WO2020181947A1 - Method for detecting one or more target nucleic acid sequences to be tested by single tube and kit thereof - Google Patents

Method for detecting one or more target nucleic acid sequences to be tested by single tube and kit thereof Download PDF

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WO2020181947A1
WO2020181947A1 PCT/CN2020/074925 CN2020074925W WO2020181947A1 WO 2020181947 A1 WO2020181947 A1 WO 2020181947A1 CN 2020074925 W CN2020074925 W CN 2020074925W WO 2020181947 A1 WO2020181947 A1 WO 2020181947A1
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seq
nucleic acid
probe
target nucleic
tested
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PCT/CN2020/074925
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Chinese (zh)
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刘利成
王华贵
韦仕卯
邹奕君
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江苏宏微特斯医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to the technical field of molecular biology, in particular to a method for detecting one or more target nucleic acid sequences to be tested in a single tube and a kit thereof.
  • the isothermal nucleic acid amplification method also known as the isothermal nucleic acid amplification method, is a rapidly developing nucleic acid amplification method since the beginning of the 21st century. Unlike PCR, which requires variable temperature and thermal denaturation to unlock double-stranded amplification, the isothermal nucleic acid amplification method is to rapidly amplify nucleic acid by adding different nucleic acid polymerases and specific primers at a constant temperature. . Isothermal amplification technology can basically achieve the detection of target nucleic acid within 1 hour, and some can even achieve detection within 20 minutes.
  • the determination methods of isothermal amplification results can basically be divided into three categories: 1. Run agarose gel electrophoresis and observe the gel image; this method of determining results is time-consuming and laborious and is not conducive to product promotion. Generally, it may only be used in the product development stage. However, this method is basically not used in the actual formation of nucleic acid isothermal detection products. 2. Visual observation method to observe the turbidity or color change of the reaction tube before and after amplification; this judgment method is simple and easy to implement, and does not require high operators. It is a common result judgment method in the market, but its existence cannot distinguish specificity Amplification and non-specific amplification, and can not do multiple amplification defects. 3.
  • this result determination method is also very simple for the grassroots personnel, it needs to be equipped with one Fluorescence detection equipment, but the equipment requirements are not high, only a simple fluorescence detector with a heating module can realize the interpretation of the results. It has higher sensitivity, faster reaction rate, more specific amplification and can be multiplexed, so this Products with this determination method are more conducive to promotion and application on the market.
  • RNaseH can specifically degrade the phosphodiester bond of RNA in the hybrid chain between DNA and RNA. Utilizing this feature of RNaseH, IDT Corporation of the United States has conducted a study on Taqman probes containing RNA bases: introduce at the 5'end of a primer Fluorescence reporter group, 3'end primer fluorescence quenching group, and RNA bases introduced into the primer.
  • this probe containing RNA bases is added to a reaction system containing RNaseH2, a high-temperature resistant RNaseH, if the target nucleic acid exists, the probe binds to the target DNA to form part of the DNA-RNA hybrid double strand, then RNaseH2 Cleavage can be performed to separate the reporter group from the quencher group and emit fluorescence, thereby determining the presence of the target nucleic acid.
  • RNaseH2 Cleavage can be performed to separate the reporter group from the quencher group and emit fluorescence, thereby determining the presence of the target nucleic acid.
  • the present invention provides a single-tube detection of one or more target nucleic acid sequences based on the principle of high temperature resistant RNaseH2 that can cleave RNA-containing base probes
  • the method and kit thereof realize real-time isothermal detection of single target or multi-target nucleic acid.
  • the present invention introduces a probe containing RNA bases (RNHP) and high temperature resistant RNaseH2 on the basis of isothermal amplification, and utilizes the characteristic of RNaseH to cleave the phosphodiester bond of RNA in the hybrid chain of DNA and RNA.
  • RNHP RNA bases
  • RNaseH2 can be cleaved to make the reporter group and quench The groups separate and emit fluorescence to determine the presence of target nucleic acid.
  • RNaseH2 can exhibit the activity of cutting DNA-RNA hybrid strands in the isothermal amplification achieved by these technologies.
  • RNaseH2 and modified primers containing RNA bases are introduced into different types of isothermal amplification techniques, and fluorescence signal detection can be achieved without hindering the isothermal amplification reaction or disturbing the sensitivity of the amplification reaction.
  • the present invention provides a single-tube method for detecting one or more target nucleic acid sequences to be tested.
  • the method includes the following steps: Step 1: Design specific isothermal amplification primers and fluorescent probes for each target nucleic acid sequence to be tested RNHP, each fluorescent probe RNHP of the target nucleic acid sequence to be tested is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be distinguished in different fluorescent signal channels; the fluorescent probe RNHP contains at least 1 RNA base, The probe base on the left side of the RNA base near the 5'end of the probe is labeled with a fluorescent group, and the probe base on the side of the RNA base near the 3'end of the probe is labeled Quenching group; said at least 1 RNA base is preferably at least 1 continuous RNA base, more preferably 1 RNA base; Step 2: in the presence of ribonuclease RNaseH, in the presence of nucleic acid polymerase Under the action of, each target nu
  • the length of the fluorescent probe RNHP is 16-45 bp, and the distance between the fluorescent group labeled on the fluorescent probe RNHP and the base of the quenching group is 5-15 bp;
  • the G+C content in the probe fragment on the left side of the RNA base is 40%-60%; and the length of the probe fragment on the right side of the RNA base is 2-4 bp.
  • the target nucleic acid sequence to be tested is a DNA sequence
  • the nucleic acid polymerase is Bst polymerase
  • the target nucleic acid sequence to be tested is an RNA sequence
  • the nucleic acid polymerase is Bst polymerase and AMV reverse transcriptase
  • the ribonuclease RNaseH is a thermostable ribonuclease RNaseH, preferably RNaseH2.
  • the isothermal amplification is loop-mediated isothermal amplification, recombinase polymerase isothermal amplification, single-primer isothermal amplification, helicase-dependent isothermal amplification, strand replacement amplification, and cross primer Amplification technology, nucleic acid-dependent amplification detection technology or nicking endonuclease nucleic acid isothermal amplification.
  • the present invention provides a single-tube test kit for detecting one or more target nucleic acid sequences to be tested.
  • the kit includes: ribonuclease RNaseH, nucleic acid polymerase, and specific Design specific isothermal amplification primers and fluorescent probe RNHP for the target nucleic acid sequence, and each target nucleic acid sequence to be tested fluorescent probe RNHP is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be distinguished in different fluorescent signal channels;
  • the fluorescent probe RNHP contains at least one RNA base, a fluorescent group is labeled on the probe base on the left side of the RNA base near the 5'end of the probe, and the RNA base is close to the probe base.
  • the probe base on the 3'end of the probe is labeled with a quenching group;
  • the at least one RNA base is preferably at least one continuous RNA base, more preferably one RNA base.
  • the kit is a loop-mediated isothermal amplification detection kit for detecting Zika virus, and the kit is formed by using a primer probe for detecting Zika virus nucleic acid and a primer probe for detecting an internal reference. The double real-time loop-mediated isothermal amplification is completed.
  • the loop-mediated isothermal amplification primers used in the kit for detecting Zika virus nucleic acid are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, the RNHP probe used is SEQ ID NO: 7; the loop-mediated isothermal expansion used to detect the human ACTB gene as an internal reference in the kit
  • the primers are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the RNHP probe used is SEQ ID NO: 14; Two kinds of RNHP probes are labeled with different fluorescence, and each sequence is as follows:
  • the kit further includes a positive quality control product and a negative blank control product;
  • the positive quality control product is a standard plasmid of internal reference gene fragments and Zika virus cRNA.
