CN103255227A - Primer-mediated cyclized constant-temperature nucleic acid rolling circle amplification method and kit - Google Patents

Primer-mediated cyclized constant-temperature nucleic acid rolling circle amplification method and kit Download PDF

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CN103255227A
CN103255227A CN2013102114430A CN201310211443A CN103255227A CN 103255227 A CN103255227 A CN 103255227A CN 2013102114430 A CN2013102114430 A CN 2013102114430A CN 201310211443 A CN201310211443 A CN 201310211443A CN 103255227 A CN103255227 A CN 103255227A
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周中人
周正峰
林忠旺
蒋庭
刘秀贵
黄知音
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QUICKING BIOTECH CO Ltd
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Abstract

本发明公开了一种引物介导环化的恒温核酸滚环扩增方法及试剂盒,包括:1)寡核苷酸序列设计;2)从靶核酸中扩增出单链DNA目标模板;3)DNA连接酶环化DNA目标模板;4)环化DNA目标模板滚环扩增。本发明不用合成高成本的长锁式探针,靶核酸的序列的长度能允许更大,能对靶核酸任意序列进行滚环扩增,并对防止样品被扩增产物污染提供了解决方法,开启了基因检测步入基层单位应用的新时代。

The invention discloses a primer-mediated circularization thermostatic nucleic acid rolling circle amplification method and a kit, comprising: 1) oligonucleotide sequence design; 2) amplifying a single-stranded DNA target template from a target nucleic acid; 3 ) DNA ligase circularizes the DNA target template; 4) circularizes the DNA target template rolling circle amplification. The present invention does not need to synthesize high-cost long lock probes, the length of the sequence of the target nucleic acid can be larger, can perform rolling circle amplification on any sequence of the target nucleic acid, and provides a solution to prevent samples from being polluted by amplified products. Opened a new era of genetic testing into the application of grassroots units.

Description

引物介导环化的恒温核酸滚环扩增方法及试剂盒Primer-mediated circularization constant temperature nucleic acid rolling circle amplification method and kit

技术领域technical field

本发明涉及一种核酸序列的恒温扩增方法及试剂盒,更具体地说,涉及引物介导环化的恒温核酸滚环扩增方法,即一种制备单链DNA链的两个引物相关序列末端连接成环并且能至少以该两个引物启动恒温滚环扩增的方法及试剂盒(Primer mediated Rolling Circle Amplification,PRCA)。The present invention relates to a nucleic acid sequence constant temperature amplification method and kit, more specifically, to a constant temperature nucleic acid rolling circle amplification method for primer-mediated circularization, that is, two primer-related sequences for preparing single-stranded DNA strands A method and a kit (Primer mediated Rolling Circle Amplification, PRCA) capable of starting constant temperature rolling circle amplification with at least two primers connected to form a circle at the ends.

背景技术Background technique

1985年美国Cetus公司用聚合酶链反应(Polymerase Chain Reaction,PCR)技术第一次实现了核酸的体外扩增,这对现代分子生物学的发展起到非常重要的作用,可以说PCR技术是生物医学领域中的一项革命性创举和里程碑。但是PCR方法仍然有不足之处如:扩增过程中需要对双链DNA进行变性,需要特制的可快速升降温的PCR仪,非特异性扩增而产生假阳性结果等。因此,近十几年来发展起来了一些新的核酸扩增技术以弥补PCR技术的不足,甚至有取代PCR技术的趋势。因此,基因片段的扩增方法现在丰富多样,成为了现代分子生物学的基本技术。In 1985, the U.S. Cetus company used Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) technology to realize the in vitro amplification of nucleic acid for the first time, which played a very important role in the development of modern molecular biology. It can be said that PCR technology is a biological A revolutionary initiative and milestone in the field of medicine. However, the PCR method still has deficiencies such as: the double-stranded DNA needs to be denatured during the amplification process, a special PCR instrument that can rapidly raise and lower the temperature is required, and false positive results are generated due to non-specific amplification. Therefore, some new nucleic acid amplification techniques have been developed in the past ten years to make up for the shortage of PCR technology, and even have a tendency to replace PCR technology. Therefore, the amplification methods of gene fragments are now abundant and diverse, and have become the basic techniques of modern molecular biology.

近些年,陆续出现了多种恒温扩增技术,它们能分别在某一个或多个特定的温度条件下(如64℃、42℃等)实现核酸(DNA或RNA)的扩增。国际上已有的恒温扩增技术如下:In recent years, a variety of constant temperature amplification technologies have emerged, which can achieve nucleic acid (DNA or RNA) amplification under one or more specific temperature conditions (such as 64°C, 42°C, etc.). The existing isothermal amplification technologies in the world are as follows:

链置换扩增技术(SDA)Strand Displacement Amplification (SDA)

依赖核酸序列的扩增技术(NASBA)Nucleic acid sequence-dependent amplification technique (NASBA)

转录介导扩增技术(TMA)Transcription Mediated Amplification (TMA)

滚环扩增技术(RCA)Rolling Circle Amplification (RCA)

连接酶链式反应(LCR)Ligase Chain Reaction (LCR)

依赖解旋酶的扩增技术(HDA)Helicase-dependent amplification (HDA)

环介导恒温扩增(LAMP)。Loop-mediated isothermal amplification (LAMP).

其中,NASBA通过一系列转录和反转录的循环过程来以避免高温变性作用,SDA则使用限制性内切酶和修饰过的模板来循环扩增。虽然它们的敏感性都很高,可以扩增低于10个拷贝数量的核酸样本,但是它们还有各自需要克服的缺点。技术要求、材料仪器要求、技术本身特异性缺陷等方面严重束缚了这些技术的推广应用。Among them, NASBA avoids high-temperature denaturation through a series of transcription and reverse transcription cycles, and SDA uses restriction enzymes and modified templates for cycle amplification. Although they are all highly sensitive and can amplify nucleic acid samples with less than 10 copies, they also have their own shortcomings that need to be overcome. Technical requirements, material and instrument requirements, and specific defects of the technology itself seriously restrict the popularization and application of these technologies.

Walker等于1991年首次提出了DNA链置换扩增(SDA)方法。该方法通过某种技术手段(如限制性内切酶)在DNA的一条链上产生碱基缺口,DNA聚合酶则从碱基缺口处3’端开始延伸反应,同时将下游的旧链剥离,因链延伸而被封闭了的碱基缺口可以重复产生,使得切割延伸链置换的过程重复进行。1992年,Walker等人简化了SDA设计,使用4条引物(B1、B2、S1、S2)与加热变性后的靶DNA退火,其中引物S1和S2是真正进行SDA扩增的引物;B1、B2分别位于S1、S2的上游和下游,作用是将S1、S2第一和第二轮延伸后的产物剥离。Walker et al first proposed the DNA strand displacement amplification (SDA) method in 1991. In this method, a base gap is generated on a strand of DNA by a certain technical means (such as a restriction endonuclease), and the DNA polymerase starts the extension reaction from the 3' end of the base gap, and at the same time, the downstream old strand is stripped, The base gaps blocked by chain extension can be repeatedly generated, so that the process of cleavage and extension chain displacement is repeated. In 1992, Walker et al. simplified the SDA design and used 4 primers (B1, B2, S1, S2) to anneal to the target DNA after heat denaturation, among which primers S1 and S2 were primers for SDA amplification; B1, B2 Located upstream and downstream of S1 and S2 respectively, the function is to strip the products after the first and second rounds of extension of S1 and S2.

2000年日本学者Notomi在Nucleic Acids Res杂志上公开了环介导等温扩增反应LAMP技术,它克服以往基因扩增方法的不足,在等温条件下能够特异、高效、快速地进行核酸的扩增,具有很多的优越性。但LAMP的核酸靶序列长度一般建议在120-180bp,最好在300bp以内,大于500bp则较难扩增。故不能进行长链DNA的扩增。此外,LAMP技术在核酸扩增产物的回收鉴定、克隆、单链分离方面均逊色于传统的PCR方法。In 2000, the Japanese scholar Notomi disclosed the loop-mediated isothermal amplification reaction LAMP technology in the journal Nucleic Acids Res. It overcomes the shortcomings of previous gene amplification methods and can perform specific, efficient and rapid nucleic acid amplification under isothermal conditions. Has many advantages. However, the length of the nucleic acid target sequence of LAMP is generally recommended to be 120-180bp, preferably within 300bp, and it is difficult to amplify if it is greater than 500bp. Therefore, the amplification of long-chain DNA cannot be performed. In addition, the LAMP technology is inferior to the traditional PCR method in terms of recovery and identification, cloning, and single-strand isolation of nucleic acid amplification products.

滚环扩增技术(RCA)是新近发展起来的一种恒温核酸扩增方法,凭借其高特异性、高灵敏度和易操作性的特点在近几年中逐渐引起人们的注意,并越来越多地用于基础研究和实际检测中。人们已经开发了各种利用RCA的扩增方法,相关文献可参考美国专利No.60/506,218、5871921A、5648377A、5854033A、6287824B1、6323009B1。Rolling circle amplification (RCA) is a newly developed constant temperature nucleic acid amplification method, which has gradually attracted people's attention in recent years due to its high specificity, high sensitivity and easy operation, and has become more and more popular. It is widely used in basic research and practical testing. People have developed various amplification methods using RCA, and related literature can refer to US Patent Nos.

滚环扩增是借鉴自然界中环状病原微生物DNA分子的滚环式的复制方式而建立的一种核酸扩增技术。人们的研究集中在如何将核酸片段进行环化,并且如何设计引物引发滚环扩增。现有大部分文献介绍的RCA反应分为锁式探针(Padlock probe)的连接和连接后扩增两部分。锁式探针的5端和3端特异性序列通过同靶序列上的互补区域结合,在连接酶的作用下连接成环。成环后的锁式探针在一个引物和合适DNA聚合酶存在的恒温下进行滚环扩增。1998年Lizardi等在线性滚环扩增技术的基础,发明了超分支滚环扩增技术(HRCA/CRCA/RAM)。HRCA是在RCA的基础上增加了一个序列同锁式探针中部分序列相同的引物,不但具有RCA的高序列特异性和简单易操作性,而且在两个引物存在下产物以超分支形式高效扩增,灵敏度极高。Rolling circle amplification is a nucleic acid amplification technology established by referring to the rolling circle replication method of DNA molecules of circular pathogenic microorganisms in nature. People's research focuses on how to circularize nucleic acid fragments and how to design primers to trigger rolling circle amplification. The RCA reaction described in most existing literatures is divided into two parts: the ligation of the padlock probe and the amplification after ligation. The 5-terminal and 3-terminal specific sequences of the padlock probe are combined with the complementary regions on the target sequence, and then connected into a circle under the action of ligase. The circularized padlock probes are subjected to rolling circle amplification at a constant temperature in the presence of a primer and a suitable DNA polymerase. In 1998, based on the linear rolling circle amplification technique, Lizardi et al. invented the hyperbranched rolling circle amplification technique (HRCA/CRCA/RAM). On the basis of RCA, HRCA adds a primer with the same sequence as that in the padlock probe. It not only has the high sequence specificity and simple operation of RCA, but also the product is highly branched in the presence of two primers. amplification with high sensitivity.

美国专利No.60/506,218中介绍了多种以RNA或DNA模板进行滚环扩增的多种技术,它们利用了核酸接头片段来连接目标核酸序列成环。US Patent No. 60/506,218 introduces a variety of technologies for rolling circle amplification using RNA or DNA templates, which utilize nucleic acid linker fragments to connect target nucleic acid sequences to form a circle.

李岩等发表的文章《一种高效扩增小片段DNA方法的建立》(首都医科大学学报2011年01期)介绍了代替合成小片段DNA的新方法,通过单链DNA连接酶直接将其连接成环,然后加入特异的正反向引物用滚环方法扩增复制,最后通过酶切得到大量同样序列的小片段。The article "Establishment of a Method for Efficiently Amplifying Small DNA Fragments" published by Li Yan et al. (Journal of Capital Medical University, Issue 01, 2011) introduces a new method instead of synthesizing small DNA fragments, which is directly connected by single-stranded DNA ligase Form a circle, then add specific forward and reverse primers to amplify and replicate by the rolling circle method, and finally obtain a large number of small fragments of the same sequence by enzyme digestion.

现有的RCA和HRCA常需要双链变性、酶连接与滚环扩增等多步反应,分别需要不同的温度,也需要多次添加反应试剂,需要添加特殊设计的引物启动滚环扩增,不适合基层单位使用。Existing RCA and HRCA often require multi-step reactions such as double-strand denaturation, enzyme ligation, and rolling circle amplification, which require different temperatures and multiple additions of reaction reagents. Specially designed primers need to be added to start rolling circle amplification. Not suitable for grassroots use.

