCGUGGCAUAGGAGAGACAUGAUAACUUGGUUAGUUUCGAUGGGGCGUCGACAUCACAAAGAAUAACUGAUGUGUCUAUAAGACUUGUUUGUAAUUAACUCUAGAAAUGGAGUUCUAAUAGUAUAUCCAGAUACAGUGAGGCUAAAUGGUAAUUAGUCAGCUCUACUCCAUUAUACCCGAUUUAUCUAGCAUAAAUAGGAUGGAUAGACAAUGAUAAGGAUAAUGACUUAACUGUGUUAUGAUAGAGGCACUUACAGGUGUCUUUAGUACGUAAGUAUUGUAUAGUGAACAGGCAAUGACUAAAGCAGAGUAUGUCGAUAGCGAAUCAAAGUUACGUUAGGGAUAACAGGGUAAUACCGCUAGAGAGUUGAUAUCGUCAGCGGUGUUUGCCACCUCGAUGUCGUCUAGGCUCAUCCUUAUGGUCGCAGCAACUAUAAAGGGUAUGACUGUUCGUCAUUUAAGGAGCUACUUGAGAUGGGUUAAUUACGGCGUGAGCCAGUAAUGUUUCUAUCAUCUAUUGCACUAUAACAACAGCUUUAUUACAUGUGUACGAGAGGAUUAUGUAAUCUAGACCAAUGGUGAUAACUAAAUGCUUAUUUAAUCGAGUAUUAAGUUAUAAGCUAAGCCUAGCAGCAAUAAGUAUUGACCGCAUAUCAAAUACGAAGUCUAUAGCUGUUUGUUGAACAAAACAUUGAAGAUAACAAUGUGCUAGUGUAAUCGGUUCAUAUGACUGUUACACUGAUCUAUGGAGCAUAUCCAUGUUAAAUGAAGGA

1.7Ckr探针(SEQ ID NO:7；针对克柔念珠菌Candida krusei)