  • the kit is a loop-mediated isothermal amplification detection kit for detecting Bunia virus, and the kit uses a primer probe for detecting Bunia virus nucleic acid and a primer probe for detecting an internal reference.
  • the formed double real-time loop-mediated isothermal amplification is completed.
  • the loop-mediated isothermal amplification primers used for detecting Bunia virus nucleic acid in the kit are SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, the RNHP used is SEQ ID NO: 21; the loop-mediated isothermal detection of the human ACTB gene used as the internal reference in the kit
  • the amplification primers are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the RNHP probe used is SEQ ID NO :14; two RNHP probes are labeled with different fluorescence,
  • the kit further includes a positive quality control product and a negative blank control product;
  • the positive quality control product is a standard plasmid of internal reference gene fragments and Bunya virus cRNA.
  • the present invention has the advantages and positive effects: on the basis of isothermal amplification technology, real-time multiplex isothermal detection can be realized only by introducing a special modified primer RNHP and combining with RNaseH2, and it can detect both DNA and nucleic acid RNA nucleic acid, compared with molecular beacons or other labeled probes used in other existing real-time isothermal amplification methods: In the presence of target nucleic acid, RNHP is a linear probe and a labeling base.
  • the cluster is simple, it is easier to bind to the target nucleic acid, thus the sensitivity is improved; compared with the single isothermal amplification method: it can be added to the detection of the internal reference, making the detection result more effective and reliable, and the result judgment method is more objective. Close the tube during the whole process to avoid amplification contamination.
  • Fig. 1 is a detection principle diagram of the method of the present invention.
  • Fig. 2 is a graph showing the amplification curve of a serial dilution solution of cRNA of a fragment of the Zika virus NS5 gene detected by the kit of the present invention.
  • Fig. 3 is a graph showing the amplification curve of a serial dilution of RNA of a Zika culture detected by the kit of the present invention.
  • Fig. 4 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a piece of RNHP designed at the position of the FIP primer to detect the cRNA of the Bunya virus S gene fragment.
  • Fig. 5 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the BIP primer to detect the cRNA of the Bunya virus S gene fragment.
  • Fig. 6 The amplification curve of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the LF primer to detect the cRNA of the Bunya virus S gene fragment.
  • Fig. 7 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the LB primer to detect the cRNA of the Bunya virus S gene fragment.
  • Fig. 8 is a graph of the amplification curve of the serial dilutions of the cRNA of the fragment of the Bunya virus S gene detected by the kit of the present invention, that is, when the design position of RNHP is between the primers F1c and B1c.
  • Kit internal reference (10 4 copies / ⁇ L) in FIG. 9 of the present invention Zika virus cRNA (10 -9 ng / ⁇ L) and Bunyaviridae cRNA (10 -9 ng / ⁇ L) in the amplification curves triplet .
  • Figure 10 The triple amplification curve of the internal control (10 5 copies/ ⁇ L), Zika virus cRNA (10 -8 ng/ ⁇ L) and Bunia virus cRNA (10 -8 ng/ ⁇ L) detected by the kit of the present invention .
  • Figure 11 The triple amplification curve diagram of the kit of the present invention for detecting internal control (10 6 copies/ ⁇ L), Zika virus cRNA (10 -7 ng/ ⁇ L) and Bunia virus cRNA (10 -7 ng/ ⁇ L) .
  • Isothermal amplification buffer, MgSO 4 , Bst DNA polymerase, and AMV enzyme were purchased from New England Biolabs, and dNTPs were purchased from Bao Bioengineering (Dalian) Co., Ltd. All primers and probes used were purchased from New England Biolabs. Synthesized by Shanghai Biological Engineering Technology Service Co., Ltd. Both the plasmid small extraction kit and the viral RNA extraction kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.
  • Embodiment 1 Basic principle of the method of the present invention
  • Fig. 1 the principle of the method for detecting one or more target nucleic acid sequences to be tested using the fluorescent signal generated by the combination of fluorescent probe (RNHP) and isothermal amplification method in the present invention is shown in Fig. 1, and the method steps are:
  • RNHP will bind to the target sequence to form a DNA-RNA hybrid chain.
  • RNaseH can specifically cleave the RNA bases of the DNA-RNA hybrid chain, thereby separating the left and right fragments of RNA bases in RNHP, and the fluorescent group and quenching group are also separated, thereby generating a fluorescent signal. If there is no target nucleic acid, as the reaction proceeds, no target sequence will be amplified, and the above-mentioned fluorescent signal will not be generated.
  • Example 2 Results of the detection of Zika virus cRNA gradient dilutions and 1 strain of Zika culture RNA gradient dilutions by the real-time LAMP kit of the present invention
  • the detection primers are designed using the relevant nucleic acid sequence of Zika virus published on NCBI and the sequence of the internal reference ACTB gene.
  • the detection primer sequences used in the examples are shown in the following table:
  • the underlined bases indicate the bases that are labeled with fluorescent groups
  • the lowercase bases indicate the bases of RNA
  • the quenching group is labeled 3' On the terminal base.
  • RNA obtained after transcription was adjusted to 100ng/ ⁇ L with sterilized double distilled water, which was used as Zika Virus positive quality control products. After preparation, the positive quality control product needs to be stored at -20°C to avoid repeated freezing and thawing. The validity period is nine months from the date of production.
  • a total of 2 positive quality control materials were constructed: ACTB and ZK-NS5.
  • the isothermal reaction system of real-time LAMP for Zika virus detection is 25 ⁇ L, and the components and final concentration of the nucleic acid reaction solution are shown in the following table:
  • Amplification reagent Final substance concentration or final enzyme activity unit 10 ⁇ isothermal amplification buffer 1 ⁇ MgSO4 6mM dNTP 1.4mM Bst enzyme 8U RNaseH2 12.5mU AMV enzyme 10U ACTB-FIP 0.8 ⁇ M ACTB-BIP 0.8 ⁇ M ACTB-LF 0.4 ⁇ M ACTB-LB 0.4 ⁇ M ACTB-OF 0.1 ⁇ M ACTB-OR 0.1 ⁇ M ZK-5S-FIP 0.8 ⁇ M ZK-5S-BIP 0.8 ⁇ M ZK-5S-LF 0.4 ⁇ M ZK-5S-LB 0.4 ⁇ M ZK-5S-OF 0.1 ⁇ M ZK-5S-OR 0.1 ⁇ M ACTB-RNHP 0.2 ⁇ M ZK-5S-RNHP 0.2 ⁇ M template 5 ⁇ L of nucleic acid to be detected and 1 ⁇ L of ACTB positive quality control material of 10 6 copies ddH 2 O Fill up the system to 25 ⁇ L mineral oil Around 40 ⁇ L
  • the real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step).
  • Applicable models ABI 7500, SLAN.
  • the ROX channel in the positive quality control reaction well has amplification signals
  • the FAM channel has amplification signals
  • other channels have no amplification signals
  • the amplification results of the positive and negative controls meet the preconditions for result determination, and then the following result determinations can be made:
  • the sensitivity of the detection of the cRNA of the NS5 gene in the gradient dilution solution can be To 10 -9 ng/ ⁇ L; a case of RNA amplification of Zika virus culture is shown in Figure 3.
  • the internal control achieves a good detection.