2012年王晓亮发表的硕士学位论文《常温SC-RCA DNA扩增技术研究》(中国海洋大学)介绍了一种改进的滚环扩增技术--自环化滚环扩增技术(Self circularization-RCA,SC-RCA),该技术利用一种具有特异酶切位点的限制性内切酶将目标DNA酶切,再通过连接酶将酶切片段和经过特异设计的接头连接成环后实现目标DNA的滚环复制。省去了长锁式探针的高成本合成,又能够保证连接成环的特异性。SC-RCA技术避免了LAMP技术难以辨别非特异性扩增的不足,又较Padlock-RCA成环简便。SC-RCA技术中,具有限制性内切酶位点的靶核苷酸序列经特异性酶切后,通过DNA连接酶将被酶切得到的片段与经过特异设计的核酸接头连接成圆环。该文章虽然可以通过通过酶切方式将靶核酸部分序列代替合成序列与特异设计的接头直接成环,省去了长锁式探针的高成本合成,但要求扩增的靶核酸序列位置有酶切位点,环化时要加入特异设计接头,滚环扩增时要加入特殊设计的引物,文章的实验需要分别进行酶切,连接,滚环扩增,无法在一个溶液体系中一次完成这些操作。In 2012, Wang Xiaoliang published his master's degree thesis "Research on SC-RCA DNA Amplification Technology at Normal Temperature" (Ocean University of China), which introduced an improved rolling circle amplification technology--Self circularization-RCA technology. , SC-RCA), this technology uses a restriction endonuclease with a specific enzyme cutting site to digest the target DNA, and then connects the digested fragment and the specially designed linker into a circle by ligase to realize the target DNA Rolling circle replication. The high-cost synthesis of long lock-type probes is omitted, and the specificity of ligation into a circle can be guaranteed. SC-RCA technology avoids the disadvantage of LAMP technology that it is difficult to distinguish non-specific amplification, and it is easier to form a circle than Padlock-RCA. In SC-RCA technology, after the target nucleotide sequence with a restriction endonuclease site is specifically digested, the digested fragment is connected to a specially designed nucleic acid linker by DNA ligase to form a circular circle. Although this article can replace the synthetic sequence of the target nucleic acid partial sequence with a specially designed adapter to directly form a circle by enzymatic digestion, which saves the high-cost synthesis of long lock probes, it requires an enzyme at the position of the amplified target nucleic acid sequence. Cutting sites, specifically designed adapters should be added for circularization, and specially designed primers should be added for rolling circle amplification. The experiments in this article need to carry out enzyme digestion, ligation, and rolling circle amplification separately, which cannot be completed in one solution system at one time. operate.

发明内容Contents of the invention

本发明要解决的技术问题在于提供一种引物介导环化的恒温核酸滚环扩增方法及试剂盒,即一种制备单链DNA链的两个引物相关序列末端连接成环且能至少以该两个引物直接启动恒温滚环扩增的方法及试剂盒。本发明的方法能在一种溶液体系中一次添加完全部试剂后恒温保持一定时间就能完成核酸扩增,本发明还将UNG酶运用到恒温的滚环扩增方法中,在开始扩增前对样品进行可能的扩增产物污染做彻底消除。另外,本发明的扩增产物还能通过免疫层析试纸对产物进行检测。因此本发明能将基因的扩增和检测变成简单操作,能开启基因诊断技术走向基层单位应用的时代。The technical problem to be solved by the present invention is to provide a constant temperature nucleic acid rolling circle amplification method and kit for primer-mediated circularization, that is, a method for preparing a single-stranded DNA chain with two primer-related sequence ends connected to form a circle and capable of at least The two primers directly start the constant temperature rolling circle amplification method and kit. The method of the present invention can complete the nucleic acid amplification after adding all the reagents in a solution system at a time and maintaining the constant temperature for a certain period of time. The present invention also applies the UNG enzyme to the constant temperature rolling circle amplification method. Thoroughly eliminate possible amplification product contamination of the sample. In addition, the amplification product of the present invention can also be detected by immunochromatographic test paper. Therefore, the present invention can turn the amplification and detection of genes into simple operations, and can open the era of the application of gene diagnosis technology towards grassroots units.

本发明按照靶核酸3’-5’序列方向依次设有四段引物:F3,FIP,BIP,B3。其中FIP为前端内引物,BIP为后端内引物,F3、B3是一对外引物,分别位于FIP、BIP的上游和下游,在带有链置换功能的DNA聚合酶、必要时可增加的逆转录酶、合适的扩增促进剂作用下,四个引物以靶核酸为模板扩增并剥离出一条单链目标模板。DNA目标模板首尾两端在恒温中被合适的DNA连接酶连接形成闭合的环化目标模板。能与环化目标模板互补的起始引物沿环化目标模板从3’端启动DNA滚环扩增,周而复始地进行环化目标模板的复制,扩增产物是以起始引物为起点的数千倍于目标模版单环长度的串联重复拷贝,每个单环序列中都有与其他引物互补的序列,这些引物杂交并启动反向的DNA链合成,可复制出终止于串联重复拷贝的扩增产物末端即起始引物5’尾端的互补链。该终止于起始引物5’尾端的互补链中包含单个或多个长度的目标模板,起始引物将与每个目标模板中的互补序列杂交而启动新的复制。包含了多个长度目标模板的互补链将在内引物的交替复制与剥离后产生单个长度的目标模板;该新复制的单个长度目标模板将不断由连接酶环化并启动新一轮的滚环扩增。如此周而复始,将扩增复制出巨量的核酸产物(如图2)。According to the 3'-5' sequence direction of the target nucleic acid, the present invention has four primers: F3, FIP, BIP, and B3. Among them, FIP is the front-end inner primer, BIP is the back-end inner primer, and F3 and B3 are a pair of outer primers, which are respectively located upstream and downstream of FIP and BIP. DNA polymerase with strand displacement function and reverse transcription can be increased if necessary. Under the action of enzymes and appropriate amplification promoters, the four primers use the target nucleic acid as a template to amplify and strip off a single-stranded target template. The first and last ends of the DNA target template are ligated by a suitable DNA ligase at a constant temperature to form a closed circularized target template. The initial primer that can be complementary to the circularized target template starts DNA rolling circle amplification from the 3' end along the circularized target template, and repeats the replication of the circularized target template. The amplification product is thousands of A tandem repeat copy that is twice the length of the single loop of the target template. Each single loop sequence has sequences complementary to other primers that hybridize and initiate DNA strand synthesis in reverse, replicating amplification that terminates in the tandem repeat copy The end of the product is the complementary strand to the 5' end of the starting primer. The complementary strand terminating at the 5' tail of the initial primer contains single or multiple lengths of target templates, and the initial primer will hybridize with the complementary sequence in each target template to initiate new replication. Complementary strands containing target templates of multiple lengths will generate a single-length target template after alternate replication and stripping of the inner primer; the newly replicated single-length target template will be continuously circularized by ligase and start a new round of rolling circle Amplify. Repeatedly, a huge amount of nucleic acid products will be amplified and copied (as shown in Figure 2).

本发明内容之一在于当单链目标模板的长度合适时,可直接通过单链DNA连接酶将其两端对接闭合成环;此后用来扩增复制单链目标模板的两个内引物启动了依次交替置换的滚环扩增;而滚环扩增中,将不断复制出新的目标模板,它将不断由连接酶环化并启动新一轮的滚环扩增。One of the contents of the present invention is that when the length of the single-stranded target template is appropriate, the two ends of the single-stranded target template can be directly docked and closed to form a circle by single-stranded DNA ligase; thereafter, the two internal primers used to amplify and copy the single-stranded target template start Rolling circle amplification with alternate replacements; in rolling circle amplification, new target templates will be copied continuously, which will be continuously circularized by ligase and start a new round of rolling circle amplification.

本发明内容之二在于设计了两条尾端与靶核酸序列互补的内引物,使得复制出的目标模板两端都形成发夹结构,它们之间没有碱基缺口而相邻着与靶核酸的部分序列杂交;在合适DNA连接酶的作用下目标模板两端被连接闭合成环;用来扩增复制单链目标模板的两个内引物启动了依次交替置换的滚环扩增。而滚环扩增中将不断复制出新的目标模板,它将不断由连接酶环化并启动新一轮的滚环扩增。The second content of the present invention is to design two internal primers whose tails are complementary to the target nucleic acid sequence, so that both ends of the copied target template form a hairpin structure, and there is no base gap between them and are adjacent to the target nucleic acid sequence. Partial sequence hybridization; under the action of a suitable DNA ligase, the two ends of the target template are ligated and closed to form a circle; the two internal primers used to amplify and copy the single-stranded target template initiate rolling circle amplification with alternate displacement in sequence. In rolling circle amplification, new target templates will be continuously copied, which will be circularized by ligase and start a new round of rolling circle amplification.

本发明内容之三在于设计了至少一条尾端不与靶核酸序列互补的内引物和一条桥式引物。复制剥离的目标模板3’端尾部序列不与靶核酸序列杂交而全部或部分与桥式引物杂交;目标模板的5’端末尾部序列全部或部分与桥式引物杂交;目标模板的两端之间没有碱基缺口而相邻着与桥式引物杂交;在合适DNA连接酶的作用下目标模板两端被连接闭合成环;用来扩增复制单链目标模板的两个内引物启动了依次交替置换的滚环扩增;而滚环扩增中,将不断复制出新的单链目标模板,它将不断由连接酶环化并启动新一轮的滚环扩增。以上说明请参考图1。The third content of the present invention is to design at least one inner primer and one bridge primer whose tail is not complementary to the target nucleic acid sequence. The 3'-end tail sequence of the replicated and stripped target template does not hybridize with the target nucleic acid sequence, but hybridizes with the bridge primer in whole or in part; the 5'-end tail sequence of the target template hybridizes with the bridge primer in whole or in part; between the two ends of the target template There is no base gap and adjacently hybridizes with the bridge primer; under the action of a suitable DNA ligase, the two ends of the target template are connected and closed to form a circle; the two internal primers used to amplify and copy the single-stranded target template start to alternate Replacement rolling circle amplification; in rolling circle amplification, new single-stranded target templates will be continuously copied, which will be continuously circularized by ligase and start a new round of rolling circle amplification. Please refer to Figure 1 for the above description.

更具体的说,本发明的一种制备单链DNA链的两个引物相关序列末端连接成环且能至少以该两个引物启动恒温滚环扩增的方法,包括步骤:More specifically, a method of the present invention for preparing a single-stranded DNA chain in which two primer-related sequence ends are connected to form a circle and at least the two primers can be used to initiate constant temperature rolling circle amplification, comprising the steps of:

1)寡核苷酸序列设计1) Oligonucleotide sequence design

寡核苷酸序列至少有四段按靶核酸3’→5’方向设计的引物:F3、FIP、BIP、B3;The oligonucleotide sequence has at least four primers designed according to the 3'→5' direction of the target nucleic acid: F3, FIP, BIP, B3;

其中,FIP为前端内引物;Wherein, FIP is the front-end inner primer;

BIP为后端内引物;BIP is the back-end inner primer;

F3、B3是一对外引物,分别位于FIP的上游和BIP的下游;F3 and B3 are a pair of outer primers located upstream of FIP and downstream of BIP respectively;

2)从靶核酸中扩增出单链DNA目标模板2) Amplify the single-stranded DNA target template from the target nucleic acid

靶核酸为DNA链或在至少含有逆转录酶的反应溶液中将RNA链靶核酸逆转录为DNA链时,在至少含有带有链置换功能的DNA聚合酶的核酸扩增反应溶液中,利用引物F3、FIP、BIP、B3,根据靶核酸序列扩增合成出单链DNA目标模板;When the target nucleic acid is a DNA strand or the RNA strand target nucleic acid is reverse-transcribed into a DNA strand in a reaction solution containing at least a reverse transcriptase, in a nucleic acid amplification reaction solution containing at least a DNA polymerase with a strand displacement function, using a primer F3, FIP, BIP, B3, synthesize single-stranded DNA target templates according to target nucleic acid sequence amplification;

3)DNA连接酶环化DNA目标模板3) DNA ligase circularizes DNA target template

在至少含有DNA连接酶的反应溶液中,DNA目标模板的首尾两端被连接形成闭合的环化DNA链;In the reaction solution containing at least DNA ligase, the first and last ends of the DNA target template are ligated to form a closed circularized DNA chain;

4)环化DNA目标模板滚环扩增4) Circularized DNA target template rolling circle amplification

在至少含有DNA连接酶和带有链置换功能的DNA聚合酶的反应溶液中,至少以步骤1)中的两个内引物启动环化DNA链的滚环扩增,得到扩增的核酸产物。In the reaction solution containing at least DNA ligase and DNA polymerase with strand displacement function, at least two internal primers in step 1) are used to initiate rolling circle amplification of circularized DNA strands to obtain amplified nucleic acid products.

所述步骤1)的寡核苷酸序列还包括:桥式引物BP、杂交探针、辅助引物;所述桥式引物BP,其全部或部分序列与一个内引物尾部序列互补,并且其全部或部分与另一内引物互补序列的尾部序列互补,使之像桥一样将这两个内引物的两端序列相邻无碱基缺口地杂交在一起;所述杂交探针,其与靶核酸在两个内引物相关序列位置之中的序列杂交;所述辅助引物来源于BIP或FIP引物中不影响其与靶核酸或桥式引物稳定结合的部分序列,或该辅助引物还来源于BIP引物与B3引物之间的序列或该FIP引物与F3引物之间的序列。The oligonucleotide sequence in step 1) also includes: a bridge primer BP, a hybridization probe, and an auxiliary primer; all or part of the bridge primer BP is complementary to the tail sequence of an inner primer, and all or The part is complementary to the tail sequence of the complementary sequence of another internal primer, so that it hybridizes the two end sequences of the two internal primers adjacent to each other without a base gap like a bridge; Sequence hybridization among the relative sequence positions of the two internal primers; the auxiliary primer is derived from the partial sequence of the BIP or FIP primer that does not affect its stable combination with the target nucleic acid or the bridge primer, or the auxiliary primer is also derived from the combination of the BIP primer and the bridge primer The sequence between the B3 primers or the sequence between the FIP primer and the F3 primer.

所述步骤1)的寡核苷酸序列还能分别进行不同或相同的修饰标记,该修饰标记包括:地高辛、荧光染料FAM、异硫氰酸荧光素、生物素、荧光淬灭基团或纳米颗粒。The oligonucleotide sequences in the step 1) can also be modified and marked with different or the same, including: digoxin, fluorescent dye FAM, fluorescein isothiocyanate, biotin, fluorescence quenching group or nanoparticles.