GAGCUACUAAUUAUAGAGGGUAAAAAAGAGUAUAUGAAGAGAUGGAAUAUACUGAGACCUAAAGGAAACUGAGAGAAAGAAACCAAGAGAACUGAAACAUCUAAGUAUCUUGAGGAAAAGAAAUAAACAUAGAUUCUAUGAAUAUUGGUGAGAGAAAAUAGAGAAGUAUGAAUAAGUAGUAGGGGUUAAAAGAUCUACGAAAAAGGGAGAACUAAUCCUAAGACUAAAUAUAAUUAGUAAGUGUAAUAAGUACCGAAAGGGAAUAAUAUAAAUAAGUGAAAUUGAGAGCAAUUAAAGGUUAAUAAAGAAGAAUUAAUUGAUAAUGUACCUUUUGUAUAAUGGGUUAGCAAGUUAUAAUAAAUAGCGAAAAAGAAAGAAUUUGAAAUGAAAAUGAGAGAAGUUAUUAAUUAUAGACCCGAAAGUAGAUGAUCUUACCGUAUACAAGUGGUUAAGAUAUAUAUUAAUAUAUAUAUAUUAAACGACUGAACAGGUUAAUGUUGCAAUAUUAUCUGAAGAUAUAUGGUAAGUAGUGAAAGACAAAUCUGAUCUACAGAUAGCUGGUUCUUUGUGAAAUAUAUUUAAGUAUAGCGUUAAUAAAGAAGUAUAAAUAUGGUAAAGCAAUUAAUAAUAAUUGGGGUAUUGUAAUAAUAUUAUCAAUAUUAUUUAAUCUCAUAAUAAUAUUUAUAUAUAAAUUAAACGUCAGACUAUUAGUGAUAAGAUUGAUAGUCAAAAGGGAAACAGCCCAGAUCAAGAGAGAAAGUUCCAAAUAAUAAUUUAAGUAUAAAUAAUAUUUAUUAAUCGUAAAACAAUAAUGGGAUUAGUUUGACAGUAAUUAUUCUAUGAUCAACGUGUAACAACGGGUUUAUUAUAAAUAUAAUAUAAUAAUAUAUAUUAUAUAAAAGAAUAUAUAUAUAUUCUGAUCGGUAAAUAAAUAGAUAAUAAUCGGAAUUAAAAAUAAUAACUGAAAUCUUGAAUAAUAGAAUUAAAAGAAUAAUAAUAAUAUUAAGGUAACAAAGCAUUUAAUGAAUAAAUAUUUUUCUAAAUGAAAAAUAAGGAAAGAAAGAAAGAAAUAGAAUUAAAUAGAGAAUGCUGACAUGAGUAACGAAUAAGAGGAAAAUCCUCUUCACUUUAUGUAUUAGGUUUUAAUAAAAAAUCGGUCCAUAAGAGAAUUUCUGAAAAGAAAUAAUUAAUAGGUAAUAAAUAAAAAAGUAUACGCCCAUAUAAAAAAAUAUAAGGUAGUAUAUGGUACCGGAAAAAGUAUUUUAUAAAAAUACCGUAAUAAAACCGACACAGGUAUACAAAUAAAAAAUGAAAGUGAAUAAGUGAAUUAUUCUAAAUGAACUCGGCAAAAUAAAAGCUCAGACGUUAGUCAAAAAGAGUGAAUAAAGUACAUGUUAUGCGACUGUUUACCAAAAACACAGCACUUUGCGAAAACGAUAAGUAUUAGAAUAAAGUGUGAAUUCUGCCCAAUGUUUAAUAAUAAAGAAAUAUAUUAAUCGAUAUAUUUAUAAAAAUUAAGUAAAUGGCGGCUUUAUACUGAGAGUCAUAAUGUAGCGAAAUUCCUUGACCGCUAAAUACGGCCCUGCAUGAAUGGAGUAACGACAUAACAACUGUAUCUAGAAUAAGCUUAGUGAAAUUGAAAUCGCUGUGAAGAUGCAGUGUACCUAAAGGUAGACGAGAAGACCCUAUGCAGCUUUACUAUGAUUUAAUAAGUUGUUAUUAUUUGAUAAAAUGUUAGUAUAUAAAGUAAUAUAUUGAUAUAAAAAUGAAAUACUUUUAUUUUAUUCCAAUUUAUUUAUAUAAAUAAAAAUAUAAAUAGAUAUAAAUAAUAACUGAAUUAAUAAUUAAUUAGUAGAAAAAAGAAUAUCCUAAUUAUAAUAUAUUUAUAAAAAUAUAUAUAUUAAUGACAGCUUUAAAUUGAUAGUUUAAAUGGGGCGUUGAUUUCACAAAAAAUAACUGAAUAUGUCCAUAUACAAUUAAAAUUAAAUGAAUUAAUGAAUUAAUCAUUAAAAAUGUUAAAAAUAAUAUGAAUUUAUUAUAAUUUUAGAAAAACCUAUGUACAGUAAAUUUGACCUGACAUCAAAUAAUUAUCUAAAAAAUAAUUUUACUAAAUUAAUAUAAUAGUGGAUAACUGUGCAAUAAUUUAACACUAACAAGUGGAAAUUAUACGUAAGUAAGGUAUAAUGAACAGAUCAUUUUAAUAGUCCAAGUGAUCGAUUAACGGAUAAAAGUUACGCUAGGGAUAACAGUGUAAUAACAUGAGAGAGUAGAUAUUGUCAAUGUUGUUUGCCACCUCGAUGUCGGCUGAGCCUAUCCUAUUGGUAGUAUAAGCUAAUAAGGGUUUGACUGUUCGUCAAUUAAAAGGCUACCUGAGCUGGGUUCAAUACGUCGUGAGACAGUAUGGUUUCUAUCUUCUUUAGGGAUUAGUUAAAAAAGGUAUAUAACUUAGUACGAAAGGAUUAGUUAUAGUUAAUCAAUGAUUUUACUGUUGUAUAUUAUGUAUAAUUAUAAUGAUACUAUCUAUUUAUAAUAUGCAGGCAGUUAAG

实施例2、分型检测的优化

1、实验分组

实施例1所述的RNA探针中，本发明人将Ca-RNL和Ca-Com两种探针混合为A组，用于检测白色念珠菌Candida albicans；将Ck、Cg、Ct、Cp、Ckr五种探针混合为B组，用于检测近平滑念珠菌Candida parapsilosis、乳酒念珠菌Candida kefyr、光滑念珠菌Candidaglabrata、热带念珠菌Candida tropicalis、克柔念珠菌Candida krusei。