  • Zika is detected
  • the sensitivity of serial dilutions of RNA from viral cultures can reach 10 -3 ng/ ⁇ L. This amplification result shows that the Zika virus real-time LAMP detection kit will not interfere with the amplification of the internal reference when it is amplifying the positive standard product or the actual sample, and the sensitivity is very high.
  • Example 3 Amplification of single-plex real-time LAMP when RNHP is designed at different positions in the LAMP amplification region
  • the detection primers are designed using the relevant nucleic acid sequences of Bunya virus published on NCBI.
  • the primers and probe sequences used in the examples are shown in the following table:
  • underlined bases indicate bases that are labeled with fluorescent groups
  • lowercase bases indicate RNA bases
  • the quencher group is labeled on the 3'end base .
  • the bacterial solution is cultured separately. After culturing the bacterial solution: Perform a small plasmid extraction on the strain of Bunia S segment gene fragment, use the small extracted plasmid as a template to perform in vitro RNA transcription, and adjust the cRNA obtained after transcription with sterilized double distilled water to 100ng/ ⁇ L, as a positive quality control for Bunya virus.
  • the prepared positive quality control products need to be stored at -20°C to avoid repeated freezing and thawing. The validity period is nine months from the date of production.
  • the constructed positive quality control is Bun-S.
  • the real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step).
  • Applicable models ABI 7500, SLAN.
  • the amplification sensitivity is 10 -8 ng/ ⁇ L; 5)
  • the probe used is Bun-RNHP, that is, when the probe is designed at a position between F1c and B1c, the amplification of Bunia positive quality control products and their gradient dilutions As shown in Figure 8, the amplification sensitivity was 10 -9 ng/ ⁇ L. This shows that the probe can be designed at any position within the target area, but the sensitivity of the design at each position is different in a test item.
  • Detection primers and probes and positive quality control materials are as described in Example 1 and Example 2, except that the Bunia virus detection probe (Bun-RNHP) is re-synthesized to a probe labeled with Cy5 signal.
  • the needle that is, the detection probe of Bunia virus is labeled with Cy5 fluorescent reporter
  • the detection probe of ACTB gene is labeled with ROX fluorescent reporter
  • the detection probe of Zika virus is labeled FAM fluorescent reporter.
  • the real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step).
  • Applicable models ABI 7500, SLAN.
  • All three templates have been well amplified; 3) Combine the 10 6 copies/ ⁇ L gradient template of ACTB positive quality control material with Zika virus positive quality control material (ZK-NS5) and Bunia virus positive quality control material (Bun-S) 10 -7
  • the amplification results of the ng/ ⁇ L template are shown in Figure 11. All three templates have been amplified well, but compared with the first two low-concentration amplified Ct values, the Ct values of some targets have become larger. , which shows that when the template concentration becomes higher, the amplification between the templates is slightly interfered, but in the triple LAMP, the detection result of each target can still reach the detection sensitivity of single-plex.

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Abstract

Provided are a method for detecting one or more target nucleic acid sequences to be tested by a single tube and a kit thereof. The method comprises the steps of: designing specific isothermal amplification primers and fluorescent probe RNHPs for each target nucleic acid sequence to be tested, and labeling the fluorescent probe RNHPs for each target nucleic acid sequence to be tested with different fluorescences; in the presence of a ribonuclease RNaseH, isothermally amplifying each target nucleic acid sequence to be tested under the action of a nucleic acid polymerase; and forming hybridization chain products labeled with different fluorescences by means of different target nucleic acid sequences to be tested, thereby achieving multiplex detection of a plurality of target nucleic acid sequences to be tested. In the present invention, on the basis of an isothermal amplification technique, real-time multiplex isothermal detection can be achieved just by introducing a special probe RNHP and combining same with RNaseH2.

Description

单管检测一种或多种待测目标核酸序列的方法及其试剂盒Method for detecting one or more target nucleic acid sequences to be tested in single tube and its kit 技术领域Technical field
本发明涉及分子生物学技术领域，特别涉及单管检测一种或多种待测目标核酸序列的方法及其试剂盒。The present invention relates to the technical field of molecular biology, in particular to a method for detecting one or more target nucleic acid sequences to be tested in a single tube and a kit thereof.
背景技术Background technique
等温扩增核酸方法，也称恒温扩增核酸方法，是进入21世纪以来快速发展的一种核酸扩增方式。不同于PCR需要变温热变性解开双链扩增的方式，等温扩增核酸方法是在一个恒定的温度下，通过添加不同的核酸聚合酶和特异性引物，来达到快速扩增核酸的目的。等温扩增技术基本都能在1小时内实现目标核酸的检出，有的甚至可以实现20min中内完成检测。The isothermal nucleic acid amplification method, also known as the isothermal nucleic acid amplification method, is a rapidly developing nucleic acid amplification method since the beginning of the 21st century. Unlike PCR, which requires variable temperature and thermal denaturation to unlock double-stranded amplification, the isothermal nucleic acid amplification method is to rapidly amplify nucleic acid by adding different nucleic acid polymerases and specific primers at a constant temperature. . Isothermal amplification technology can basically achieve the detection of target nucleic acid within 1 hour, and some can even achieve detection within 20 minutes.
等温扩增结果的判定方式基本可以分为三大类：一、跑琼脂糖凝胶电泳，观察胶图；此种结果判定方式费时费力不利于产品推广，一般只在产品研发阶段可能会使用，而在实际形成的核酸等温检测的产品中基本不使用此方式进行结果判定。二、目测法观察反应管在扩增前后的浊度或者颜色变化；此种判定方式简单易行，对操作人员要求不高，是市场上常见的一种结果判定方式，但是其存在不能区分特异扩增与非特异扩增且也不能做多重扩增的缺陷。三、在反应液中引入荧光信号，在简易的可读取荧光信号的设备上进行扩增，读取荧光信号的变化；这一结果判定方式对于基层人员来说也非常简单，它需要配置一个荧光检测设备，但是设备要求不高，只需要是一个有加热模块的简单荧光检测仪就可以实现结果的判读，其灵敏度较高、反应速率快、扩增更特异且可以做多重，所以采用此种判定方式的产品更利于在市面上进行推广及应用。The determination methods of isothermal amplification results can basically be divided into three categories: 1. Run agarose gel electrophoresis and observe the gel image; this method of determining results is time-consuming and laborious and is not conducive to product promotion. Generally, it may only be used in the product development stage. However, this method is basically not used in the actual formation of nucleic acid isothermal detection products. 2. Visual observation method to observe the turbidity or color change of the reaction tube before and after amplification; this judgment method is simple and easy to implement, and does not require high operators. It is a common result judgment method in the market, but its existence cannot distinguish specificity Amplification and non-specific amplification, and can not do multiple amplification defects. 3. Introduce the fluorescent signal into the reaction solution, perform amplification on a simple device that can read the fluorescent signal, and read the change of the fluorescent signal; this result determination method is also very simple for the grassroots personnel, it needs to be equipped with one Fluorescence detection equipment, but the equipment requirements are not high, only a simple fluorescence detector with a heating module can realize the interpretation of the results. It has higher sensitivity, faster reaction rate, more specific amplification and can be multiplexed, so this Products with this determination method are more conducive to promotion and application on the market.