所述步骤1)中,FIP只包含F2序列,F2与靶核酸序列F2c互补;所述BIP只包含B2序列,B2与靶核酸部分序列相同;步骤2)中,利用引物F3、FIP、BIP、B3从靶核酸扩增出单链DNA目标模板后,其两端分别是两个内引物的相关序列F2c、B2,其互补链两端分别是F2、B2c;其中,B2c是B2的互补序列;步骤3)中,DNA目标模板及其互补链能被DNA连接酶连接形成一个闭合的环化DNA链;步骤4)中,在至少含有带有链置换功能的DNA聚合酶的反应溶液中,至少以FIP和BIP这两个引物启动环化DNA链的滚环扩增。In the step 1), the FIP only includes the F2 sequence, and F2 is complementary to the target nucleic acid sequence F2c; the BIP only includes the B2 sequence, and B2 is identical to the partial sequence of the target nucleic acid; in the step 2), using primers F3, FIP, BIP, After B3 amplifies the single-stranded DNA target template from the target nucleic acid, its two ends are the relevant sequences F2c and B2 of the two inner primers respectively, and the two ends of the complementary chain are F2 and B2c respectively; wherein, B2c is the complementary sequence of B2; In step 3), the DNA target template and its complementary strand can be joined by DNA ligase to form a closed circularized DNA strand; in step 4), in the reaction solution containing at least a DNA polymerase with strand displacement function, at least Rolling circle amplification of circularized DNA strands was initiated with two primers, FIP and BIP.

所述步骤1)中,寡核苷酸序列还设计有辅助引物;所述辅助引物为若干个:该辅助引物来源于BIP引物中不影响B2与靶核酸序列稳定结合的5’端序列或BIP引物与B3引物之间的序列,或该辅助引物还同时来源于FIP引物中不影响F2与靶核酸序列稳定结合的3’端序列或FIP引物与F3引物之间的序列。In the step 1), the oligonucleotide sequence is also designed with auxiliary primers; the auxiliary primers are several: the auxiliary primers are derived from the 5' end sequence or BIP that does not affect the stable binding of B2 to the target nucleic acid sequence in the BIP primer The sequence between the primer and the B3 primer, or the auxiliary primer is also derived from the 3' end sequence of the FIP primer that does not affect the stable combination of F2 and the target nucleic acid sequence or the sequence between the FIP primer and the F3 primer.

所述步骤1)中,前端内引物FIP按5’→3’序列方向至少包含2段序列F1c、F2,其中,F2与F2c互补,F2c是靶核酸序列F3c在靶核酸5’方向的部分序列;FIP引物中的F1c是F1的互补序列,F1c与处在靶核酸序列F2c的5’方向的部分序列相同,F3c是F3的互补序列;所述后端内引物BIP按3’→5’序列方向至少包含2段序列B2、B1c,其中,BIP引物中的B2与靶核酸部分序列相同,B1c是与处在靶核酸B2序列的3’方向的部分序列B1互补;所述F1c与B1在靶核酸序列上是相邻无碱基间隔的前后两段序列;步骤2)中,利用引物F3、FIP、BIP、B3从靶核酸扩增出单链DNA目标模板后,DNA目标模板及其互补链两端分别形成发夹结构;其中,F1、B1c之间或F1c、B1之间形成了没有碱基缺口的杂交缝隙;步骤3)中,DNA目标模板及其互补链两端之间的间隙能被DNA连接酶连接闭合,形成环化DNA链;步骤4)中,在至少含有带有链置换功能的DNA聚合酶的反应溶液中,至少以FIP和BIP两个内引物启动环化DNA链的滚环扩增。In the step 1), the front-end internal primer FIP contains at least two sequences F1c and F2 in the sequence direction of 5'→3', wherein F2 and F2c are complementary, and F2c is a partial sequence of the target nucleic acid sequence F3c in the 5' direction of the target nucleic acid ; F1c in the FIP primer is the complementary sequence of F1, F1c is identical with the partial sequence in the 5' direction of the target nucleic acid sequence F2c, and F3c is the complementary sequence of F3; the primer BIP in the back end is in the sequence of 3'→5' The direction includes at least two sequences B2 and B1c, wherein B2 in the BIP primer is identical to the partial sequence of the target nucleic acid, and B1c is complementary to the partial sequence B1 in the 3' direction of the target nucleic acid B2 sequence; The nucleic acid sequence is two sequences before and after adjacent abasic intervals; in step 2), after using primers F3, FIP, BIP, and B3 to amplify the single-stranded DNA target template from the target nucleic acid, the DNA target template and its complementary strand The two ends respectively form a hairpin structure; among them, a hybridization gap without a base gap is formed between F1 and B1c or between F1c and B1; in step 3), the gap between the two ends of the DNA target template and its complementary strand can be DNA ligase joins and closes to form a circularized DNA chain; in step 4), in a reaction solution containing at least a DNA polymerase with strand displacement function, at least two internal primers, FIP and BIP, are used to initiate the rolling of the circularized DNA chain Circle amplification.

所述步骤1)中,寡核苷酸序列还设计有辅助引物;所述辅助引物为若干个;该辅助引物来源于BIP引物中不影响B2及B1c与靶核酸序列稳定结合的中间序列或BIP引物与B3引物之间的序列,或该辅助引物还同时来源于FIP引物中不影响F2及F1c与靶核酸序列稳定结合的中间序列或FIP引物与F3引物之间的序列。In the step 1), the oligonucleotide sequence is also designed with auxiliary primers; the auxiliary primers are several; the auxiliary primers are derived from the intermediate sequence or BIP in the BIP primers that do not affect the stable combination of B2 and B1c with the target nucleic acid sequence The sequence between the primer and the B3 primer, or the auxiliary primer is also derived from the intermediate sequence in the FIP primer that does not affect the stable combination of F2 and F1c with the target nucleic acid sequence, or the sequence between the FIP primer and the F3 primer.

所述步骤1)中,前端内引物FIP按5’→3’序列方向至少包含2段序列F4、F2,其中,F2与靶核酸序列F2c互补杂交;F4序列是与单链DNA目标模板不相同且不互补的DNA序列;所述后端内引物BIP按3’→5’序列方向至少包含2段序列B2、B4,其中,B2与靶核酸部分序列相同;B4序列是与单链DNA目标模板不相同且不互补的DNA序列;步骤1)中,还设有桥式引物BP,它按5’→3’序列方向分别与FIP中F4的互补序列F4c和BIP中B4的两个序列全部或部分碱基杂交互补,或者按5’→3’序列方向分别与BIP中B4的互补序列B4c和FIP中F4的两个序列全部或部分碱基杂交互补;FIP和BIP中最多有一条内引物中有部分序列不与桥式引物互补,而与靶核酸部分序列相同或互补,其位置在内引物F2或B2与靶核酸相同或互补位置的3’端方向;步骤2)中,利用引物F3、FIP、BIP、B3从靶核酸扩增出单链DNA目标模板后,DNA目标模板或其互补链的两端以桥式引物为互补链形成了没有碱基缺口的杂交缝隙;步骤3)中,DNA目标模板或其互补链的两端能被DNA连接酶连接为环化DNA链;步骤4)中,在至少含有带有链置换功能的DNA聚合酶的反应溶液中,至少以FIP和BIP两个内引物启动环化DNA链的滚环扩增。In the step 1), the front-end internal primer FIP contains at least two sequences F4 and F2 in the sequence direction of 5'→3', wherein, F2 is complementary to the target nucleic acid sequence F2c and hybridizes; the F4 sequence is different from the single-stranded DNA target template and a non-complementary DNA sequence; the back-end internal primer BIP contains at least two sequences B2 and B4 in the sequence direction of 3'→5', wherein, B2 is identical to the partial sequence of the target nucleic acid; the sequence of B4 is identical to the single-stranded DNA target template Different and non-complementary DNA sequences; in step 1), there is also a bridge primer BP, which is the complementary sequence F4c of F4 in FIP and the two sequences of B4 in BIP respectively in the 5'→3' sequence direction or all or Partial base hybridization and complementarity, or hybridization and complementarity with all or part of the bases of the complementary sequence B4c of B4 in BIP and F4 in FIP in the sequence direction of 5'→3'; FIP and BIP have at most one inner primer Part of the sequence is not complementary to the bridge primer, but is identical or complementary to the partial sequence of the target nucleic acid, and its position is in the direction of the 3' end of the same or complementary position of the internal primer F2 or B2 to the target nucleic acid; in step 2), use primers F3, After FIP, BIP, and B3 amplify the single-stranded DNA target template from the target nucleic acid, the two ends of the DNA target template or its complementary strand form a hybridization gap without base gaps with the bridge primer as the complementary strand; step 3), The two ends of the DNA target template or its complementary strand can be connected into a circularized DNA strand by DNA ligase; in step 4), in the reaction solution containing at least a DNA polymerase with strand displacement function, at least two FIP and BIP Each internal primer initiates rolling circle amplification of the circularized DNA strand.

所述桥式引物BP,能通过另外设计的四个引物序列从靶核酸序列中与单链DNA目标模板序列完全无关的序列中扩增剥离出来;其中,所述四个引物序列,为:重新设计的不同于F3、FIP、BIP、B3的内引物和外引物的各2条序列。The bridge primer BP can be amplified and stripped from the sequence completely unrelated to the single-stranded DNA target template sequence in the target nucleic acid sequence through four additionally designed primer sequences; wherein, the four primer sequences are: re- Two sequences of inner primers and outer primers different from F3, FIP, BIP, and B3 were designed.

所述步骤1)中寡核苷酸序列还设计有辅助引物;所述辅助引物来源于BIP引物中不影响B2与靶核酸序列稳定结合、B4与桥式引物稳定结合的中间序列或BIP引物与B3引物之间的序列,或该辅助引物还同时来源于FIP引物中不影响F2与靶核酸序列稳定结合、F4c与桥式引物稳定结合的中间序列或FIP引物与F3引物之间的序列。The oligonucleotide sequence in the step 1) is also designed with an auxiliary primer; the auxiliary primer is derived from the intermediate sequence in the BIP primer that does not affect the stable binding of B2 to the target nucleic acid sequence, and the stable binding of B4 to the bridge primer or the BIP primer and The sequence between the B3 primers, or the auxiliary primer is also derived from the intermediate sequence in the FIP primer that does not affect the stable binding of F2 to the target nucleic acid sequence, the stable binding of F4c to the bridge primer, or the sequence between the FIP primer and the F3 primer.

所述步骤2)中,逆转录酶包括:AMV或M-MLV;带有链置换功能的DNA聚合酶包括:Bst DNA聚合酶或Phi29DNA聚合酶;步骤2)中,至少含有带有链置换功能的DNA聚合酶的核酸扩增反应溶液,其组分还至少包括:扩增促进剂和核酸染料;其中,扩增促进剂包括以下的一个或多个成分的组合:甜菜碱,海藻糖、脯氨酸,二甲基亚砜、三甲胺-N-氧化物、氯化四甲基铵、甲酰胺、BSA、单链结合蛋白、T4Gene32Protein、homoectoine、Zn2+-cyclen(cyclen为1,4,7,10-tetraazacyclododecane);所述核酸染料包括:SYBR Green I、钙黄绿素、GELGREEN和GELRED。In the step 2), the reverse transcriptase includes: AMV or M-MLV; the DNA polymerase with strand displacement function includes: Bst DNA polymerase or Phi29 DNA polymerase; in step 2), at least the DNA polymerase with strand displacement function The nucleic acid amplification reaction solution of the DNA polymerase, its component also comprises at least: amplification promoter and nucleic acid dye; Wherein, the amplification promoter comprises the combination of following one or more components: betaine, trehalose, promethazine amino acid, dimethyl sulfoxide, trimethylamine-N-oxide, tetramethylammonium chloride, formamide, BSA, single-chain binding protein, T4Gene32Protein, homoectoine, Zn 2+ -cyclen (cyclen is 1,4, 7,10-tetraazacyclododecane); the nucleic acid dyes include: SYBR Green I, Calcein, GELGREEN and GELRED.

所述步骤2)中,在根据靶核酸扩增出单链DNA目标模板前,还能进行样品的靶核酸扩增产物污染消除处理:样品完成核酸提取后,加入尿嘧啶-N-糖基化酶,将可能对样品造成了污染的靶核酸扩增产物中的尿嘧啶进行糖苷键水解,此后,将溶液温度升高到55-98℃并保持最多10分钟,溶液中的尿嘧啶-N-糖基化酶将被灭活。In the step 2), before the single-stranded DNA target template is amplified according to the target nucleic acid, the target nucleic acid amplification product contamination elimination treatment of the sample can also be carried out: after the nucleic acid extraction of the sample is completed, uracil-N-glycosylated The enzyme hydrolyzes the glycosidic bond of uracil in the target nucleic acid amplification product that may have caused contamination to the sample. After that, the temperature of the solution is raised to 55-98°C and maintained for a maximum of 10 minutes. The uracil-N- in the solution Glycosylases will be inactivated.

所述污染消除处理、扩增单链目标模板、DNA连接酶环化目标模板、环化目标模板滚环扩增四个步骤中,各步骤能设置不同的温度条件,或将其中的若干步骤设置同一个温度;这四个步骤中的相关试剂成分能分别多次添加或一次添加完成。In the four steps of pollution elimination treatment, amplification of single-stranded target template, DNA ligase circularization target template, and circularization target template rolling circle amplification, different temperature conditions can be set for each step, or several steps can be set to The same temperature; the relevant reagent components in these four steps can be added multiple times or completed in one addition.

所述步骤3)、4)中,所述DNA连接酶包括:T4DNA ligase、Taq DNA Ligase、Ampligase和ssDNAligase。In the steps 3) and 4), the DNA ligase includes: T4DNA ligase, Taq DNA Ligase, Ampligase and ssDNAligase.