A组混合体系包括：Ca-RNL和Ca-Com探针各250ng/mL；

B组混合体系包括：Ck、Cg、Ct、Cp、Ckr探针各500ng/mL；

所有DNA样本购自美国ATCC，为已经完成提取的核酸。经紫外分光光度法测定浓度后，用纯化水稀释至如下浓度：

白色念珠菌10^7拷贝/ml、10^6拷贝/ml、10^5拷贝/ml

热带念珠菌10^5拷贝/ml、10^4拷贝/ml、10^7拷贝/ml

乳酒念珠菌10^7拷贝/ml、10^5拷贝/ml

光滑念珠菌10^7拷贝/ml、10^5拷贝/ml

近平滑念珠菌10^7拷贝/ml、10^5拷贝/ml

克柔念珠菌10^7拷贝/ml、10^5拷贝/ml

2、杂交捕获操作的一般流程

(a)取50μL待测DNA样本，加入25μL 2M NaOH 5mM EDTA，65℃孵育30min，进行解链变性。

(b)在变性后的样本中加入25μL 3M醋酸-醋酸钠缓冲液，pH 4.0和5mM EDTA(其中含RNA探针)，65℃孵育60min，形成DNA-RNA杂合体。

(c)将上述溶液(共100μL)转入固定有抗DNA-RNA杂合体抗体(山羊抗DNA-RNA杂合体抗体)的固相载体(微孔板)中，室温1100rpm振荡60min，从而捕获DNA-RNA杂合体。

(d)弃去微孔板中液体，加入75μL含20ng/mL抗DNA-RNA杂合体抗体-碱性磷酸酶偶联体的0.1M Tris-HCl pH7.4，0.6M NaCl，1mM MgCl₂，0.1mM ZnCl₂缓冲液，室温孵育45min。

(e)弃去微孔板中液体，加入300μL 0.1M Tris-HCl pH7.4，0.6M NaCl缓冲液，清洗6次。

(f)加入75μL碱性磷酸酶发光底物CDP-Star，室温避光孵育15min后于化学发光仪读取信号。

3、杂交捕获的优化步骤A

参照前述一般流程的(a)～(f)，不同点在于：将(c)和(d)步骤合并。具体如下：

将上述溶液(共100μL)转入固定有抗DNA-RNA杂合体抗体的固相载体(微孔板)中，加入75μL含20ng/mL抗DNA-RNA杂合体抗体-碱性磷酸酶偶联体的0.1M Tris-HCl pH7.4，0.6M NaCl，1mM MgCl₂，0.1mM ZnCl₂缓冲液，室温1100rpm振荡60min，形成双抗体夹心复合物。

4、杂交捕获的优化步骤B

参照前述一般流程的(a)～(f)，不同点在于：将(c)和(d)步骤合并，并添加RNaseA至终浓度0.1mg/ml。具体如下：

将上述溶液(共100μL)转入固定有抗DNA-RNA杂合体抗体的固相载体(微孔板)中，加入75μL含20ng/mL抗DNA-RNA杂合体抗体-碱性磷酸酶偶联体的0.1M Tris-HCl pH7.4，0.6M NaCl，1mM MgCl₂，0.1mM ZnCl₂缓冲液，和5μL 3.6mg/ml(终浓度0.1mg/ml)的RNase A，室温1100rpm振荡60min，形成双抗体夹心复合物。

5、结果判读

两组探针分别对同一样本(两份)进行检测，根据各自的阴阳性结果可以判断样本中含有的念珠菌类型：

A组阳性、B组阴性：判断为含白色念珠菌；

定义信号/噪声(Signal/Noise)比例(信噪比)：“样本与空白对照的信号值的比”大于2为检出。

6、实验结果

运用上述改进的捕获-检测一步法，并与杂交捕获操作的一般流程进行比较，针对不同的浓度的样本或空白对照，加入探针B组，结果如表1～表3所示。

表1

表2

表3

现有杂交捕获技术的步骤下，探针B组对白色念珠菌在10^6拷贝/ml时就有阳性结果(信噪比2.09)，说明产生了假阳性。

使用改进的步骤(优化步骤B)，探针B组对白色念珠菌在10^7拷贝/ml的浓度下仍为阴性，无假阳性的现象；同时10^4拷贝/ml的热带念珠菌结果保持阳性，灵敏度高。