RNaseH可以特异地降解DNA与RNA杂交链中的RNA的磷酸二酯键，利用RNaseH这一特性，美国IDT公司进行过包含有RNA碱基的Taqman探针的研究：在一条引物的5’端引入荧光报告基团，3’端引物荧光淬灭基团，引物内部引入RNA碱基。将这种包含有RNA碱基而形成的探针加入含有RNaseH2这种耐高温的RNaseH的反应体系里时，如果目标核酸存在，探针与目标DNA结合形成部分DNA-RNA杂交双链，则RNaseH2可以进行切 割，使得报告基团与淬灭基团分离而发出荧光，从而判定目标核酸的存在。鉴于目前临床上对简便快速，灵敏度高，特异性好及多靶标检测技术的需求，而现有技术都有其局限性，不能完全满足临床的需求，因此我们亟需开发出一种快速，准确且低成本的多重检测技术。RNaseH can specifically degrade the phosphodiester bond of RNA in the hybrid chain between DNA and RNA. Utilizing this feature of RNaseH, IDT Corporation of the United States has conducted a study on Taqman probes containing RNA bases: introduce at the 5'end of a primer Fluorescence reporter group, 3'end primer fluorescence quenching group, and RNA bases introduced into the primer. When this probe containing RNA bases is added to a reaction system containing RNaseH2, a high-temperature resistant RNaseH, if the target nucleic acid exists, the probe binds to the target DNA to form part of the DNA-RNA hybrid double strand, then RNaseH2 Cleavage can be performed to separate the reporter group from the quencher group and emit fluorescence, thereby determining the presence of the target nucleic acid. In view of the current clinical needs for simple and rapid, high sensitivity, good specificity and multi-target detection technology, and the existing technologies have their limitations and cannot fully meet the clinical needs, so we urgently need to develop a rapid and accurate And low-cost multiple detection technology.
发明内容Summary of the invention
为了解决高灵敏度的实时等温扩增检测核酸的问题，本发明提供了一种基于耐高温的RNaseH2可酶切含RNA碱基探针的原理来进行单管检测一个或多个待测目标核酸序列的方法及其试剂盒，其实现了实时等温检测单靶标或者多靶标核酸。In order to solve the problem of high-sensitivity real-time isothermal amplification and detection of nucleic acids, the present invention provides a single-tube detection of one or more target nucleic acid sequences based on the principle of high temperature resistant RNaseH2 that can cleave RNA-containing base probes The method and kit thereof realize real-time isothermal detection of single target or multi-target nucleic acid.
本发明在等温扩增的基础上引入了一条含RNA碱基的探针(RNHP)与耐高温的RNaseH2，利用RNaseH可酶切DNA与RNA杂交链中的RNA的磷酸二酯键的特性，当待测样本中含有靶标核酸时，通过等温扩增出大量靶标DNA/cDNA，RNHP会与目标DNA/cDNA结合形成部分DNA-RNA杂交双链，则RNaseH2可以进行切割，使得报告基团与淬灭基团分离而发出荧光，从而判定目标核酸的存在。目前存在的多种等温扩增技术，其扩增所需的恒定温度都处于RNaseH2的酶活性温度范围内，RNaseH2在这些技术实现的等温扩增中都可以发挥出切割DNA-RNA杂交链的活性。理论上将RNaseH2与包含有RNA碱基的修饰引物引入不同类型的等温扩增技术中，在不阻碍等温扩增反应或者干扰扩增反应灵敏度的情况下，都可以实现荧光信号的检测。The present invention introduces a probe containing RNA bases (RNHP) and high temperature resistant RNaseH2 on the basis of isothermal amplification, and utilizes the characteristic of RNaseH to cleave the phosphodiester bond of RNA in the hybrid chain of DNA and RNA. When the sample to be tested contains target nucleic acid, a large amount of target DNA/cDNA is amplified by isothermal method. RNHP will combine with the target DNA/cDNA to form part of the DNA-RNA hybrid double-stranded. Then RNaseH2 can be cleaved to make the reporter group and quench The groups separate and emit fluorescence to determine the presence of target nucleic acid. There are a variety of isothermal amplification technologies, and the constant temperature required for amplification is within the range of the enzyme activity temperature of RNaseH2. RNaseH2 can exhibit the activity of cutting DNA-RNA hybrid strands in the isothermal amplification achieved by these technologies. . Theoretically, RNaseH2 and modified primers containing RNA bases are introduced into different types of isothermal amplification techniques, and fluorescence signal detection can be achieved without hindering the isothermal amplification reaction or disturbing the sensitivity of the amplification reaction.
本发明提供一种单管检测一种或多种待测目标核酸序列的方法，所述方法包括以下步骤：步骤1：针对每种待测目标核酸序列设计特异性等温扩增引物和荧光探针RNHP，每种待测目标核酸序列荧光探针RNHP标记不同的荧光，使得不同待测目标核酸序列能够在不同荧光信号通道中区分；所述荧光探针RNHP包含至少1个RNA碱基，在所述RNA碱基左侧靠近所述探针5’端一侧的探针碱基上标记荧光基团，在所述RNA碱基靠近所述探针3’端一侧的探针碱基上标记淬灭基团；所述至少1个RNA碱基优选地为至少1个连续的RNA碱基，更优选地为1个RNA碱基；步骤2：在核糖核酸酶RNaseH存在下，在核酸聚合酶的作用下等温扩增每种待测目标核酸序列，每种荧光探针RNHP结合到相应的待测目标核酸序列上，形成探针-目标核酸杂交双链；所述核糖核酸酶RNaseH切割探针-目标核酸杂交双链中的RNA碱基，使得所述RNA碱基右侧的含有淬灭基团的探针片段游离出去，而在RNA碱基左侧的含有荧光基团的片段仍然保持形成杂 交链且荧光基团发出荧光；和步骤3：通过待测目标核酸序列形成的带不同荧光标记的杂交链产物，实现待测目标核酸序列的检测。The present invention provides a single-tube method for detecting one or more target nucleic acid sequences to be tested. The method includes the following steps: Step 1: Design specific isothermal amplification primers and fluorescent probes for each target nucleic acid sequence to be tested RNHP, each fluorescent probe RNHP of the target nucleic acid sequence to be tested is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be distinguished in different fluorescent signal channels; the fluorescent probe RNHP contains at least 1 RNA base, The probe base on the left side of the RNA base near the 5'end of the probe is labeled with a fluorescent group, and the probe base on the side of the RNA base near the 3'end of the probe is labeled Quenching group; said at least 1 RNA base is preferably at least 1 continuous RNA base, more preferably 1 RNA base; Step 2: in the presence of ribonuclease RNaseH, in the presence of nucleic acid polymerase Under the action of, each target nucleic acid sequence to be tested is amplified isothermally, and each fluorescent probe RNHP binds to the corresponding target nucleic acid sequence to be tested to form a probe-target nucleic acid hybrid double strand; the ribonuclease RNaseH cuts the probe -The target nucleic acid hybridizes to the RNA bases in the duplex, so that the probe fragment containing the quencher group on the right side of the RNA base is freed, while the fragment containing the fluorescent group on the left side of the RNA base remains formed The hybridized chain and the fluorescent group emits fluorescence; and Step 3: The detection of the target nucleic acid sequence to be tested is achieved through the hybridization chain products with different fluorescent labels formed by the target nucleic acid sequence to be tested.
在一种实施方式中，所述荧光探针RNHP长度为16-45bp，所述荧光探针RNHP上标记的荧光基团和淬灭基团的碱基之间的距离为5-15bp；所述RNA碱基的左侧所述探针片段中G+C含量为40％-60％；和所述RNA碱基的右侧所述探针片段的长度为2-4bp。In one embodiment, the length of the fluorescent probe RNHP is 16-45 bp, and the distance between the fluorescent group labeled on the fluorescent probe RNHP and the base of the quenching group is 5-15 bp; The G+C content in the probe fragment on the left side of the RNA base is 40%-60%; and the length of the probe fragment on the right side of the RNA base is 2-4 bp.