所述步骤4)中,带有链置换功能的DNA聚合酶包括:Bst DNA聚合酶或Phi29DNA聚合酶;所述含有DNA连接酶和带有链置换功能的DNA聚合酶的反应溶液,其组分还至少包括:扩增促进剂或核酸染料;其中,扩增促进剂包括以下的一个或多个成分的组合:甜菜碱,海藻糖、脯氨酸,二甲基亚砜、三甲胺-N-氧化物、氯化四甲基铵、甲酰胺、BSA、单链结合蛋白、T4Gene32Protein、homoectoine、Zn2+-cyclen;所述核酸染料包括:SYBR Green I、钙黄绿素、GELGREEN和GELRED。In step 4), the DNA polymerase with strand displacement function includes: Bst DNA polymerase or Phi29 DNA polymerase; the reaction solution containing DNA ligase and DNA polymerase with strand displacement function, its components It also includes at least: an amplification accelerator or a nucleic acid dye; wherein, the amplification accelerator includes a combination of one or more of the following components: betaine, trehalose, proline, dimethyl sulfoxide, trimethylamine-N- oxide, tetramethylammonium chloride, formamide, BSA, single-chain binding protein, T4Gene32Protein, homoectoine, Zn 2+ -cyclen; the nucleic acid dyes include: SYBR Green I, calcein, GELGREEN and GELRED.

所述步骤2)的扩增的单链DNA目标模板、步骤3)的环化DNA链和步骤4)中的核酸产物,能通过免疫层析试纸或荧光信号进行检测。如步骤2)的扩增的单链DNA目标模板、步骤3)的环化DNA链和步骤4)中的核酸产物可带有或结合了修饰标记分子的寡核苷酸序列;其中,该修饰标记分子能够被相应的配体识别,并能通过免疫层析试纸对产物进行检测;步骤2)的扩增的单链DNA目标模板、步骤3)的环化DNA链和步骤4)中的核酸产物还能够导致荧光信号的上升或下降,并能通过荧光信号对产物进行检测。The amplified single-stranded DNA target template in step 2), the circularized DNA strand in step 3) and the nucleic acid product in step 4) can be detected by immunochromatographic test paper or fluorescent signals. For example, the amplified single-stranded DNA target template in step 2), the circularized DNA strand in step 3), and the nucleic acid product in step 4) may carry or be combined with an oligonucleotide sequence of a modified labeling molecule; wherein, the modification The labeled molecule can be recognized by the corresponding ligand, and the product can be detected by immunochromatographic test paper; the amplified single-stranded DNA target template in step 2), the circularized DNA strand in step 3) and the nucleic acid in step 4) The product can also cause the rise or fall of the fluorescent signal, and the product can be detected by the fluorescent signal.

另外,根据上述方法,本发明还公开了一种应用于所述方法的试剂盒,至少包括上述的寡核苷酸序列。In addition, according to the above method, the present invention also discloses a kit applied to the method, comprising at least the above oligonucleotide sequence.

所述试剂盒还包括以下组分中的至少一种:The kit also includes at least one of the following components:

(1)上述的带有链置换功能DNA聚合酶;(1) The above-mentioned DNA polymerase with strand displacement function;

(2)上述的DNA连接酶;(2) The above-mentioned DNA ligase;

(3)上述的扩增促进剂;(3) The above-mentioned amplification promoters;

(4)上述的核酸染料;(4) The above-mentioned nucleic acid dyes;

(5)尿嘧啶-N-糖基化酶;(5) Uracil-N-glycosylase;

(6)上述的逆转录酶。(6) The above-mentioned reverse transcriptase.

本发明,通过内外各2条引物从靶核酸恒温扩增并剥离出的DNA单链目标模板两端可被合适的DNA连接酶在合适条件下连接形成一个闭合的环化目标模板。带有链置换功能的DNA聚合酶在恒温条件下,以闭合环状DNA为模板启动滚环扩增。滚环扩增产物中不断出现新的DNA单链目标模板,它将不断由DNA连接酶环化并启动新一轮滚环扩增。本发明能在一种溶液体系中一次添加完全部试剂后恒温保持一定时间后完成扩增。In the present invention, the two ends of the DNA single-strand target template that are amplified and stripped from the target nucleic acid by two internal and external primers can be ligated under suitable conditions by a suitable DNA ligase to form a closed circularized target template. DNA polymerase with strand displacement function initiates rolling circle amplification using closed circular DNA as a template under constant temperature conditions. New single-stranded DNA target templates constantly appear in the rolling circle amplification product, which will be continuously circularized by DNA ligase and start a new round of rolling circle amplification. The invention can complete the amplification after adding all the reagents in a solution system at a time and keeping the constant temperature for a certain period of time.

与现有技术相比,本发明的有益效果在于:Compared with prior art, the beneficial effect of the present invention is:

1)相比现有的锁式探针滚环扩增技术,本发明不用再合成高成本的长锁式探针,靶核酸的序列的长度能允许更大;1) Compared with the existing padlock probe rolling circle amplification technology, the present invention does not need to synthesize high-cost long padlock probes, and the sequence length of the target nucleic acid can be larger;

2)相比现有的酶切式滚环扩增技术,本发明通过引物的设计,能对靶核酸任意序列进行滚环扩增;2) Compared with the existing enzyme-cut rolling circle amplification technology, the present invention can carry out rolling circle amplification on any sequence of target nucleic acid through the design of primers;

3)相比现有的滚环扩增技术及其他的恒温扩增技术(LAMP除外)都需要分步在不同溶液体系中不同温度操作,本发明能在一种溶液体系中一次添加完全部试剂后恒温保持一定时间就能完成扩增,这为扩增产物的封闭扩增,并对防止样品被扩增产物污染提供了解决方法;3) Compared with the existing rolling circle amplification technology and other constant temperature amplification technologies (except LAMP), which need to be operated at different temperatures in different solution systems step by step, the present invention can add all the reagents in one solution system at one time The amplification can be completed by keeping the constant temperature for a certain period of time, which provides a solution for the closed amplification of the amplification product and to prevent the sample from being contaminated by the amplification product;

4)相比目前能在一种溶液体系中一次添加全部试剂并恒温扩增的LAMP技术,本发明能提供数量更少更简单的引物设计,有的方法中只要与靶核酸相关的四个引物,有的方法中需要5个与靶核酸相关的引物。鉴于RNA病毒很容易基因变异,即使在保守序列上也存在点突变,数量少的引物就能更便于进行种属范围的特异性设计;4) Compared with the current LAMP technology that can add all the reagents in one solution system at one time and amplify at a constant temperature, the present invention can provide fewer and simpler primer designs. In some methods, only four primers related to the target nucleic acid are required , some methods require 5 primers related to the target nucleic acid. In view of the fact that RNA viruses are easy to mutate genes, and there are point mutations even in conserved sequences, a small number of primers can be more convenient for species-specific design;

5)相比现有的恒温扩增技术,本发明将UNG酶运用到恒温的滚环扩增方法中,在开始扩增前将样品可能的扩增产物污染进行彻底消除。这就从技术上解决了基层单位无法做到检测分区操作而无法使用现有核酸试剂的问题,真正开启了基因检测步入基层单位应用的新时代。5) Compared with the existing constant temperature amplification technology, the present invention applies UNG enzyme to the constant temperature rolling circle amplification method to completely eliminate possible amplification product contamination of the sample before starting the amplification. This technically solves the problem that grassroots units cannot use existing nucleic acid reagents due to the inability to perform detection partition operations, and truly opens a new era of genetic testing entering grassroots units.

附图说明Description of drawings

图1是本发明的恒温滚环扩增示意图;Fig. 1 is the schematic diagram of constant temperature rolling circle amplification of the present invention;

图1中,按照靶核酸3’-5’序列方向依次设有四段引物:F3,FIP,BIP,B3。其中FIP为前端内引物,BIP为后端内引物,F3、B3是一对外引物,分别位于FIP、BIP的上游和下游,在带有链置换功能的DNA聚合酶、必要时可增加的逆转录酶、合适的扩增促进剂作用下,四个引物以靶核酸为模板扩增并剥离出一条单链目标模板。In Figure 1, there are four primers in sequence according to the 3'-5' sequence direction of the target nucleic acid: F3, FIP, BIP, B3. Among them, FIP is the front-end inner primer, BIP is the back-end inner primer, and F3 and B3 are a pair of outer primers, which are respectively located upstream and downstream of FIP and BIP. DNA polymerase with strand displacement function and reverse transcription can be increased if necessary. Under the action of enzymes and appropriate amplification promoters, the four primers use the target nucleic acid as a template to amplify and strip off a single-stranded target template.

根据两条内引物设计的不同,分别衍生出三种扩增方式:According to the difference in the design of the two internal primers, three amplification methods are derived respectively:

第一种方式是自接引物滚环扩增(SP-RCA):FIP前端内引物FIP只包含F2序列;后端内引物BIP内只有B2序列;单链DNA目标模板被复制合成后,其两端分别是F2c、B2,可被DNA连接酶连接形成一个闭合的环化DNA链;所述环化DNA链将在FIP引物的启动下进行滚环扩增。The first method is self-primed rolling circle amplification (SP-RCA): the FIP front-end primer FIP only contains the F2 sequence; the back-end primer BIP only contains the B2 sequence; after the single-stranded DNA target template is copied and synthesized, the two The ends are F2c and B2 respectively, which can be ligated by DNA ligase to form a closed circularized DNA chain; the circularized DNA chain will undergo rolling circle amplification under the initiation of FIP primers.

第二种方式是发夹引物滚环扩增(HP-RCA):前端内引物FIP按5’→3’序列方向包含F1c、F2;后端内引物BIP按3’→5’序列方向包含B2、B1c。同时F1c与B1在靶核酸序列上是相邻无碱基间隔的前后两段序列;目标模板被复制合成后,其两端分别是两个内引物的相关序列F1、B1c,而F1与F1c互补,B1与B1c互补,目标模板两端分别形成发夹结构,其中F1、B1c之间形成了没有碱基缺口的缝隙;目标模板两端之间的间隙被DNA连接酶连接闭合,及其互补链变成了环化DNA链。与环化DNA链互补的FIP内引物启动了滚环扩增The second method is hairpin primer rolling circle amplification (HP-RCA): the front-end internal primer FIP contains F1c and F2 in the 5'→3' sequence direction; the back-end internal primer BIP contains B2 in the 3'→5' sequence direction , B1c. At the same time, F1c and B1 are the two sequences before and after the adjacent abasic interval on the target nucleic acid sequence; after the target template is copied and synthesized, its two ends are the related sequences F1 and B1c of the two internal primers, and F1 and F1c are complementary , B1 and B1c are complementary, and the two ends of the target template form a hairpin structure respectively, and a gap without a base gap is formed between F1 and B1c; the gap between the two ends of the target template is connected and closed by DNA ligase, and its complementary strand into a circularized DNA strand. Inner FIP primers complementary to circularized DNA strands initiate rolling circle amplification

第三种方式是桥式发夹引物滚环扩增(BH-RCA):前端内引物FIP按5’→3’序列方向包含F4、F2。后端内引物BIP按3’→5’序列方向包含B2、B4。桥式引物BP按5’→3’序列方向分别与F4的互补序列F4c和B4两个序列全部杂交互补。目标模板被复制合成后,其两端分别是F4c、B4;桥式引物的5’端部分序列与B4全部杂交,它3’端部分序列与F4c全部杂交;目标模板的两端F4c与B4都以桥式引物为互补链形成了没有碱基缺口的杂交缝隙,被合适的DNA连接酶连接为环化目标模板;FIP内引物的中部序列不与桥式引物互补,而与靶核酸部分序列相同。复制的目标模板中FIP内引物的这个中部序列的互补序列能与靶核酸杂交,因此环化后的目标模板呈哑铃状结构。与环化目标模板互补的内引物与BP同时作为起始引物启动滚环扩增。The third method is bridge hairpin primer rolling circle amplification (BH-RCA): the front-end inner primer FIP includes F4 and F2 in the sequence direction of 5'→3'. The primer BIP in the back end contains B2 and B4 in the sequence direction of 3'→5'. The bridge primer BP was hybridized and complementary to the two complementary sequences of F4, F4c and B4, respectively in the 5'→3' sequence direction. After the target template is copied and synthesized, its two ends are F4c and B4 respectively; the 5' end part sequence of the bridge primer is fully hybridized with B4, and its 3' end part sequence is fully hybridized with F4c; both ends of the target template are F4c and B4. The bridge primer is used as the complementary strand to form a hybridization gap without base gaps, which is connected by a suitable DNA ligase as a circularized target template; the middle sequence of the primer in FIP is not complementary to the bridge primer, but is identical to the partial sequence of the target nucleic acid . The complementary sequence of the middle sequence of the primer in the FIP in the copied target template can hybridize with the target nucleic acid, so the circularized target template has a dumbbell-shaped structure. The inner primer complementary to the circularized target template and BP are simultaneously used as initial primers to initiate rolling circle amplification.

图2是本发明的扩增方法示意图。Fig. 2 is a schematic diagram of the amplification method of the present invention.

图2中,对自接引物滚环扩增(SP-RCA)的一个图示说明:按照靶核酸3’-5’序列方向依次设有四段引物:F3,FIP,BIP,B3。其中FIP为前端内引物,BIP为后端内引物,F3、B3是一对外引物,分别位于FIP、BIP的上游和下游,在带有链置换功能的DNA聚合酶、必要时可增加的逆转录酶、合适的扩增促进剂作用下,四个引物以靶核酸为模板扩增并剥离出一条单链目标模板。DNA目标模板首尾两端在恒温中被合适的DNA连接酶连接形成闭合的环化目标模板。In Figure 2, a schematic illustration of self-primer rolling circle amplification (SP-RCA): four primers are set in sequence according to the 3'-5' sequence direction of the target nucleic acid: F3, FIP, BIP, B3. Among them, FIP is the front-end inner primer, BIP is the back-end inner primer, and F3 and B3 are a pair of outer primers, which are respectively located upstream and downstream of FIP and BIP. DNA polymerase with strand displacement function and reverse transcription can be increased if necessary. Under the action of enzymes and appropriate amplification promoters, the four primers use the target nucleic acid as a template to amplify and strip off a single-stranded target template. The first and last ends of the DNA target template are ligated by a suitable DNA ligase at a constant temperature to form a closed circularized target template.