热带念珠菌与白色念珠菌同源性高、容易交叉，而利用RNase A之后，则两者可被良好地区分。

发明人分析发现，优化的体系(优化步骤B)中，RNase A的添加可对非特异性杂交产生的DNA-RNA杂合体上的单链RNA部分进行切割，减少抗体对其的结合。其工作原理如图2所示：当DNA-RNA杂合体正常杂交时，其不对此类杂合体进行降低；而当DNA-RNA杂合体为非特异性杂交的情形时，RNase A可对其进行降解。因此，RNase A的运用大大提高了检测的准确性，以及提高了检测的灵敏度。

实施例3、探针A组和B组对念珠菌型别的鉴定准确性

1、实验分组及实验步骤

实施例1所述的RNA探针中，本发明人将Ca-RNL和Ca-Com两种探针混合为A组，用于检测白色念珠菌Candida albicans；将Ck、Cg、Ct、Cp四种探针混合为B组，用于检测近平滑念珠菌Candida parapsilosis、乳酒念珠菌Candida kefyr、光滑念珠菌Candidaglabrata、热带念珠菌Candida tropicalis、克柔念珠菌Candida krusei。

A组混合体系包括：Ca-RNL和Ca-Com探针各250ng/mL；

B组混合体系包括：Ck、Cg、Ct、Cp、Ckr探针各500ng/mL；

白色念珠菌10^5拷贝/ml、10^7拷贝/ml；

热带念珠菌10^7拷贝/ml、10^5拷贝/ml；

乳酒念珠菌10^7拷贝/ml、10^5拷贝/ml；

光滑念珠菌10^7拷贝/ml、10^5拷贝/ml；

近平滑念珠菌10^7拷贝/ml、10^5拷贝/ml。

克柔念珠菌10^7拷贝/ml、10^5拷贝/ml

2、杂交捕获操作的一般流程

同前述实施例2。

3、杂交捕获的优化步骤B

同前述实施例2。

4、结果判读

同前述实施例2。

5、实验结果

运用改进的捕获-检测一步法，并与杂交捕获操作的一般流程进行比较，针对不同的浓度的样本或空白对照，加入探针A组或B组，结果如表4～表5。

表4

表5

探针A组：能够良好地测出白色念珠菌型别，对于其它念珠菌则为阴性。没有发现任何假阳性或假阴性情况。

探针B组：能够良好地测出热带念珠菌、乳酒念珠菌、光滑念珠菌、近平滑念珠菌、克柔念珠菌型别；而对于白色念珠菌，即使在高浓度下也没有检出。没有发现任何假阳性或假阴性情况。

实施例4、念珠菌探针全混实验

本实施例中，将所有念珠菌探针于纯化水中共混，研究本发明的检测方法的特异性和敏感性。

将Ca-RNL、Ca-Com、Cp、Cg、Ck、Ct、Ckr探针全部混合，其中Ca-RNL、Ca-Com浓度为各250ng/mL，Cp、Cg、Ck、Ct、Ckr浓度为各500ng/mL。

利用所述的混合探针进行检测，样本为各种念珠菌DNA10^5拷贝/mL(每种DNA单独实验)。检测方法采用前述杂交捕获的优化步骤B。

结果如表6。

表6

结果显示，所述混合探针可以特异性检出特定的念珠菌，即使将它们混合为较为复杂的体系，检测特异性和灵敏度优异。

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

序列表

<110> 杭州德同生物技术有限公司

<120> 念珠菌检测及分型的方法和试剂盒

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uuuuuaauua uaauuaucua acugaauauc uauauaauau uauauauaaa uaaauguaau 960

auuaugguag uggaacauuu aaugaauaau aaguauauua auauuuacgg auuaauauau 1020

gauauauaaa uuaaauagag aaugcugaca ugaguaacga aaaaaagguu uaauccuuuu 1080

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uauauuguac gauagguaaa aucuaaauau auauauuaua ucuauauuau auauauuaua 1200