在一种实施方式中，所述待测目标核酸序列为DNA序列，所述核酸聚合酶为Bst聚合酶，或所述待测目标核酸序列为RNA序列，所述核酸聚合酶为Bst聚合酶与AMV逆转录酶；和所述核糖核酸酶RNaseH是耐热核糖核酸酶RNaseH，优选地为RNaseH2。In one embodiment, the target nucleic acid sequence to be tested is a DNA sequence, the nucleic acid polymerase is Bst polymerase, or the target nucleic acid sequence to be tested is an RNA sequence, and the nucleic acid polymerase is Bst polymerase and AMV reverse transcriptase; and the ribonuclease RNaseH is a thermostable ribonuclease RNaseH, preferably RNaseH2.
在一种实施方式中，所述等温扩增为环介导等温扩增、重组酶聚合酶等温扩增、单引物等温扩增、依赖解旋酶的等温扩增、链替代扩增、交叉引物扩增技术、核酸依赖性扩增检测技术或切刻内切酶核酸等温扩增。In one embodiment, the isothermal amplification is loop-mediated isothermal amplification, recombinase polymerase isothermal amplification, single-primer isothermal amplification, helicase-dependent isothermal amplification, strand replacement amplification, and cross primer Amplification technology, nucleic acid-dependent amplification detection technology or nicking endonuclease nucleic acid isothermal amplification.
在一种实施方式中，本发明提供一种单管检测一种或多种待测目标核酸序列的试剂盒，所述试剂盒包括：核糖核酸酶RNaseH，核酸聚合酶，和针对每种待测目标核酸序列设计特异性等温扩增引物和荧光探针RNHP，每种待测目标核酸序列荧光探针RNHP标记不同的荧光，使得不同待测目标核酸序列能够在不同荧光信号通道中区分；所述荧光探针RNHP包含至少1个RNA碱基，在所述RNA碱基左侧靠近所述探针5’端一侧的探针碱基上标记荧光基团，在所述RNA碱基靠近所述探针3’端一侧的探针碱基上标记淬灭基团；所述至少1个RNA碱基优选地为至少1个连续的RNA碱基，更优选地为1个RNA碱基。In one embodiment, the present invention provides a single-tube test kit for detecting one or more target nucleic acid sequences to be tested. The kit includes: ribonuclease RNaseH, nucleic acid polymerase, and specific Design specific isothermal amplification primers and fluorescent probe RNHP for the target nucleic acid sequence, and each target nucleic acid sequence to be tested fluorescent probe RNHP is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be distinguished in different fluorescent signal channels; The fluorescent probe RNHP contains at least one RNA base, a fluorescent group is labeled on the probe base on the left side of the RNA base near the 5'end of the probe, and the RNA base is close to the probe base. The probe base on the 3'end of the probe is labeled with a quenching group; the at least one RNA base is preferably at least one continuous RNA base, more preferably one RNA base.
在一种实施方式中，所述试剂盒是检测寨卡病毒的环介导等温扩增检测试剂盒，所述试剂盒利用检测寨卡病毒核酸的引物探针搭配检测内参的引物探针形成的二重实时环介导等温扩增来完成，所述试剂盒内检测寨卡病毒核酸所用的环介导等温扩增引物分别是SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6，所用的RNHP探针是SEQ ID NO：7；所述试剂盒内检测作为内参的人类ACTB基因所用的环介导等温扩增引物分别是SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13，所用的RNHP探针是SEQ ID NO：14；两种RNHP探针分别标记不同的荧光，各个序列如下：In one embodiment, the kit is a loop-mediated isothermal amplification detection kit for detecting Zika virus, and the kit is formed by using a primer probe for detecting Zika virus nucleic acid and a primer probe for detecting an internal reference. The double real-time loop-mediated isothermal amplification is completed. The loop-mediated isothermal amplification primers used in the kit for detecting Zika virus nucleic acid are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, the RNHP probe used is SEQ ID NO: 7; the loop-mediated isothermal expansion used to detect the human ACTB gene as an internal reference in the kit The primers are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the RNHP probe used is SEQ ID NO: 14; Two kinds of RNHP probes are labeled with different fluorescence, and each sequence is as follows:
Figure PCTCN2020074925-appb-000001
Figure PCTCN2020074925-appb-000001
Figure PCTCN2020074925-appb-000002
Figure PCTCN2020074925-appb-000002
在一种实施方式中，所述试剂盒还包括阳性质控品和阴性空白对照品；所述阳性质控品为内参基因片段的标准质粒和寨卡病毒cRNA。In one embodiment, the kit further includes a positive quality control product and a negative blank control product; the positive quality control product is a standard plasmid of internal reference gene fragments and Zika virus cRNA.
在一种实施方式中，所述试剂盒是检测布尼亚病毒的环介导等温扩增检测试剂盒，所述试剂盒利用检测布尼亚病毒核酸的引物探针搭配检测内参的引物探针形成的二重实时环介导等温扩增来完成，所述试剂盒内检测布尼亚病毒核酸所用的环介导等温扩增引物分别是SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，所用的RNHP是SEQ ID NO：21；所述试剂盒内检测作为内参的人类ACTB基因所用的环介导等温扩增引物分别是SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13，所用的RNHP探针是SEQ ID NO：14；两种RNHP探针分别标记不同的荧光，In one embodiment, the kit is a loop-mediated isothermal amplification detection kit for detecting Bunia virus, and the kit uses a primer probe for detecting Bunia virus nucleic acid and a primer probe for detecting an internal reference. The formed double real-time loop-mediated isothermal amplification is completed. The loop-mediated isothermal amplification primers used for detecting Bunia virus nucleic acid in the kit are SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, the RNHP used is SEQ ID NO: 21; the loop-mediated isothermal detection of the human ACTB gene used as the internal reference in the kit The amplification primers are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the RNHP probe used is SEQ ID NO ：14; two RNHP probes are labeled with different fluorescence,
Figure PCTCN2020074925-appb-000003
Figure PCTCN2020074925-appb-000003
Figure PCTCN2020074925-appb-000004
Figure PCTCN2020074925-appb-000004
在一种实施方式中，所述试剂盒还包括阳性质控品和阴性空白对照品；所述阳性质控品为内参基因片段的标准质粒和布尼亚病毒cRNA。In one embodiment, the kit further includes a positive quality control product and a negative blank control product; the positive quality control product is a standard plasmid of internal reference gene fragments and Bunya virus cRNA.
本发明具有的优点和积极效果是：在等温扩增技术的基础上，只是通过引入一条特殊的修饰引物RNHP并结合RNaseH2就可实现实时多重的等温检测，且其既可以检测DNA核酸也可以检测RNA核酸，与现有其它实时等温扩增方式中用到的分子信标或者其它标记类探针相比：在有目标核酸存在的情况下，RNHP因为其本身是一种线性探针且标记基团简单，它更容易与目标核酸结合，从而灵敏度得到提高；与单重等温扩增方式相比：其可以加入内参的检测，使得检测结果更加有效可靠，且结果判断方式也更加客观。整个过程闭管操作，避免扩增污染。The present invention has the advantages and positive effects: on the basis of isothermal amplification technology, real-time multiplex isothermal detection can be realized only by introducing a special modified primer RNHP and combining with RNaseH2, and it can detect both DNA and nucleic acid RNA nucleic acid, compared with molecular beacons or other labeled probes used in other existing real-time isothermal amplification methods: In the presence of target nucleic acid, RNHP is a linear probe and a labeling base. The cluster is simple, it is easier to bind to the target nucleic acid, thus the sensitivity is improved; compared with the single isothermal amplification method: it can be added to the detection of the internal reference, making the detection result more effective and reliable, and the result judgment method is more objective. Close the tube during the whole process to avoid amplification contamination.