能与环化目标模板互补的起始引物FIP沿环化目标模板从3’端启动DNA滚环扩增,周而复始地进行环化目标模板的复制,扩增产物是以起始引物为起点的数千倍于目标模版单环长度的串联重复拷贝,每个单环序列中都有与BIP引物互补的序列,BIP引物杂交并启动反向的DNA链合成,可复制出终止于串联重复拷贝的扩增产物末端即FIP引物5’尾端的互补链。该终止于FIP引物5’尾端的互补链中包含单个或多个长度的目标模板,FIP引物将与每个目标模板中的互补序列杂交而启动新的复制。包含了多个长度目标模板的互补链将在FIP和BIP内引物的交替复制与剥离后产生单个长度的目标模板;该新复制的单个长度目标模板将不断由连接酶环化并启动新一轮的滚环扩增。如此周而复始,将扩增复制出巨量的核酸产物The initial primer FIP, which can be complementary to the circularized target template, starts DNA rolling circle amplification from the 3' end of the circularized target template, and repeats the replication of the circularized target template. A tandem repeat copy thousands of times the length of the target template single loop, each single loop sequence has a sequence complementary to the BIP primer, the BIP primer hybridizes and initiates reverse DNA strand synthesis, and can replicate the amplified terminating in the tandem repeat copy The end of the amplified product is the complementary strand at the 5' end of the FIP primer. The complementary strand terminating at the 5' end of the FIP primer contains target templates of single or multiple lengths, and the FIP primer will hybridize with the complementary sequence in each target template to initiate new replication. Complementary strands containing target templates of multiple lengths will generate a single-length target template after alternate replication and stripping of primers in FIP and BIP; this newly replicated single-length target template will be continuously circularized by ligase and start a new round rolling circle amplification. Repeatedly, a huge amount of nucleic acid products will be amplified and copied

具体实施方式Detailed ways

下面通过实施例对本发明作进一步详细说明。The present invention will be described in further detail below by way of examples.

以下实施例中涉及的化学试剂如未特别说明,则是采用市售的商业化产品。The chemical reagents involved in the following examples are commercially available commercial products unless otherwise specified.

实施例1:桥式发夹引物BHP-RCA检测双链DNAExample 1: Bridge hairpin primer BHP-RCA detects double-stranded DNA

该实施例采用单增李斯特(Listeria monocytogenes)菌细菌培养物作为样本。菌种购自ATCC(ATCC19116),取1μL原种菌液到3mlTSBYE培养基中,于35℃振荡培养24小时,取200μL细菌悬浮液,5000rpm离心2分钟,收集菌体用做后续DNA抽提。根据NCBI公布的序列信息,以单增李斯特菌较保守的溶血素编码基因hlyA为靶序列,部分序列如下(GenBank NO.AB566375):In this embodiment, a bacterial culture of Listeria monocytogenes is used as a sample. Bacteria were purchased from ATCC (ATCC19116). Take 1 μL of the original seed solution into 3 ml of TSBYE medium, shake and culture at 35°C for 24 hours, take 200 μL of bacterial suspension, centrifuge at 5000 rpm for 2 minutes, and collect the bacteria for subsequent DNA extraction. According to the sequence information released by NCBI, the relatively conserved hemolysin coding gene hlyA of Listeria monocytogenes was used as the target sequence, and the partial sequence is as follows (GenBank No.AB566375):

CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCATATGGCCGTCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAGAACTGACAAATATCAT(如SEQ ID NO.1所示)CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATCTCCAAGTGTGGCATATGGCCGTCCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAGAACTGACAAATATCAT (shown in SEQ ID NO.1)

1、据上述序列,设计所需的五种引物,并经由引物合成公司完成,引物序列如下:1. According to the above sequences, design the required five primers and complete them through the primer synthesis company. The primer sequences are as follows:

外引物F3:CGGCAAAGCTGTTACTA(如SEQ ID NO.2所示)Outer primer F3: CGGCAAAGCTGTTACTA (as shown in SEQ ID NO.2)

外引物B3:GTCAGTTCTACATCACC(如SEQ ID NO.3所示)Outer primer B3: GTCAGTTTCATCATCACC (as shown in SEQ ID NO.3)

内引物FIP:TGAATCCGTTAGTTTTTATATGCAGGAGGGCAGTTGCAAGCGCTTGG(如SEQ ID NO.4所示)Internal primer FIP: TGAATCCGTTAGTTTTTTATATGCAGGAGGGCAGTTGCAAGCGCTTGG (as shown in SEQ ID NO.4)

内引物BIP:AATAAGGAGGCGGCTGCTGG GACAGATTTCCCACTTACG(如SEQ ID NO.5所示)Internal primer BIP: AATAAGGAGGCGGCTGCTGG GACAGATTTCCCACTTACG (as shown in SEQ ID NO.5)

桥式引物BP:CTAACGGATTCAAATAAGGAGG(如SEQ ID NO.6所示)Bridge primer BP: CTAACGGATTCAAATAAGGAGG (as shown in SEQ ID NO.6)

2、样品处理:2. Sample processing:

采用商品化的细菌核酸提取试剂盒进行DNA抽提,得到单增李斯特菌的DNA模板,浓度为320ng/μL。A commercial bacterial nucleic acid extraction kit was used for DNA extraction to obtain a DNA template of Listeria monocytogenes at a concentration of 320 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

体系配制如下:The system configuration is as follows:

10×BST Buffer(NEB公司)                       1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(Thermo公司)            1μL10×Taqe ligase Buffer (Thermo Company) 1 μL

dNTP(25mM,Takara公司)                        1μLdNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                            0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                            0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                           0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                           0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                          0.2μLBridge primer BP (100μM) 0.2μL

DNA模板                                         1μLDNA template 1 μL

Bst酶(NEB公司,稀释成8U/μl)                   1μLBst enzyme (NEB company, diluted to 8U/μl) 1 μL

Taqe ligase(Thermo公司,40U/μL)            0.5μLTaqe ligase (Thermo, 40U/μL) 0.5μL

ddH2O                                        13.7μLddH 2 O 13.7 μL

将上述反应体系配制好后,混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100mL)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。结果表明,用本发明所述方法,经过1小时恒温扩增反应可很好地检测出目标模板。After the above reaction system is prepared, mix well, centrifuge at 3000rpm for 30 seconds, and amplify at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100mL) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band. The results show that the target template can be well detected after 1 hour constant temperature amplification reaction by using the method of the present invention.

另外,根据上述的恒温扩增反应体系的组分,也可制备成相应的检测试剂盒,以便广泛使用,即该检测试剂盒(不含有DNA模板),其组分含有:外引物F3、外引物B3、内引物FIP、内引物BIP、桥式引物BP;另外,该检测试剂盒的组分还可含有10×BST Buffer、10×Taqe ligase Buffer、dNTP、Bst酶、Taqeligase和ddH2O。In addition, according to the components of the above constant temperature amplification reaction system, a corresponding detection kit can also be prepared for wide use, that is, the detection kit (without DNA template), its components include: outer primer F3, outer Primer B3, inner primer FIP, inner primer BIP, bridge primer BP; in addition, the components of the detection kit may also contain 10×BST Buffer, 10×Taqe ligase Buffer, dNTP, Bst enzyme, Taqeligase and ddH 2 O.

4、扩增反应时间对结果的影响:4. The effect of amplification reaction time on the result:

步骤3所述反应体系中,在其它条件不变的情况下,仅改变恒温扩增反应时间,分别在20min、30min、50min进行64℃扩增反应,试验结果表明,用本发明所述方法,经过30分钟恒温扩增反应,即可很好地检测出目标模板。In the reaction system described in step 3, under the condition that other conditions remain unchanged, only the constant temperature amplification reaction time is changed, and the amplification reaction at 64°C is carried out at 20min, 30min, and 50min respectively. The test results show that with the method of the present invention, After 30 minutes of constant temperature amplification reaction, the target template can be well detected.

5、扩增反应温度对结果的影响:5. Effect of amplification reaction temperature on the result:

步骤3所述反应体系中,在其它条件不变的情况下,仅改变反应温度,在45℃、48℃、50℃、52℃、55℃、58℃、60℃、62℃、65℃、70℃温度条件下,恒温扩增1小时,试验结果表明:本发明最适反应温度为:62-64℃。In the reaction system described in step 3, when other conditions remain unchanged, only the reaction temperature is changed, at 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C, 62°C, 65°C, Under the temperature condition of 70°C, constant temperature amplification was carried out for 1 hour, and the test results showed that the optimum reaction temperature of the present invention was: 62-64°C.

实施例2:桥式发夹引物BHP-RCA检测单链DNAExample 2: Bridge hairpin primer BHP-RCA detects single-stranded DNA

本实施案例采用猪圆环病毒(Porcine Circovirus,PCV)灭活疫苗作为样本。疫苗购自哈尔滨维科生物技术开发公司,该疫苗灭活前含毒量不低于105.5TCID50。PCV是单链闭合环状DNA病毒,基因组全长约1.76kb。PCV2具有较高的致病性,是仔猪断奶后多系统衰竭综合症的主要病原。根据NCBI公布的序列信息,以PCV2较保守复制相关基因Rep为靶序列,部分序列如下(GenBank NO. AF207700):This implementation case uses porcine circovirus (Porcine Circovirus, PCV) inactivated vaccine as a sample. The vaccine was purchased from Harbin Veken Biotechnology Development Company, and the virus content of the vaccine was not less than 105.5 TCID50 before inactivation. PCV is a single-stranded closed circular DNA virus with a full-length genome of about 1.76kb. PCV2 has high pathogenicity and is the main pathogen of post-weaning multisystemic wasting syndrome in piglets. According to the sequence information published by NCBI, the relatively conservative replication-related gene Rep of PCV2 is used as the target sequence, and the partial sequence is as follows (GenBank No. AF207700):

CTAGATCTCAAGGACAACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGGCCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACGAATGTACACGTCATTGTGGGGCCACCTGGGTGTG(如SEQ ID NO.7所示)CTAGATCTCAAGGACAACGGAGTGACCTTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGGCCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACGAATGTACACGTCATTGTGGGGCCACNOGGGTGTG (as shown in SEQ ID 7)

1、据上述序列,设计所需的五种引物,并经由引物合成公司完成,引物序列如下:1. According to the above sequences, design the required five primers and complete them through the primer synthesis company. The primer sequences are as follows:

外引物F3:AGATCTCAAGGACAACG(如SEQ ID NO.8所示)Outer primer F3: AGATCTCAAGGACAACG (as shown in SEQ ID NO.8)

外引物B3:GTCATTGTGGGGCCACCT(如SEQ ID NO.9所示)Outer primer B3: GTCATTGTGGGGCCACCT (as shown in SEQ ID NO.9)

内引物FIP: TGAATCCGTTAGTTTTTCTCCCGCTCTCCGACCTGTCTACTGCTGTGAG (如SEQ ID NO.10所示)Internal primer FIP: TGAATCCGTTAGTTTTTCTCCCGCTCTCCGACCTGTCTACTGCTGTGAG (as shown in SEQ ID NO.10)

内引物BIP:AATAAGGAGGCGGCTGCTGG- CATTCGTCTTCCAATCACG (如SEQ ID NO.11所示)Internal primer BIP: AATAAGGAGGCGGCTGCTGG- CATTCGTCTTCCAATCACG (as shown in SEQ ID NO.11)

桥式引物BP:CTAACGGATTCAAATAAGGAGG(如SEQ ID NO.12所示)Bridge primer BP: CTAACGGATTCAAATAAGGAGG (as shown in SEQ ID NO.12)

2、样品处理:2. Sample processing:

采用商品化的病毒核酸提取试剂盒进行核酸抽提,得到PCV2的DNA模板,浓度为130ng/μL。A commercial viral nucleic acid extraction kit was used for nucleic acid extraction to obtain a PCV2 DNA template with a concentration of 130 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

体系配制如下:The system configuration is as follows:

10×BST Buffer(NEB公司)                1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(Thermo公司)     1μL10×Taqe ligase Buffer (Thermo Company) 1 μL

dNTP(25mM,Takara公司)                 1μLdNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                     0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                     0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                    0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                    0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                   0.2μLBridge primer BP (100μM) 0.2μL

DNA模板                                  1μLDNA template 1 μL

Bst酶(NEB公司,稀释成8U/μl)           1μLBst enzyme (NEB company, diluted to 8U/μl) 1 μL

Taq ligase(Thermo公司,40U/μL)      0.5μLTaq ligase (Thermo, 40U/μL) 0.5μL

ddH2O                                 13.7μLddH 2 O 13.7 μL

将上述反应体系配制好后混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100ml)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。结果表明,用本发明所述方法,经过1小时恒温扩增反应可很好地检测出猪伪狂犬病毒DNA。The above reaction system was prepared and mixed evenly, centrifuged at 3000rpm for 30 seconds, and amplified at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100ml) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band. The result shows that the porcine pseudorabies virus DNA can be well detected through the 1-hour constant temperature amplification reaction by using the method of the present invention.