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aacuguuuac uaaaaacacc gcacuuugca aaaacguuaa guuauaguau aaggugugaa 1440

cucugcucca ugcuuaauua auaaauauga auaauuauua ugauuauuau auuaaauuua 1500

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ugaauucgca gugaagaugc uguguaccua uaguaagacg aaaagacccu uugcagcuuu 1680

acuguaauua gauagaucga auuauuaugu auuuuguuca guauauuaag uaauagauua 1740

uauauaaaug aaauacuuau cuaaauaucu uaaauauuau uuuuaauaau auuuuauaau 1800

aauucuuauc uuuauaauau auuuaauuga uauauauucu uaaauauaua uuauauuaua 1860

uaauauuuau auuguauaau auaauuaaag agaaaaucuc uaauugguag uuuugauggg 1920

gcgucauuuu cagcaaaagu aucugaauaa guccauauau aaauuauaau guuauugauu 1980

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auaaacaaaa uugaacaggg uaauauaacg aaagaguaga uauuguaagu uauguuugcc 2700

accucgaugu cgacucuacc uau 2723

<210> 5

<211> 2564

<212> RNA

<213> 探针(Probe)

<400> 5

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uaauuuauaa auuuaauuua uaaauaauaa uaagugaaaa uaauauuaaa auauuauuaa 840

auaaucaguu aauggguuug acaauaacca uuuuuuaaug aacauguaac aaugcacuga 900

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uuaaaccuuu ucaccuaaaa cauaagguuu uacuauaaaa guacggcacc uaauauaaau 1200

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cguaauguau accgacucag guauguuagu agagaauaug aagguguauu agauaauuaa 1380

ggauaaggaa cucggcaaaa auagcucuaa cguuagucaa uaaagagaua uaaaaacaag 1440

guuguacaac uguuuacuaa aaacaccgca cuuugcaaaa acuauuuaag uuauaguaua 1500

aggugugaac ucugcucaau gcuuaauaua uaaauuauau auuuauuaaa gaauauauaa 1560

auaaauuuaa guaaauagca gccuuauuau gaggguuaua auguagcgaa auuccuuggc 1620

ugauaauuac agucccgcau gaaugacgua augauacaac aacugucuca uccuuaaacu 1680

aagugaaauu gaauuugcag ugaagaugcu guauauuugu aguuagacaa gaagacccua 1740

ugcagcuuua cuauaauuag auagaucgaa uuauuauuua uuauauucag cauauuaagu 1800

aauuuauaua uauauauaua uauauaauaa ucguuuagau auaaaugaga uacuuauuau 1860

aauaauauaa uaauucuaau cuuuaauaaa uauauauaua uuuaauagaa aauuaauaua 1920

uucuuaaaua uauuuuauaa uauuauuauu auuaauaaua uuauauauua agagacaauc 1980

ucuaauuggu aguuuugaug gggcgucauu uucagcaaaa guaucugaau aaguccauau 2040

auaaauuauu auaaaauuuu auuuauaaua uaaaguauau auuauauaua cuaauuauaa 2100

aauauauuau auauuuuuau auauauaaua uaauaauaga aaaauuauau acaguaaaau 2160

ugucuggaaa acaauaauau uuauuuauuu uuaauaaaua uuaaauuaau auaauaguau 2220

aacuguguaa agauuaugac acaaacaagu ggaauaagua cguaaguaug guauaaugaa 2280

caaauaauac ugauuguaaa gguuauugau aacgaauaaa aguuacgcua gggauaacag 2340

gguaauauaa cgaaagagua gaaauuguaa guuauguuug ccaccucgau gucgacucua 2400

ccuauccuau ugguuguaaa agcuaauaag gguucgacug uucgucgauu aaaagguaac 2460

gugaguuggg uuaaauacga cgugagucag uaugguuuuu aucuacuaca aauaauauua 2520

ucuuauuaaa ucucauuauu aguacgcaag gaccauaaug aauu 2564

<210> 6

<211> 2547

<212> RNA

<213> 探针(Probe)