附图说明Description of the drawings
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。In order to more clearly describe the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only some of the embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.
图1是本发明方法的检测原理图。Fig. 1 is a detection principle diagram of the method of the present invention.
图2本发明中的试剂盒检测寨卡病毒NS5基因的片段的cRNA的梯度稀释液的扩增曲线图。Fig. 2 is a graph showing the amplification curve of a serial dilution solution of cRNA of a fragment of the Zika virus NS5 gene detected by the kit of the present invention.
图3本发明中的试剂盒检测一例寨卡培养物的RNA梯度稀释液的扩增曲线图。Fig. 3 is a graph showing the amplification curve of a serial dilution of RNA of a Zika culture detected by the kit of the present invention.
图4本发明中检测布尼亚病毒的引物搭配一条设计在FIP引物位置的RNHP去检测布尼亚病毒S基因的片段的cRNA的梯度稀释液的扩增曲线图。Fig. 4 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a piece of RNHP designed at the position of the FIP primer to detect the cRNA of the Bunya virus S gene fragment.
图5本发明中检测布尼亚病毒的引物搭配一条设计在BIP引物位置的RNHP去检测布尼亚病毒S基因的片段的cRNA的梯度稀释液的扩增曲线图。Fig. 5 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the BIP primer to detect the cRNA of the Bunya virus S gene fragment.
图6本发明中检测布尼亚病毒的引物搭配一条设计在LF引物位置的RNHP去检测布尼亚病毒S基因的片段的cRNA的梯度稀释液的扩增曲线图。Fig. 6 The amplification curve of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the LF primer to detect the cRNA of the Bunya virus S gene fragment.
图7本发明中检测布尼亚病毒的引物搭配一条设计在LB引物位置的RNHP去检测布尼亚病毒S基因的片段的cRNA的梯度稀释液的扩增曲线图。Fig. 7 Amplification curve diagram of the primers for detecting Bunia virus in the present invention with a RNHP designed at the position of the LB primer to detect the cRNA of the Bunya virus S gene fragment.
图8本发明中的试剂盒检测布尼亚病毒S基因的片段的cRNA的梯度稀释液的扩增曲线图，即RNHP的设计位置位于引物F1c与B1c之间时。Fig. 8 is a graph of the amplification curve of the serial dilutions of the cRNA of the fragment of the Bunya virus S gene detected by the kit of the present invention, that is, when the design position of RNHP is between the primers F1c and B1c.
图9本发明中的试剂盒检测内参(10 4copies/μL)、寨卡病毒cRNA(10 -9ng/μL)与布尼亚病毒cRNA(10 -9ng/μL)的三重扩增曲线图。 Kit internal reference (10 4 copies / μL) in FIG. 9 of the present invention, Zika virus cRNA (10 -9 ng / μL) and Bunyaviridae cRNA (10 -9 ng / μL) in the amplification curves triplet .
图10本发明中的试剂盒检测内参(10 5copies/μL)、寨卡病毒cRNA(10 -8ng/μL)与布尼亚病毒cRNA(10 -8ng/μL)的三重扩增曲线图。 Figure 10: The triple amplification curve of the internal control (10 5 copies/μL), Zika virus cRNA (10 -8 ng/μL) and Bunia virus cRNA (10 -8 ng/μL) detected by the kit of the present invention .
图11本发明中的试剂盒检测内参(10 6copies/μL)、寨卡病毒cRNA(10 -7ng/μL)与布尼亚病毒cRNA(10 -7ng/μL)的三重扩增曲线图。 Figure 11 The triple amplification curve diagram of the kit of the present invention for detecting internal control (10 6 copies/μL), Zika virus cRNA (10 -7 ng/μL) and Bunia virus cRNA (10 -7 ng/μL) .
具体实施方式detailed description
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结合实施例对本发明作进一步说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都应当属于本申请保护的范围。In order to enable those skilled in the art to better understand the technical solutions in the present application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. . Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work should fall within the protection scope of this application.
下述实施例中，如无特殊说明，均为本领域常规方法。等温扩增缓冲液、MgSO 4、Bst DNA聚合酶、AMV酶均购于纽英伦生物技术有限公司(New England Biolabs)，dNTP购于宝生物工程(大连)有限公司，所用引物及探针均由上海生物工程技术服务有限公司合成。质粒小提试剂盒与病毒RNA提取试剂盒均购自北京天根生物科技有限公司。 In the following embodiments, unless otherwise specified, they are all conventional methods in the art. Isothermal amplification buffer, MgSO 4 , Bst DNA polymerase, and AMV enzyme were purchased from New England Biolabs, and dNTPs were purchased from Bao Bioengineering (Dalian) Co., Ltd. All primers and probes used were purchased from New England Biolabs. Synthesized by Shanghai Biological Engineering Technology Service Co., Ltd. Both the plasmid small extraction kit and the viral RNA extraction kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.
实施例一 本发明方法的基本原理 Embodiment 1 Basic principle of the method of the present invention
如图1所示，本发明利用荧光探针(RNHP)与等温扩增方法相结合产生的荧光信号检测一个或多个待测靶标核酸序列的方法原理如图1所示，其方法步骤为：As shown in Fig. 1, the principle of the method for detecting one or more target nucleic acid sequences to be tested using the fluorescent signal generated by the combination of fluorescent probe (RNHP) and isothermal amplification method in the present invention is shown in Fig. 1, and the method steps are:
1)针对每个待测靶标核酸序列设计一条RNHP，根据采用的等温扩增方法的不同，针对每个待测靶标核酸序列设计特异性等温扩增引物，将引物与RNHP加入采用的等温扩增方法的反应体系中。1) Design an RNHP for each target nucleic acid sequence to be tested. According to the different isothermal amplification methods used, design specific isothermal amplification primers for each target nucleic acid sequence to be tested, and add the primers and RNHP to the used isothermal amplification Method of the reaction system.
2)在有靶标核酸存在的情况下，随着扩增反应的进行，有大量的靶标核酸被扩出，RNHP会结合到靶标序列上去，从而形成一段DNA-RNA杂交链。RNaseH可以特异性切豁DNA-RNA杂交链的RNA碱基，从而使RNHP中RNA碱基的左侧与右侧的片段分离，荧光基团与淬灭基团也分离，从而产生荧光信号。若没有靶标核酸存在的情况下，随着反应进行，就不会有靶标序列被扩增出，也就不会产生上述的荧光信号。2) In the presence of target nucleic acid, as the amplification reaction progresses, a large amount of target nucleic acid is expanded, and RNHP will bind to the target sequence to form a DNA-RNA hybrid chain. RNaseH can specifically cleave the RNA bases of the DNA-RNA hybrid chain, thereby separating the left and right fragments of RNA bases in RNHP, and the fluorescent group and quenching group are also separated, thereby generating a fluorescent signal. If there is no target nucleic acid, as the reaction proceeds, no target sequence will be amplified, and the above-mentioned fluorescent signal will not be generated.