实施例3:桥式发夹引物BHP-RCA检测RNAExample 3: Bridge hairpin primer BHP-RCA detects RNA

本实施例采用人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)为检测对象。病毒RNA样本由厦门万泰沧海生物科技有限公司徐飞海博士惠赠。HIV为单链RNA逆转录病毒,绝大多数艾滋病由HIV-1型引起。根据NCBI公布的序列信息,以HIV-1比较保守的结构蛋白gag基因为靶序列,部分序列如下In this embodiment, human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is used as the detection object. Viral RNA samples were donated by Dr. Xu Feihai from Xiamen Wantai Canghai Biotechnology Co., Ltd. HIV is a single-stranded RNA retrovirus, and the vast majority of AIDS is caused by HIV-1. According to the sequence information published by NCBI, the gag gene, a relatively conserved structural protein of HIV-1, is used as the target sequence, and the partial sequence is as follows

(GenBank NO.:AF128998):(GenBank NO.: AF128998):

TCGACGGAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAATTTTTTACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATAAATGGGAGAAAATTCGGTTAAGGCCAGGAGGAAAGAAAACATATCAGTTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAA(如SEQ ID NO.13所示)TCGACGGAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAATTTTTACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATAAATGGGAGAAAATTCGGTTAAGGCCAGGAGGAAAGAAAACATATCAGTNOAAACATATAGTAT as shown in GGGCGA1GC

1、据上面的序列,设计了所需的五种引物,引物序列如下:1. According to the above sequence, design the required five primers, the primer sequence is as follows:

外引物F3:CGACGGAGGACTCGGCTTG(如SEQ ID NO.14所示)Outer primer F3: CGACGGAGGACTCGGCTTG (as shown in SEQ ID NO.14)

外引物B3:GCTTGCCCATACTATATG(如SEQ ID NO.15所示)Outer primer B3: GCTTGCCCATACTATATG (as shown in SEQ ID NO.15)

内引物FIP:TGAATCCGTTAGTTTTTCGTACTCACCAGAAGCGCGCACGGCAAG (如SEQ ID NO.16所示)Internal primer FIP: TGAATCCGTTAGTTTTTCGTACTCACCAGAAGCGCGCACGGCAAG (as shown in SEQ ID NO.16)

内引物BIP:AATAAGGAGGCGGCTGCTGGCTGATATGTTTTCTTTCCTCC (如SEQ ID NO.17所示)Internal primer BIP: AATAAGGAGGCGGCTGCTGGCTGATATGTTTTTCTTTCCTCC (as shown in SEQ ID NO.17)

桥式引物BP:CTAACGGATTCAAATAAGGAGG(如SEQ ID NO.18所示)Bridge primer BP: CTAACGGATTCAAATAAGGAGG (as shown in SEQ ID NO.18)

2、样品处理:2. Sample processing:

采用商品化的病毒核酸提取试剂盒进行核酸抽提得到HIV的RNA模板,浓度为210ng/μL。A commercial viral nucleic acid extraction kit was used for nucleic acid extraction to obtain an HIV RNA template at a concentration of 210 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

体系配制如下:The system configuration is as follows:

10×BST Buffer(NEB公司)                 1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(Thermo公司)      1μL10×Taqe ligase Buffer (Thermo Company) 1 μL

dNTP(25mM,Takara公司)                  1μLdNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                      0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                      0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                     0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                     0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                    0.2μLBridge primer BP (100μM) 0.2μL

RNA模板                                   1μLRNA template 1 μL

Bst酶(NEB,稀释成8U/μl)                1μLBst enzyme (NEB, diluted to 8U/μl) 1 μL

AMV酶(30U/μL,Takara)                  1μLAMV enzyme (30U/μL, Takara) 1 μL

Taq ligase(40U/μL,Thermo)           0.5μLTaq ligase (40U/μL, Thermo) 0.5μL

ddH2O                                  12.7μLddH 2 O 12.7 μL

将上述反应体系配制好后混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100ml)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。结果表明,用本发明所述方法,经过1小时恒温扩增反应可很好地检测出HIV-1。The above reaction system was prepared and mixed evenly, centrifuged at 3000rpm for 30 seconds, and amplified at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100ml) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band. The results show that HIV-1 can be well detected after 1 hour constant temperature amplification reaction by using the method of the present invention.

实施例4:桥式发夹引物BHP-RCA结合UNG酶防污染系统检测犬埃利希Example 4: Bridge hairpin primer BHP-RCA combined with UNG enzyme anti-pollution system to detect Ehrlich canis

本发明方法灵敏度高,非常容易造成气溶胶污染而造成假阳性结果。本发明方法可结合UNG酶(尿嘧啶-N-糖基化酶)防污染系统,避免假阳性结果的发生。埃利希是专性细胞内寄生物,造成犬免疫力低下、极度消瘦,犬埃利希病也被称为犬的艾滋病。犬埃利希基因组大小约为1000kbp,根据NCBI公布的序列信息,以犬埃利希较保守16S rDNA基因为靶序列进行检测,部分序列如下(GenBank NO.:AF162860)The method of the invention has high sensitivity and is very easy to cause aerosol pollution and cause false positive results. The method of the present invention can be combined with the UNG enzyme (uracil-N-glycosylase) anti-pollution system to avoid the occurrence of false positive results. Ehrlichi is an obligate intracellular parasite, causing low immunity and extreme emaciation in dogs. Canine Ehrlichi disease is also known as canine AIDS. The genome size of Ehrlich canis is about 1000kbp. According to the sequence information released by NCBI, the relatively conserved 16S rDNA gene of Ehrlich canis was used as the target sequence for detection. The partial sequence is as follows (GenBank NO.: AF162860)

GAGGGGGAAAGATTTATCGCTATTAGATGAGCCTACGTTAGATTAGCTAGTTGGTGAGGTAATGGCTTACCAAGGCTATGATCTATAGCTGGTCTGAGAGGACGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCTATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAACTC(如SEQ ID NO.19所示)GAGGGGGAAAGATTTATCGCTATTAGATGAGCCTACGTTAGATTAGCTAGTTGGTGAGGTAATGGCTTACCAAGGCTATGATCTATAGCTGGTCTGAGAGGACGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCTATGTCGT.GTGAGTGAAGAAGGTCTAG

1、据上面的序列,设计了所需的五种引物,引物序列如下:1. According to the above sequence, design the required five primers, the primer sequence is as follows:

外引物F3:GAAAGATTTATCGCTAT(如SEQ ID NO.20所示)Outer primer F3: GAAAGATTTATCGCTAT (as shown in SEQ ID NO.20)

外引物B3:CGAAGGCCTTCTTCACT(如SEQ ID NO.21所示)Outer primer B3: CGAAGGCCTTCTTCACT (as shown in SEQ ID NO.21)

内引物FIP:TGAATCCGTTAGTTTTTGCCTTGGTAAGCATGAGCCTACGTTAGAT (如SEQ ID NO.22所示)Internal primer FIP: TGAATCCGTTAGTTTTTTGCCTTGGTAAGCATGAGCCTACGTTAGAT (as shown in SEQ ID NO.22)

内引物BIP:AATAAGGAGGGCATAGCTGGATCAGGCT(如SEQ ID NO.23所示)Internal primer BIP: AATAAGGAGGGCATAGCTGGATCAGGCT (as shown in SEQ ID NO.23)

桥式引物BP:CTAACGGATTCAAATAAGGAGG(如SEQ ID NO.24所示)Bridge primer BP: CTAACGGATTCAAATAAGGAGG (as shown in SEQ ID NO.24)

2、样品处理:2. Sample processing:

临床确诊为犬艾利希病的病犬,取全血样本,采用商品化的全血基因组提取试剂盒进行核酸抽提,得到DNA模板,浓度为600ng/μL。Whole blood samples were taken from dogs clinically diagnosed with Ehrlich's disease, and nucleic acid was extracted using a commercial whole blood genome extraction kit to obtain DNA templates at a concentration of 600 ng/μL.

3、UNG酶处理、恒温扩增反应及检测:3. UNG enzyme treatment, constant temperature amplification reaction and detection:

体系配制如下:The system configuration is as follows:

10×BST Buffer(NEB公司)                1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(NEB公司)        1μL10×Taqe ligase Buffer (NEB Company) 1 μL

dUNTP(25mM,Takara公司)                1μLdUNTP (25mM, Takara company) 1μL

外引物F3(100μM)                     0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                     0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                    0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                    0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                   0.2μLBridge primer BP (100μM) 0.2μL

DNA模板                                  1μLDNA template 1 μL

Bst酶(NEB,稀释成8U/μl)               1μLBst enzyme (NEB, diluted to 8U/μl) 1 μL

Taq ligase(40U/μL,Thermo)          0.5μLTaq ligase (40U/μL, Thermo) 0.5μL

ddH2O                                 12.7μLddH 2 O 12.7 μL

将上述反应体系配制好后混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100ml)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。The above reaction system was prepared and mixed evenly, centrifuged at 3000rpm for 30 seconds, and amplified at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100ml) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band.

取上述扩增反应液0.1μL作为模板,结合UNG酶体系,验证其去污染效果。反应体系和方法如下:Take 0.1 μL of the above-mentioned amplification reaction solution as a template and combine it with the UNG enzyme system to verify its decontamination effect. Reaction system and method are as follows:

反应体系:reaction system:

10×BST Buffer(NEB公司)                 1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(Thermo公司)      1μL10×Taqe ligase Buffer (Thermo Company) 1 μL

dUNTP(25mM,Takara公司)                 1μLdUNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                      0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                      0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                     0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                     0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                    0.2μLBridge primer BP (100μM) 0.2μL

UNG酶(2U/μL)                         0.5μLUNG enzyme (2U/μL) 0.5 μL

上述扩增反应液                          0.1μLThe above amplification reaction solution 0.1μL

ddH2O                                   14.1μLddH 2 O 14.1 μL

上述反应体系在25℃温育10分钟,95℃作用2分钟使UNG酶失活。在反应体系中补入1μL Bst酶(NEB,稀释成8U/μl),0.5μL Taq ligase(Thermo,40U/μL)混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。同时做一管不经UNG酶处理的对照。反应产物经2%(2g/100ml)的琼脂糖凝胶电泳,结果表明:未经UNG酶处理的反应体系,最终扩增产物呈现明亮的Marker状的梯形条带,经过UNG酶处理的反应体系,扩增产物不产生任何条带,有效地避免了假阳性结果的产生。用本发明所述方法结合UNG酶防污染体系能有效避免假阳性结果的产生。The above reaction system was incubated at 25°C for 10 minutes, then acted at 95°C for 2 minutes to inactivate UNG enzyme. Add 1 μL Bst enzyme (NEB, diluted to 8 U/μl) and 0.5 μL Taq ligase (Thermo, 40 U/μL) into the reaction system, mix well, centrifuge at 3000 rpm for 30 seconds, and amplify at 64°C for 1 hour. At the same time, make a control tube without UNG enzyme treatment. The reaction product was subjected to 2% (2g/100ml) agarose gel electrophoresis, and the results showed that: the reaction system without UNG enzyme treatment, the final amplified product presented a bright Marker-like trapezoidal band, and the reaction system treated with UNG enzyme , the amplified product does not produce any bands, which effectively avoids the generation of false positive results. Using the method of the invention in combination with the UNG enzyme pollution prevention system can effectively avoid the generation of false positive results.

实施例5:自连引物SP-RCA检测双链DNAEmbodiment 5: Self-ligating primer SP-RCA detects double-stranded DNA

本发明方法可以不需要桥式引物,在单链DNA聚合酶的作用下使第一轮扩增产物直接成环,而得到RCA扩增的模板序列。以检测单增李斯特(Listeria monocytogenes)菌为例说明如下:The method of the present invention can directly form a circle of the first-round amplification product under the action of the single-stranded DNA polymerase without the bridge primer, so as to obtain the template sequence amplified by RCA. Take the detection of Listeria monocytogenes as an example to illustrate as follows:

采用单增李斯特菌细菌培养物作为样本。菌种购自ATCC(ATCC19116),取1μL原种菌液到3mlTSBYE培养基中,于35℃振荡培养24小时,取200μL细菌悬浮液,5000rpm离心2分钟,收集菌体用做后续DNA抽提。根据NCBI公布的序列信息,以单增李斯特菌较保守的溶血素编码基因hlyA为靶序列,部分序列如下(GenBank NO. AB566375):A bacterial culture of Listeria monocytogenes was used as the sample. Bacteria were purchased from ATCC (ATCC19116). Take 1 μL of the original seed solution into 3 ml of TSBYE medium, shake and culture at 35°C for 24 hours, take 200 μL of bacterial suspension, centrifuge at 5000 rpm for 2 minutes, and collect the bacteria for subsequent DNA extraction. According to the sequence information published by NCBI, the relatively conserved hemolysin coding gene hlyA of Listeria monocytogenes was used as the target sequence, and the partial sequence is as follows (GenBank No. AB566375):

CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCATATGGCCGTCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAGAACTGACAAATATCAT(如SEQ ID NO.1所示)CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATCTCCAAGTGTGGCATATGGCCGTCCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAGAACTGACAAATATCAT (shown in SEQ ID NO.1)

1、据上述序列,设计所需的四种引物,并经由引物合成公司完成,引物序列如下:1. According to the above sequences, design the four required primers and complete them through the primer synthesis company. The primer sequences are as follows:

外引物F3:CGGCAAAGCTGTTACTA(如SEQ ID NO.2所示)Outer primer F3: CGGCAAAGCTGTTACTA (as shown in SEQ ID NO.2)

外引物B3:GTCAGTTCTACATCACC(如SEQ ID NO.3所示)Outer primer B3: GTCAGTTTCATCATCACC (as shown in SEQ ID NO.3)

内引物FIP:GCAGTTGCAAGCGCTTGG(如SEQ ID NO.25所示)Internal primer FIP: GCAGTTGCAAGCGCTTGG (as shown in SEQ ID NO.25)