<400> 6

uagagugagg ggaguaaggg agagggaucc uaaugggagu aggagaggua auugaaagac 60

uacauuuaau aacaaugucu aaguauggac guaaagacua ugauuaaccu agacaaagga 120

aaggguccuu aaugggaccg uucuaagcua gaaauaguga cgauauugga ggcaucugaa 180

uugaaacauc uuaguaagau gcuaguaaac caauagggag cacuucagua ccggugaggg 240

aaaaagugcu aguauauaag uaauaucaug ugauaugaau auaguagugc uacuacuaau 300

acuaaugccu aaucauaagu aacaaguacc gugagggaaa uacauacauu acugcuuaau 360

uaaucggaau auaauauuaa gguuauauaa agacaguaaa caaauacuua gauaaggagc 420

agcuauagaa uagcguaccu cuuguauaau gggucaguga guuagcuuca uuagcgaaag 480

aaacauugga uuuaaucgga ugauauaguu aauggggcua gacccgaaag uauaugaucu 540

auacaugacc agguuaugua guuaacacuu uauugacuau guagaccuaa caggugggug 600

uugcaauacc cucuggagag uuguguaucg augugaaagg cuacucucau auacagauag 660

cugguugucu gugaaauaua ucuuaguaua gcgauuaaau augcauuacu agguaaauca 720

cuagauuuga acauaucucu uaugucaugg acauuaauug auauuuaugg gagcuauuag 780

agcuuaucau acgaaucuaa caucugaacu aguuaaguag cauuaauuug aucagucaau 840

cuaggugcga uaagguaccu agaugagaag ggaauaaccu agauuacucg guguaagucc 900

uuaauaacaa cuaaguggua uuaauccuau cccuuaagca uuauguuuaa gaauuucuaa 960

uaagacaguu aaucagugga uaugauagua aucaucugcu agagaacgug uaacaacgca 1020

uuaacugaua cauagauagu aggaguagau auacgggccu aaguuguuga cugaacagua 1080

aauaguuacu cuaauaguaa cuaugguaac agaccacacc auacuauuaa uggugcagaa 1140

uaugcugacu ugaguaacgu aaggagggau aagcccuucc ccuauaugua caagguuuua 1200

cuacaaauua acggccucua aucguaucua ugaagauaga uaggagauga guaauaugac 1260

uagaaauagu gcuaaccgua cuguagaccg acacuggugu acuauaucuu gauuauauaa 1320

gauaaaauag gguaugagau aagccauaug aaugaacucg gcaauaagag uauaaccucu 1380

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ucuuaucaug aggauccgaa gguagcgaaa uuccuagacg uuuaauuggc guccugcaug 1560

aaugaguaca cgaugcuaua gauguaucca uaugguucuc agugaaauag uaauugcagu 1620

gaagauacug uaucagcaau agauugacga gaagacccua uacagcuuua cuauagaguu 1680

accuugcuau ucacuacccu uuauaagccu auguacucgu acagcuuagu aagcguaguc 1740

cguagcuaau cuccauagaa auaugcuuac uuaaucgugg cauaggagag acaugauaac 1800

uugguuaguu ucgauggggc gucgacauca caaagaauaa cugauguguc uauaagacuu 1860

guuuguaauu aacucuagaa auggaguucu aauaguauau ccagauacag ugaggcuaaa 1920

ugguaauuag ucagcucuac uccauuauac ccgauuuauc uagcauaaau aggauggaua 1980

gacaaugaua aggauaauga cuuaacugug uuaugauaga ggcacuuaca ggugucuuua 2040

guacguaagu auuguauagu gaacaggcaa ugacuaaagc agaguauguc gauagcgaau 2100

caaaguuacg uuagggauaa caggguaaua ccgcuagaga guugauaucg ucagcggugu 2160

uugccaccuc gaugucgucu aggcucaucc uuauggucgc agcaacuaua aaggguauga 2220

cuguucguca uuuaaggagc uacuugagau ggguuaauua cggcgugagc caguaauguu 2280

ucuaucaucu auugcacuau aacaacagcu uuauuacaug uguacgagag gauuauguaa 2340

ucuagaccaa uggugauaac uaaaugcuua uuuaaucgag uauuaaguua uaagcuaagc 2400

cuagcagcaa uaaguauuga ccgcauauca aauacgaagu cuauagcugu uuguugaaca 2460

aaacauugaa gauaacaaug ugcuagugua aucgguucau augacuguua cacugaucua 2520

uggagcauau ccauguuaaa ugaagga 2547

<210> 7

<211> 2546

<212> RNA

<213> 探针(Probe)