实施例二 本发明中实时LAMP试剂盒检测寨卡病毒cRNA梯度稀释液及1株寨卡培养物RNA梯度稀释液的结果Example 2 Results of the detection of Zika virus cRNA gradient dilutions and 1 strain of Zika culture RNA gradient dilutions by the real-time LAMP kit of the present invention
1.检测引物的设计1. Design of detection primers
使用发表在NCBI上的寨卡病毒的相关核酸序列及内参ACTB基因的序列设计检测引物，实施例中所用的检测引物序列具体见下表：The detection primers are designed using the relevant nucleic acid sequence of Zika virus published on NCBI and the sequence of the internal reference ACTB gene. The detection primer sequences used in the examples are shown in the following table:
Figure PCTCN2020074925-appb-000005
Figure PCTCN2020074925-appb-000005
注释：在上述表中SEQ ID NO：7和SEQ ID NO：14中，带下划线碱基表示是标记荧光基团的碱基，小写碱基表示是RNA碱基，淬灭基团标记在3’末端碱基上。Note: In SEQ ID NO: 7 and SEQ ID NO: 14 in the above table, the underlined bases indicate the bases that are labeled with fluorescent groups, the lowercase bases indicate the bases of RNA, and the quenching group is labeled 3' On the terminal base.
2.阳性质控品的制备2. Preparation of positive quality control products
从NCBI上分别下载人的ACTB基因的序列片段及寨卡病毒的NS5基因序列的片段，分别设计这两个片段扩增的引物：ACTB-F、ACTB-R；ZK-NS5-F、ZK-NS5-R(见下表)。将扩增出的ACTB目的片段与寨卡病毒的目的片段分别***到pMD19-T载体中，挑单克隆经测序验证后，选择***成功并且序列正确的单克隆进行保菌。每次制备阳性质控品时，首先分别活化构建好的内参ACTB基因片段的菌株与寨卡病毒NS5基因片段的菌株，然后提取活化后的菌株的质粒并测序，确定序列正确后再分别培养菌液。培养菌液后：对ACTB基因片段的菌株进行质粒大提，用无菌的DNA稀释液将提取的质粒浓度调至10 4copies/μL，以此作为ACTB基因片段的阳性质控品；对寨卡NS5基因片段的菌株进行质粒小提，以小提后的质粒做模板，进行体外RNA转录，将转录后得到的RNA用灭菌后的双蒸水调至100ng/μL，以此作为寨卡病毒的阳性质控品。制备完后的阳性质控品需要-20℃储存，避免反复冻融，有效期为自生产之日起九个月。共构建了2个阳性质控品： ACTB、ZK-NS5。 Download the human ACTB gene sequence fragment and the Zika virus NS5 gene sequence fragment from NCBI, respectively, design the primers for the amplification of these two fragments: ACTB-F, ACTB-R; ZK-NS5-F, ZK- NS5-R (see the table below). The amplified target fragment of ACTB and the target fragment of Zika virus were inserted into the pMD19-T vector. After the single clone was selected and verified by sequencing, the clone with the successful insertion and the correct sequence was selected for bacteria preservation. Each time a positive quality control product is prepared, first activate the constructed strain with the internal control ACTB gene fragment and the strain with the Zika virus NS5 gene fragment respectively, and then extract the plasmid of the activated strain and sequence it, and then culture the bacteria separately after confirming the sequence is correct liquid. After culturing bacteria: Strains ACTB gene fragments for plasmid mention large, with a sterile diluent extracted DNA plasmid concentration was adjusted to 10 4 copies / μL, as a positive control materials ACTB gene fragments; of Village The NS5 gene fragment of the NS5 strain was subjected to a small plasmid extraction, and the small extracted plasmid was used as a template for in vitro RNA transcription. The RNA obtained after transcription was adjusted to 100ng/μL with sterilized double distilled water, which was used as Zika Virus positive quality control products. After preparation, the positive quality control product needs to be stored at -20°C to avoid repeated freezing and thawing. The validity period is nine months from the date of production. A total of 2 positive quality control materials were constructed: ACTB and ZK-NS5.
Figure PCTCN2020074925-appb-000006
Figure PCTCN2020074925-appb-000006
3.实时LAMP反应体系与反应程序3. Real-time LAMP reaction system and reaction procedures
检测寨卡病毒的实时LAMP的等温反应体系为25μL，其核酸反应液的组分与终浓度如下表所示：The isothermal reaction system of real-time LAMP for Zika virus detection is 25 μL, and the components and final concentration of the nucleic acid reaction solution are shown in the following table:
扩增试剂Amplification reagent 终物质的量浓度或者终酶活单位Final substance concentration or final enzyme activity unit
10×等温扩增缓冲液10× isothermal amplification buffer 1×1×
MgSO4MgSO4 6mM6mM
dNTPdNTP 1.4mM1.4mM
Bst酶Bst enzyme 8U8U
RNaseH2RNaseH2 12.5mU12.5mU
AMV酶AMV enzyme 10U10U
ACTB-FIPACTB-FIP 0.8μM0.8μM
ACTB-BIPACTB-BIP 0.8μM0.8μM
ACTB-LFACTB-LF 0.4μM0.4μM
ACTB-LBACTB-LB 0.4μM0.4μM
ACTB-OFACTB-OF 0.1μM0.1μM
ACTB-ORACTB-OR 0.1μM0.1μM
ZK-5S-FIPZK-5S-FIP 0.8μM0.8μM
ZK-5S-BIPZK-5S-BIP 0.8μM0.8μM
ZK-5S-LFZK-5S-LF 0.4μM0.4μM
ZK-5S-LBZK-5S-LB 0.4μM0.4μM
ZK-5S-OFZK-5S-OF 0.1μM0.1μM
ZK-5S-ORZK-5S-OR 0.1μM0.1μM
ACTB-RNHPACTB-RNHP 0.2μM0.2μM
ZK-5S-RNHPZK-5S-RNHP 0.2μM0.2μM
模板template 5μL待检测核酸及1μL 10 6copies的ACTB阳性质控品 5μL of nucleic acid to be detected and 1μL of ACTB positive quality control material of 10 6 copies
ddH 2O ddH 2 O 补齐体系至25μLFill up the system to 25μL
矿物油mineral oil 40μL左右Around 40μL
反应程序：Reaction procedure:
实时LAMP扩增反应程序为：90cycle:63℃,15s；63℃,30s(此步收集荧光)。适用机型：ABI 7500、SLAN。The real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step). Applicable models: ABI 7500, SLAN.