内引物BIP:GACAGATTTCCCACTTACG(如SEQ ID NO.26所示)Internal primer BIP: GACAGATTTCCCACTTACG (as shown in SEQ ID NO.26)

2、样品处理:2. Sample processing:

采用商品化的细菌核酸提取试剂盒进行DNA抽提,得到单增李斯特菌的DNA模板,浓度为320ng/μL。A commercial bacterial nucleic acid extraction kit was used for DNA extraction to obtain a DNA template of Listeria monocytogenes at a concentration of 320 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

20μL体系配制如下:The 20μL system was prepared as follows:

10×BST Buffer(NEB公司)                        1μL10×BST Buffer (NEB Company) 1 μL

10×ssDNA Ligase Buffer(Epicentre公司)         1μL10×ssDNA Ligase Buffer (Epicentre) 1 μL

dNTP(25mM,Takara公司)                         1μLdNTP (25mM, Takara company) 1 μL

ATP(1mM,Epicentre公司)                        1μLATP (1mM, Epicentre Company) 1 μL

MnCl2(50mM)                                      1μL MnCl2 (50mM) 1μL

外引物F3(100μM)                             0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                             0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                            0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                            0.2μLInternal primer BIP (100μM) 0.2μL

DNA模板                                          1μLDNA template 1 μL

Bst酶(NEB公司,稀释成8U/μl)                    1μLBst enzyme (NEB company, diluted to 8U/μl) 1 μL

ssDNA Ligase(100U/μL,Epicentre公司)          1μLssDNA Ligase (100U/μL, Epicentre Company) 1 μL

ddH2O                                         11.4μLddH 2 O 11.4 μL

将上述反应体系配好后混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100ml)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。结果表明,自连引物HP-RCA扩增方法,结合单链DNA连接酶,在没有桥式引物的情况下,经过1小时恒温扩增反应可很好地检测出目标模板。The above reaction system was prepared and mixed evenly, centrifuged at 3000rpm for 30 seconds, and amplified at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100ml) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band. The results showed that the self-ligating primer HP-RCA amplification method combined with single-strand DNA ligase can detect the target template well after 1 hour constant temperature amplification reaction without bridge primer.

实施例6:发夹引物HP-RCA检测双链DNAEmbodiment 6: Hairpin primer HP-RCA detects double-stranded DNA

本发明方法可以不需要桥式引物,利用特殊设计的FIP和BIP,在扩增之后形成发夹结构,扩增产物的两端直接紧密相连,在连接酶的作用下形成一个闭合环状结构,以此为模板进行后续RCA扩增。据此方法,以检测单增李斯特(Listeria monocytogenes)菌为例说明如下:The method of the present invention can use specially designed FIP and BIP without bridge primers to form a hairpin structure after amplification, the two ends of the amplified product are directly connected closely, and a closed circular structure is formed under the action of ligase, This was used as a template for subsequent RCA amplification. According to this method, take the detection of Listeria monocytogenes as an example to illustrate as follows:

采用单增李斯特菌细菌培养物作为样本。菌种购自ATCC(ATCC 19116),取1μL原种菌液到3mlTSBYE培养基中,于35℃振荡培养24小时,取200μL细菌悬浮液,5000rpm离心2分钟,收集菌体用做后续DNA抽提。根据NCBI公布的序列信息,以单增李斯特菌较保守的溶血素编码基因hlyA为靶序列,部分序列如下(GenBank NO. AB566375):A bacterial culture of Listeria monocytogenes was used as the sample. Bacteria were purchased from ATCC (ATCC 19116). Take 1 μL of the original seed liquid into 3ml TSBYE medium, shake and culture at 35°C for 24 hours, take 200 μL of bacterial suspension, centrifuge at 5000rpm for 2 minutes, and collect the bacteria for subsequent DNA extraction . According to the sequence information published by NCBI, the relatively conserved hemolysin coding gene hlyA of Listeria monocytogenes was used as the target sequence, and the partial sequence is as follows (GenBank No. AB566375):

CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCATATGGCCGTCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAG(如SEQ ID NO.27所示)CAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATCTCCAAGTGTGGCATATGGCCGTCCAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGACGCTGCCGTAAGTGGGAAATCTGTCTCAGGTGATGTAG (shown in SEQ ID NO. 27)

1、据上述序列,设计所需的四种引物,并经由引物合成公司完成,引物序列如下:1. According to the above sequences, design the four required primers and complete them through the primer synthesis company. The primer sequences are as follows:

外引物F3:CGGCAAAGCTGTTACTA(如SEQ ID NO.2所示)Outer primer F3: CGGCAAAGCTGTTACTA (as shown in SEQ ID NO.2)

外引物B3:CTTTTACTTTAGTACTATGG(如SEQ ID NO.28所示)Outer primer B3: CTTTTACTTTAGTACTATGG (as shown in SEQ ID NO.28)

内引物FIP:ATATGCAGGAGG GCAGTTGCAAGCGCTTGG(如SEQ ID NO.29所示)Internal primer FIP: ATATGCAGGAGG GCAGTTGCAAGCGCTTGG (as shown in SEQ ID NO.29)

内引物BIP:ATCTCAAGTGTGGTTGATAATTTCAAATAAACTTG(如SEQ ID NO.30所示)Internal primer BIP: ATCTCAAGTGTGGTTGATAATTTCAAATAAACTTG (as shown in SEQ ID NO.30)

2、样品处理:2. Sample processing:

采用商品化的细菌核酸提取试剂盒进行DNA抽提,得到单增李斯特菌的DNA模板,浓度为320ng/μL。A commercial bacterial nucleic acid extraction kit was used for DNA extraction to obtain a DNA template of Listeria monocytogenes at a concentration of 320 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

20μL体系配制如下:The 20μL system was prepared as follows:

10×BST Buffer(NEB公司)                        1μL10×BST Buffer (NEB Company) 1 μL

10×Taq DNA Ligase Buffer(Thermo公司)        1μL10×Taq DNA Ligase Buffer (Thermo Company) 1 μL

dNTP(25mM,Takara公司)                       1μLdNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                           0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                           0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                          0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                          0.2μLInternal primer BIP (100μM) 0.2μL

DNA模板                                        1μLDNA template 1 μL

Bst酶(NEB公司,稀释成8U/μl)                  1μLBst enzyme (NEB company, diluted to 8U/μl) 1 μL

Taq DNA ligase(Thermo公司,40U/μL)        0.5μLTaq DNA ligase (Thermo, 40U/μL) 0.5μL

ddH2O                                        13.9μLddH 2 O 13.9 μL

将上述反应体系配好后混合均匀,3000rpm离心30秒,64℃恒温扩增1小时。扩增产物经2%(2g/100ml)的琼脂糖凝胶电泳,呈现明亮的Marker状的梯形条带。结果表明,利用HP-RCA扩增方法,经过1小时恒温扩增反应,可很好地检测出目标核酸模板。The above reaction system was prepared and mixed evenly, centrifuged at 3000rpm for 30 seconds, and amplified at a constant temperature of 64°C for 1 hour. The amplified product was subjected to 2% (2g/100ml) agarose gel electrophoresis, showing a bright Marker-like ladder-shaped band. The results show that the target nucleic acid template can be well detected by using the HP-RCA amplification method after a 1-hour constant temperature amplification reaction.

实施例7:桥式发夹引物BHP-RCA结合分子信标实时检测目标模板Example 7: Bridge hairpin primer BHP-RCA combined with molecular beacons for real-time detection of target templates

本发明方法可以通过分子信标,实现对扩增产物的实时监测。本实施例以人类免疫缺陷病毒(HumanImmunodeficiency Virus,HIV)为例说明。HIV为单链RNA逆转录病毒,绝大多数艾滋病由HIV-1型引起。根据NCBI公布的序列信息,以HIV-1比较保守的结构蛋白gag基因为靶序列,部分序列如下(GenBank NO.:AF128998):The method of the present invention can realize the real-time monitoring of the amplification product through the molecular beacon. This embodiment is illustrated by taking human immunodeficiency virus (Human Immunodeficiency Virus, HIV) as an example. HIV is a single-stranded RNA retrovirus, and the vast majority of AIDS is caused by HIV-1. According to the sequence information released by NCBI, the HIV-1 conservative structural protein gag gene is used as the target sequence, and the partial sequence is as follows (GenBank NO.: AF128998):

TCGACGGAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAATTTTTTACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATAAATGGGAGAAAATTCGGTTAAGGCCAGGAGGAAAGAAAACATATCAGTTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAA(如SEQ ID NO.13所示)TCGACGGAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAATTTTTACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATAAATGGGAGAAAATTCGGTTAAGGCCAGGAGGAAAGAAAACATATCAGTNOAAACATATAGTAT as shown in GGGCGA1GC

1、据上面的序列,设计了所需的四种引物及一条检测用探针,引物序列如下:1. According to the above sequence, four required primers and a detection probe were designed. The primer sequence is as follows:

外引物F3:CGACGGAGGACTCGGCTTG(如SEQ ID NO.14所示)Outer primer F3: CGACGGAGGACTCGGCTTG (as shown in SEQ ID NO.14)

外引物B3:GCTTGCCCATACTATATG(如SEQ ID NO.15所示)Outer primer B3: GCTTGCCCATACTATATG (as shown in SEQ ID NO.15)

内引物FIP:TGAATCCGTTAGTTTTTCGTACTCACCAGAAGCGCGCACGGCAAG (如SEQ ID NO.16所示)Internal primer FIP: TGAATCCGTTAGTTTTTCGTACTCACCAGAAGCGCGCACGGCAAG (as shown in SEQ ID NO.16)

内引物BIP:AATAAGGAGGCGGCTGCTGGCTGATATGTTTTCTTTCCTCC (如SEQ ID NO.17所示)Internal primer BIP: AATAAGGAGGCGGCTGCTGGCTGATATGTTTTTCTTTCCTCC (as shown in SEQ ID NO.17)

桥式引物BP:CTAACGGATTCAAATAAGGAGG(如SEQ ID NO.18所示)Bridge primer BP: CTAACGGATTCAAATAAGGAGG (as shown in SEQ ID NO.18)

分子信标(检测用探针):FAM-cacctcGATGGGTGCGAGAGCGTCAGgaggtg-DABCYL(如SEQ ID NO.31所示);其中,FAM、DABCYL中文名称分别是羧基荧光素、4-(4-恶烷氨基苯偶氮)苯甲酸,它们分别是荧光基团和荧光淬灭基团,是分子信标最常用的一对组合。Molecular beacon (probe for detection): FAM-cacctcGATGGGTGCGAGAGCGTCAGgaggtg-DABCYL (as shown in SEQ ID NO.31); wherein, the Chinese names of FAM and DABCYL are carboxyfluorescein and 4-(4-oxanaminophenylazo ) Benzoic acid, which are fluorescent groups and fluorescent quenching groups, respectively, is the most commonly used pair combination of molecular beacons.

2、样品处理:2. Sample processing:

HIV病毒RNA样本由厦门万泰沧海生物科技有限公司徐飞海博士惠赠。采用商品化的病毒核酸提取试剂盒进行核酸抽提得到HIV的RNA模板,浓度为210ng/μL。The HIV virus RNA sample was donated by Dr. Xu Feihai from Xiamen Wantai Canghai Biotechnology Co., Ltd. A commercial viral nucleic acid extraction kit was used for nucleic acid extraction to obtain an HIV RNA template at a concentration of 210 ng/μL.

3、恒温扩增反应及检测:3. Isothermal amplification reaction and detection:

20μL体系配制如下:The 20μL system was prepared as follows:

10×BST Buffer(NEB公司)                    1μL10×BST Buffer (NEB Company) 1 μL

10×Taqe ligase Buffer(NEB公司)            1μL10×Taqe ligase Buffer (NEB Company) 1 μL

dNTP(25mM,Takara公司)                   1μLdNTP (25mM, Takara company) 1 μL

外引物F3(100μM)                        0.1μLOuter primer F3 (100μM) 0.1μL

外引物B3(100μM)                        0.1μLOuter primer B3 (100μM) 0.1μL

内引物FIP(100μM)                       0.2μLInternal primer FIP (100μM) 0.2μL

内引物BIP(100μM)                       0.2μLInternal primer BIP (100μM) 0.2μL

桥式引物BP(100μM)                      0.2μLBridge primer BP (100μM) 0.2μL

上述的分子信标(10μM)                   0.2μLThe above molecular beacon (10 μM) 0.2 μL

ROX Dye(150μM)                         0.2μLROX Dye (150μM) 0.2μL

DNA模板                                     1μLDNA template 1 μL

Bst酶(NEB,稀释成8U/μl)                  1μLBst enzyme (NEB, diluted to 8U/μl) 1 μL

AMV酶(30U/μL,Takara公司)                1μLAMV enzyme (30U/μL, Takara company) 1 μL

Taq ligase(40U/μL,Thermo公司)         0.5μLTaq ligase (40U/μL, Thermo Company) 0.5μL

ddH2O                                     12.3μLddH 2 O 12.3 μL

将上述反应体系配制好后,混合均匀,3000rpm离心30秒,64℃恒温扩增1小时,用ABI7500荧光定量PCR进行荧光收集,每30秒收集一次荧光信号。荧光曲线显示,在25分钟左右进入指数扩增期。结果表明,用本发明所述方法可以通过分子信标,实现对扩增产物的实时监测。After preparing the above reaction system, mix well, centrifuge at 3000rpm for 30 seconds, amplify at a constant temperature of 64°C for 1 hour, use ABI7500 fluorescent quantitative PCR to collect fluorescence, and collect fluorescence signals every 30 seconds. The fluorescence curve shows that it enters the exponential amplification phase at about 25 minutes. The result shows that the real-time monitoring of the amplified product can be realized through the molecular beacon by using the method of the present invention.