<400> 7

gagcuacuaa uuauagaggg uaaaaaagag uauaugaaga gauggaauau acugagaccu 60

aaaggaaacu gagagaaaga aaccaagaga acugaaacau cuaaguaucu ugaggaaaag 120

aaauaaacau agauucuaug aauauuggug agagaaaaua gagaaguaug aauaaguagu 180

agggguuaaa agaucuacga aaaagggaga acuaauccua agacuaaaua uaauuaguaa 240

guguaauaag uaccgaaagg gaauaauaua aauaagugaa auugagagca auuaaagguu 300

aauaaagaag aauuaauuga uaauguaccu uuuguauaau ggguuagcaa guuauaauaa 360

auagcgaaaa agaaagaauu ugaaaugaaa augagagaag uuauuaauua uagacccgaa 420

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gacugaacag guuaauguug caauauuauc ugaagauaua ugguaaguag ugaaagacaa 540

aucugaucua cagauagcug guucuuugug aaauauauuu aaguauagcg uuaauaaaga 600

aguauaaaua ugguaaagca auuaauaaua auugggguau uguaauaaua uuaucaauau 660

uauuuaaucu cauaauaaua uuuauauaua aauuaaacgu cagacuauua gugauaagau 720

ugauagucaa aagggaaaca gcccagauca agagagaaag uuccaaauaa uaauuuaagu 780

auaaauaaua uuuauuaauc guaaaacaau aaugggauua guuugacagu aauuauucua 840

ugaucaacgu guaacaacgg guuuauuaua aauauaauau aauaauauau auuauauaaa 900

agaauauaua uauauucuga ucgguaaaua aauagauaau aaucggaauu aaaaauaaua 960

acugaaaucu ugaauaauag aauuaaaaga auaauaauaa uauuaaggua acaaagcauu 1020

uaaugaauaa auauuuuucu aaaugaaaaa uaaggaaaga aagaaagaaa uagaauuaaa 1080

uagagaaugc ugacaugagu aacgaauaag aggaaaaucc ucuucacuuu auguauuagg 1140

uuuuaauaaa aaaucggucc auaagagaau uucugaaaag aaauaauuaa uagguaauaa 1200

auaaaaaagu auacgcccau auaaaaaaau auaagguagu auaugguacc ggaaaaagua 1260

uuuuauaaaa auaccguaau aaaaccgaca cagguauaca aauaaaaaau gaaagugaau 1320

aagugaauua uucuaaauga acucggcaaa auaaaagcuc agacguuagu caaaaagagu 1380

gaauaaagua cauguuaugc gacuguuuac caaaaacaca gcacuuugcg aaaacgauaa 1440

guauuagaau aaagugugaa uucugcccaa uguuuaauaa uaaagaaaua uauuaaucga 1500

uauauuuaua aaaauuaagu aaauggcggc uuuauacuga gagucauaau guagcgaaau 1560

uccuugaccg cuaaauacgg cccugcauga auggaguaac gacauaacaa cuguaucuag 1620

aauaagcuua gugaaauuga aaucgcugug aagaugcagu guaccuaaag guagacgaga 1680

agacccuaug cagcuuuacu augauuuaau aaguuguuau uauuugauaa aauguuagua 1740

uauaaaguaa uauauugaua uaaaaaugaa auacuuuuau uuuauuccaa uuuauuuaua 1800

uaaauaaaaa uauaaauaga uauaaauaau aacugaauua auaauuaauu aguagaaaaa 1860

agaauauccu aauuauaaua uauuuauaaa aauauauaua uuaaugacag cuuuaaauug 1920