另外,根据实施例2-7的恒温扩增反应体系的组分,按照实施例1中的制备检测试剂盒方法,也可将实施例2-7的恒温扩增反应体系的组分(除去DNA模板外的组分)制备成相应的应用于制备单链DNA链的两个引物相关序列末端连接成环且能至少以该两个引物启动恒温滚环扩增的方法的检测试剂盒,并按照各实施例中的反应条件和检测方法进行检测。In addition, according to the components of the constant temperature amplification reaction system of Example 2-7, according to the method for preparing the detection kit in Example 1, the components of the constant temperature amplification reaction system of Example 2-7 (removing DNA Components outside the template) to prepare a corresponding detection kit for the method of connecting the two primer-related sequence ends of a single-stranded DNA strand to form a circle and at least using the two primers to initiate constant temperature rolling circle amplification, and according to The reaction conditions and detection methods in each embodiment are tested.

Figure IDA00003277402700011
Figure IDA00003277402700011

Figure IDA00003277402700021
Figure IDA00003277402700021

Figure IDA00003277402700041
Figure IDA00003277402700041

Figure IDA00003277402700051
Figure IDA00003277402700051

Figure IDA00003277402700061
Figure IDA00003277402700061

Figure IDA00003277402700071
Figure IDA00003277402700071

Figure IDA00003277402700081
Figure IDA00003277402700081

Figure IDA00003277402700091
Figure IDA00003277402700091

Figure IDA00003277402700101
Figure IDA00003277402700101

Claims (18)

1. two primer correlated series ends that prepare the single stranded DNA chain connect into the method for encircling and starting the constant temperature rolling circle amplifications at least with these two primers, it is characterized in that, comprise step:
1) oligonucleotide sequence design
Oligonucleotide sequence has four sections primers that design by target nucleic acid 3 ' → 5 ' direction at least: F3, FIP, BIP, B3;
Wherein, FIP is the front end inner primer;
BIP is the rear end inner primer;
F3, B3 are a pair of outer primers, lay respectively at the upstream of FIP and the downstream of BIP;
2) from target nucleic acid, amplify the single stranded DNA To Template
Target nucleic acid is DNA chain or when in the reaction soln that contains reversed transcriptive enzyme at least RNA chain target nucleic acid reverse transcription being the DNA chain, in containing the nucleic acid amplification reaction solution of the archaeal dna polymerase that has the strand displacement function at least, utilize primers F 3, FIP, BIP, B3, amplification synthesizes the single stranded DNA To Template according to target nucleic acid sequence;
3) dna ligase cyclized DNA To Template
In the reaction soln that contains dna ligase at least, the head and the tail two ends of DNA To Template are connected to form closed cyclized DNA chain;
4) cyclized DNA To Template rolling circle amplification
In the reaction soln that contains dna ligase and the archaeal dna polymerase that has the strand displacement function at least, start the rolling circle amplification of cyclized DNA chains at least with two inner primers in the step 1), the nucleic acid product that obtains increasing.
2. the method for claim 1, it is characterized in that: the oligonucleotide sequence of described step 1) also comprises: bridge-type primer BP, hybridization probe, auxiliary primer;
Described bridge-type primer BP, its all or part of sequence and the complementation of an inner primer afterbody sequence, and its all or part of and afterbody sequence complementation another inner primer complementary sequence makes it as bridge the adjacent no base breach of the two terminal sequences ground of these two inner primers to be hybridized together;
Described hybridization probe, itself and the sequence hybridization of target nucleic acid among two inner primer correlated series positions;
Described auxiliary primer derives from the partial sequence that does not influence itself and target nucleic acid or bridge-type primer stable bond in BIP or the FIP primer, maybe should auxiliary primer also derives from sequence between BIP primer and the B3 primer or the sequence between this FIP primer and the F3 primer.
3. the method for claim 1, it is characterized in that: the oligonucleotide sequence of described step 1) can also carry out similar and different modification mark respectively, and this modification mark comprises: digoxin, fluorescence dye FAM, fluorescein isothiocyanate, vitamin H, fluorescent quenching group or nano particle.
4. the method for claim 1, it is characterized in that: in the described step 1), FIP only comprises the F2 sequence, F2 and target nucleic acid sequence F2c complementation; Described BIP only comprises the B2 sequence, and B2 is identical with the target nucleic acid partial sequence;
Step 2) in, utilize primers F 3, FIP, BIP, B3 to amplify the single stranded DNA To Template from target nucleic acid after, its two ends are respectively correlated series F2c, the B2 of two inner primers, its complementary strand two ends are respectively F2, B2c; Wherein, B2c is the complementary sequence of B2;
In the step 3), DNA To Template and complementary strand thereof can be connected to form the cyclized DNA chain of a closure by dna ligase;
In the step 4), in the reaction soln that contains the archaeal dna polymerase that has the strand displacement function at least, start the rolling circle amplification of cyclized DNA chain at least with FIP and these two primers of BIP.
5. method as claimed in claim 4, it is characterized in that: in the described step 1), oligonucleotide sequence has also designed auxiliary primer;
Described auxiliary primer is several: this auxiliary primer derives from the 5 ' terminal sequence that do not influence B2 and target nucleic acid sequence stable bond in the BIP primer or the sequence between BIP primer and the B3 primer, maybe should auxiliary primer also derives from the 3 ' terminal sequence that do not influence F2 and target nucleic acid sequence stable bond in the FIP primer or the sequence between FIP primer and the F3 primer simultaneously.
6. the method for claim 1, it is characterized in that: in the described step 1), front end inner primer FIP comprises 2 sections sequence F1c, F2 at least by 5 ' → 3 ' sequence direction, wherein, F2 and F2c complementation, F2c is that target nucleic acid sequence F3c is in the partial sequence of target nucleic acid 5 ' direction; F1c in the FIP primer is the complementary sequence of F1, and F1c is identical with the partial sequence of the 5 ' direction that is in target nucleic acid sequence F2c, and F3c is the complementary sequence of F3;
Described rear end inner primer BIP comprises 2 sections sequence B 2, B1c at least by 3 ' → 5 ' sequence direction, and wherein, the B2 in the BIP primer is identical with the target nucleic acid partial sequence, and B1c is the partial sequence B1 complementation with the 3 ' direction that is in target nucleic acid B2 sequence;
Described F1c and B1 are adjacent no base two sections sequences in front and back at interval at target nucleic acid sequence;
Step 2) in, utilize primers F 3, FIP, BIP, B3 to amplify the single stranded DNA To Template from target nucleic acid after, DNA To Template and complementary strand two ends thereof form hairpin structure respectively; Wherein, formed between F1, the B1c or between F1c, the B1 and do not had the hybridization of base breach slit;
In the step 3), the gap between DNA To Template and the complementary strand two ends thereof can be connected closed by dna ligase, forms the cyclized DNA chain;
In the step 4), in the reaction soln that contains the archaeal dna polymerase that has the strand displacement function at least, start the rolling circle amplification of cyclized DNA chain at least with FIP and two inner primers of BIP.
7. method as claimed in claim 6, it is characterized in that: in the described step 1), oligonucleotide sequence has also designed auxiliary primer;
Described auxiliary primer is several; Should auxiliary primer derive from the intermediate sequence that do not influence B2 and B1c and target nucleic acid sequence stable bond in the BIP primer or the sequence between BIP primer and the B3 primer, maybe this auxiliary primer also derives from the intermediate sequence that do not influence F2 and F1c and target nucleic acid sequence stable bond in the FIP primer or the sequence between FIP primer and the F3 primer simultaneously.
8. the method for claim 1, it is characterized in that: in the described step 1), front end inner primer FIP comprises 2 sections sequence F4, F2 at least by 5 ' → 3 ' sequence direction, wherein, the complementary hybridization of F2 and target nucleic acid sequence F2c; The F4 sequence is the dna sequence dna inequality and not complementary with the single stranded DNA To Template;
Described rear end inner primer BIP comprises 2 sections sequence B 2, B4 at least by 3 ' → 5 ' sequence direction, and wherein, B2 is identical with the target nucleic acid partial sequence; The B4 sequence is the dna sequence dna inequality and not complementary with the single stranded DNA To Template;
In the step 1), also be provided with bridge-type primer BP, it by 5 ' → 3 ' sequence direction respectively with FIP among the complementary sequence F4c of F4 and the BIP all or part of base hybridization of two sequences of B4 complementary, perhaps by 5 ' → 3 ' sequence direction respectively with BIP among the complementary sequence B4c of B4 and the FIP all or part of base hybridization of two sequences of F4 complementary;
Have at most among FIP and the BIP have in the inner primer partial sequence not with the complementation of bridge-type primer, and identical with the target nucleic acid partial sequence or complementary, its position is at inner primer F2 or B2 is identical with target nucleic acid or 3 ' extreme direction of complimentary positions;
Step 2) in, utilize primers F 3, FIP, BIP, B3 to amplify the single stranded DNA To Template from target nucleic acid after, the two ends of DNA To Template or its complementary strand are that complementary strand has formed and do not have the hybridization of base breach slit with the bridge-type primer;
In the step 3), the two ends of DNA To Template or its complementary strand can be connected to the cyclized DNA chain by dna ligase;
In the step 4), in the reaction soln that contains the archaeal dna polymerase that has the strand displacement function at least, start the rolling circle amplification of cyclized DNA chain at least with FIP and two inner primers of BIP.
9. method as claimed in claim 8 is characterized in that: described bridge-type primer BP, four primer sequences that can be by other design from target nucleic acid sequence with the irrelevant fully sequence of single stranded DNA To Template sequence in amplification peel off out; Wherein, described four primer sequences, for: each 2 sequence of the inner primer that is different from F3, FIP, BIP, B3 of redesign and outer primer.
10. method as claimed in claim 8, it is characterized in that: oligonucleotide sequence has also designed auxiliary primer in the described step 1);
Described auxiliary primer derives from the intermediate sequence that do not influence B2 and target nucleic acid sequence stable bond, B4 and bridge-type primer stable bond in the BIP primer or the sequence between BIP primer and the B3 primer, maybe should auxiliary primer also derives from the intermediate sequence that do not influence F2 and target nucleic acid sequence stable bond, F4c and bridge-type primer stable bond in the FIP primer or the sequence between FIP primer and the F3 primer simultaneously.
11. the method for claim 1 is characterized in that: described step 2), reversed transcriptive enzyme comprises: AMV or M-MLV; The archaeal dna polymerase that has the strand displacement function comprises: Bst archaeal dna polymerase or Phi29DNA polysaccharase;
Step 2) in, contain the nucleic acid amplification reaction solution of the archaeal dna polymerase that has the strand displacement function at least, its component also comprises at least: amplification promotor and nucleic acid dye;
Wherein, amplification promotor comprises the combination of following one or more compositions: trimethyl-glycine, trehalose, proline(Pro), dimethyl sulfoxide (DMSO), Trimethylamine 99-N-oxide compound, Tetramethylammonium chloride, methane amide, BSA, single strand binding protein, T4Gene32Protein, homoectoine, Zn 2+-cyclen;
Described nucleic acid dye comprises: SYBR Green I, fluorexon, GELGREEN and GELRED.
12. the method for claim 1 is characterized in that: described step 2), before amplifying the single stranded DNA To Template according to target nucleic acid, can also carry out the target nucleic acid amplified production abatement of pollution of sample and handle:
After sample is finished nucleic acid extraction, add uridylic-N-glycosylase, may cause the uridylic in the target nucleic acid amplified production that pollutes to carry out the glycosidic link hydrolysis to sample, after this, solution temperature is elevated to 55-98 ℃ and kept maximum 10 minutes, and the uridylic in the solution-N-glycosylase will be inactivated.
13. method as claimed in claim 12, it is characterized in that: described abatement of pollution is handled, is increased in strand To Template, dna ligase cyclisation To Template, four steps of cyclisation To Template rolling circle amplification, each step can arrange different temperature condition, or some steps are wherein arranged same temperature; Related reagent composition in these four steps can repeatedly add respectively or once add and finish.
14. the method for claim 1 is characterized in that: described step 3), 4), described dna ligase comprises: T4DNA ligase, Taq DNA Ligase, Ampligase and ssDNA ligase.
15. method as claimed in claim 2 is characterized in that: in the described step 4), the archaeal dna polymerase that has the strand displacement function comprises: Bst archaeal dna polymerase or Phi29DNA polysaccharase;
The described reaction soln that contains dna ligase and have the archaeal dna polymerase of strand displacement function, its component also comprises at least: amplification promotor or nucleic acid dye;
Wherein, amplification promotor comprises the combination of following one or more compositions: trimethyl-glycine, trehalose, proline(Pro), dimethyl sulfoxide (DMSO), Trimethylamine 99-N-oxide compound, Tetramethylammonium chloride, methane amide, BSA, single strand binding protein, T4Gene32Protein, homoectoine, Zn 2+-cyclen;
Described nucleic acid dye comprises: SYBR Green I, fluorexon, GELGREEN and GELRED.
16. the method for claim 1 is characterized in that: the nucleic acid product in the single stranded DNA To Template of amplification described step 2), the cyclized DNA chain of step 3) and the step 4), can detect by immune chromatography test paper or fluorescent signal.
17. one kind is applied to the test kit of method according to claim 1, it is characterized in that: comprise oligonucleotide sequence as claimed in claim 1 at least.
18. test kit as claimed in claim 17 is characterized in that: described test kit also comprises at least a in the following component:
(1) the strand displacement function DNA polysaccharase that has as claimed in claim 11;
(2) dna ligase as claimed in claim 14;
(3) amplification promotor as claimed in claim 11;
(4) nucleic acid dye as claimed in claim 11;
(5) uridylic-N-glycosylase;
(6) reversed transcriptive enzyme as claimed in claim 11.
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