auaguuuaaa uggggcguug auuucacaaa aaauaacuga auauguccau auacaauuaa 1980

aauuaaauga auuaaugaau uaaucauuaa aaauguuaaa aauaauauga auuuauuaua 2040

auuuuagaaa aaccuaugua caguaaauuu gaccugacau caaauaauua ucuaaaaaau 2100

aauuuuacua aauuaauaua auaguggaua acugugcaau aauuuaacac uaacaagugg 2160

aaauuauacg uaaguaaggu auaaugaaca gaucauuuua auaguccaag ugaucgauua 2220

acggauaaaa guuacgcuag ggauaacagu guaauaacau gagagaguag auauugucaa 2280

uguuguuugc caccucgaug ucggcugagc cuauccuauu gguaguauaa gcuaauaagg 2340

guuugacugu ucgucaauua aaaggcuacc ugagcugggu ucaauacguc gugagacagu 2400

augguuucua ucuucuuuag ggauuaguua aaaaagguau auaacuuagu acgaaaggau 2460

uaguuauagu uaaucaauga uuuuacuguu guauauuaug uauaauuaua augauacuau 2520

cuauuuauaa uaugcaggca guuaag 2546

Claims

1.一种检测念珠菌的方法，其特征在于，所述方法包括：

(1)将待测的念珠菌DNA样本解链处理，得到单链DNA样本；

(2)将步骤(1)获得的单链DNA样本、特异性识别目标DNA的探针以及检测抗体、RNase A共同加样到包被有捕获抗体的固相载体上；所述捕获抗体与检测抗体为特异性抗DNA-RNA杂合体的抗体，所述检测抗体携带可检测信号；当DNA样本存在目标DNA时，在固相载体上形成捕获抗体-双链杂交体-检测抗体三元复合物；

(3)根据检测抗体的可检测信号，得知目标DNA的存在与否或存在量。

2.如权利要求1所述的方法，其特征在于，所述的RNase A在混合体系中的终浓度为0.02～0.5mg/ml；较佳地0.05～0.3mg/ml。

3.如权利要求1所述的方法，其特征在于，所述的念珠菌包括：白色念珠菌，热带念珠菌，近平滑念珠菌，乳酒念珠菌，光滑念珠菌，克柔念珠菌；较佳地，所述特异性识别目标DNA的探针包括：

(a)针对白色念珠菌的SEQ ID NO:1，SEQ ID NO:2探针；

(b)针对近平滑念珠菌的SEQ ID NO:3探针；

(c)针对乳酒念珠菌的SEQ ID NO:4探针；

(d)针对光滑念珠菌的SEQ ID NO:5探针；

(e)针对热带念珠菌的SEQ ID NO:6探针；

(f)针对克柔念珠菌的SEQ ID NO:7探针。

4.如权利要求3所述的方法，其特征在于，通过分组法进行分型检测，包括：将(a)的探针作为一组，将(b)～(f)的探针作为一组，两组分别在独立的体系中进行反应，从而检测后将念珠菌分型为：白色念珠菌或其它念珠菌；该其它念珠菌为热带念珠菌、近平滑念珠菌、乳酒念珠菌、光滑念珠菌、克柔念珠菌或其组合；和/或

探针共混后进行检测；较佳地，按照重量体积比，(a)的任一探针与(b)～(f)中任一探针的比例为1:(1.2～3)；更佳地为1:(1.5～2.5)(例如1:1.8，1:2，1:2.2等)。

5.如权利要求3所述的方法，其特征在于，所述的探针上，还设置荧光基团，针对不同型的念珠菌的探针其荧光基团不同，从而进行念珠菌的分型。

6.如权利要求1所述的方法，其特征在于，所述的可检测信号包括：碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶；和/或

(3)中，所述检测抗体的可检测信号的检测包括：加入识别所述可检测信号的底物，测定底物的显色情况；较佳地，采用化学发光法测定底物的显色情况。

7.一种用于检测念珠菌的检测试剂盒，其中含有：

(4)RNase A。

8.如权利要求7所述的检测试剂盒，其特征在于，所述的念珠菌包括：白色念珠菌，热带念珠菌，近平滑念珠菌，乳酒念珠菌，光滑念珠菌，克柔念珠菌；较佳地，所述特异性识别目标DNA的探针包括：

(a)针对白色念珠菌的SEQ ID NO:1，SEQ ID NO:2探针；

(b)针对近平滑念珠菌的SEQ ID NO:3探针；

(c)针对乳酒念珠菌的SEQ ID NO:4探针；

(d)针对光滑念珠菌的SEQ ID NO:5探针；

(e)针对热带念珠菌的SEQ ID NO:6探针；

(f)针对克柔念珠菌的SEQ ID NO:7探针。

9.如权利要求8所述的检测试剂盒，其特征在于，所述的所述的探针被分组保存，其中，(a)的探针作为一组，(b)～(f)的探针作为一组，两组分别在独立的体系中保存。

10.如权利要求7～9任一所述的检测试剂盒，其特征在于，其中还含有：

核酸解链试剂；

洗涤试剂；

识别所述可检测信号的底物；或

显色剂。