4.实验结果：4. Experimental results:
先参看阳性质控品与阴性空白对照孔内的扩增结果：阳性质控品反应孔内的ROX通道有扩增信号，FAM通道有扩增信号，其它通道无扩增信号；阴性反应孔内每个通道都没有扩增信号。阳性与阴性质控品的扩增结果符合可以进行结果判定的前提，然后可进行以下结果判定：First refer to the amplification results in the positive control wells and the negative blank control wells: the ROX channel in the positive quality control reaction well has amplification signals, the FAM channel has amplification signals, and other channels have no amplification signals; in the negative reaction wells There is no amplification signal in each channel. The amplification results of the positive and negative controls meet the preconditions for result determination, and then the following result determinations can be made:
因为在寨卡病毒的阳性质控品的梯度稀释液(10 -12ng/μL～10 -6ng/μL)与一例寨卡培养物提取的RNA及其稀释液中(10 -5ng/μL～RNA原液)不含内参基因，所以在二者的梯度稀释液的每个反应孔内人为地都加入了10 6copies的内参模板。寨卡病毒NS5基因的 cRNA的梯度稀释液扩增如图2所示，在ROX通道内，内参实现了很好的检出，在FAM通道内，检测NS5基因的cRNA的梯度稀释液的灵敏度可至10 -9ng/μL；一例寨卡病毒培养物的RNA的梯度稀释液扩增如图3所示，在ROX通道内，内参实现了很好的检出，在FAM通道内，检测寨卡病毒培养物的RNA的梯度稀释液的灵敏度可至10 -3ng/μL。这个扩增结果表明，寨卡病毒实时LAMP检测试剂盒无论是在扩阳性标准品还是在扩实际样本时，其与内参的扩增都不会相互干扰，且灵敏度都非常高。 Because in the Zika virus positive quality control product gradient dilution (10 -12 ng/μL ~ 10 -6 ng/μL) and a case of Zika culture extracted RNA and its dilution (10 -5 ng/μL) ~RNA stock solution) does not contain internal reference genes, so 10 6 copies of internal reference template are artificially added to each reaction well of the two gradient dilutions. The amplification of the cRNA of the Zika virus NS5 gene in gradient dilutions is shown in Figure 2. In the ROX channel, the internal reference achieves a good detection. In the FAM channel, the sensitivity of the detection of the cRNA of the NS5 gene in the gradient dilution solution can be To 10 -9 ng/μL; a case of RNA amplification of Zika virus culture is shown in Figure 3. In the ROX channel, the internal control achieves a good detection. In the FAM channel, Zika is detected The sensitivity of serial dilutions of RNA from viral cultures can reach 10 -3 ng/μL. This amplification result shows that the Zika virus real-time LAMP detection kit will not interfere with the amplification of the internal reference when it is amplifying the positive standard product or the actual sample, and the sensitivity is very high.
实施例三 将RNHP设计在LAMP扩增区域内不同位置时进行单重实时LAMP的扩增Example 3 Amplification of single-plex real-time LAMP when RNHP is designed at different positions in the LAMP amplification region
1.检测引物与探针的设计1. Design of detection primers and probes
使用发表在NCBI上的布尼亚病毒的相关核酸序列设计检测引物，实施例中所使用的引物与探针序列具体见下表：The detection primers are designed using the relevant nucleic acid sequences of Bunya virus published on NCBI. The primers and probe sequences used in the examples are shown in the following table:
Figure PCTCN2020074925-appb-000007
Figure PCTCN2020074925-appb-000007
注释：在上述表中SEQ ID NO：21-25中，带下划线碱基表示是标记荧光基团的碱基，小写碱基表示是RNA碱基，淬灭基团标记在3’末端碱基上。Note: In SEQ ID NO: 21-25 in the above table, underlined bases indicate bases that are labeled with fluorescent groups, lowercase bases indicate RNA bases, and the quencher group is labeled on the 3'end base .
2.阳性质控品的制备2. Preparation of positive quality control products
从NCBI上下载布尼亚病毒的S段基因序列的片段，设计这个片段扩增的引物为Bun-S-F(SEQ ID NO：30，ATTGCTGCTTACAGGTTTCT)与Bun-S-R(SEQ NO：31，AGGAAAGACGCAGAGGAGTG)。将扩增出的布尼亚病毒的目的片段***到pMD19-T载体中，挑单克隆经测序验证后，选择***成功并且序列正确的单克隆进行保菌。每次制备阳性质控品时，首先活化构建好的布尼亚病毒S段基因片段的菌株，然后提取活化后的菌株的质粒并测序，确定序列正确后再分别培养菌液。培养菌液后：对布尼亚S段基因片段的菌株进行质粒小提，以小提后的质粒做模板，进行体外RNA转录，将转录后得到的cRNA用灭菌后的双蒸水调至100ng/μL，以此作为布尼亚病毒的阳性质控品。制 备完后的阳性质控品需要-20℃储存，避免反复冻融，有效期为自生产之日起九个月。构建的阳性质控品为：Bun-S。Download a fragment of the S segment gene sequence of Bunia virus from NCBI, and design the primers for amplification of this fragment as Bun-S-F (SEQ ID NO: 30, ATTGCTGCTTACAGGTTTCT) and Bun-S-R (SEQ NO: 31, AGGAAAGACGCAGAGGAGTG). Insert the amplified target fragment of Bunia virus into the pMD19-T vector, pick a single clone and verify it by sequencing, then select the clone with the successful insertion and the correct sequence to preserve the bacteria. Each time a positive quality control product is prepared, the strain of the constructed bunya virus S segment gene fragment is first activated, and then the plasmid of the activated strain is extracted and sequenced. After the sequence is confirmed, the bacterial solution is cultured separately. After culturing the bacterial solution: Perform a small plasmid extraction on the strain of Bunia S segment gene fragment, use the small extracted plasmid as a template to perform in vitro RNA transcription, and adjust the cRNA obtained after transcription with sterilized double distilled water to 100ng/μL, as a positive quality control for Bunya virus. The prepared positive quality control products need to be stored at -20°C to avoid repeated freezing and thawing. The validity period is nine months from the date of production. The constructed positive quality control is Bun-S.
3.布尼亚病毒单重实时LAMP检测反应体系如下表所示：3. Bunia virus single real-time LAMP detection reaction system is shown in the following table:
Figure PCTCN2020074925-appb-000008
Figure PCTCN2020074925-appb-000008
实时LAMP扩增反应程序为：90cycle:63℃,15s；63℃,30s(此步收集荧光)。适用机型：ABI 7500、SLAN。The real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step). Applicable models: ABI 7500, SLAN.
4.实验结果：在扩增布尼亚病毒的阳性质控品的梯度稀释液(10 -9ng/μL～10 -5ng/μL)时1)当所用探针为Bun-RNHFIP，即探针设计在FIP引物所在的位置时，布尼亚阳性质控品及其梯度稀释液的扩增如图4所示，扩增灵敏度至10 -6ng/μL；2)当所用探针为Bun-RNHBIP，即探针设计在BIP引物所在的位置时，布尼亚阳性质控品及其梯度稀释液的扩增如图5所示，扩增灵敏度至10 -6ng/μL；3)当所用探针为Bun-RNHLF，即探针设计在F2与F1c之间的位置时，布尼亚阳性质控品及其梯度稀释液的扩增如图6所示，扩增灵敏度至10 -7ng/μL；4)当所用探针为Bun-RNHLB，即探针设计在B2与B1c之间的位置时，布尼亚阳性质控品及其梯度稀释液的扩增如图7所示，扩增灵敏度至10 -8ng/μL；5)当所用探针为Bun-RNHP，即探针设计在F1c与B1c之间的位置时，布尼亚阳性质控品及其梯度稀释液的扩增如图8所示，扩增灵敏度至10 -9ng/μL。这说明探针可以设计在靶标区域内的任何一个位置，但在一个检测项目中设计在各个位置的灵敏度有差别。 4. Experimental results: When amplifying the positive quality control product of Bunia virus in a serial dilution (10 -9 ng/μL～10 -5 ng/μL) 1) When the probe used is Bun-RNHFIP, the probe When the needle is designed at the position where the FIP primer is located, the amplification of Bunia positive quality control product and its serial dilutions is shown in Figure 4, and the amplification sensitivity is 10 -6 ng/μL; 2) When the probe used is Bunia -RNHBIP, that is, when the probe is designed at the position of the BIP primer, the amplification of Bunia positive quality control and its serial dilution is shown in Figure 5, and the amplification sensitivity is 10 -6 ng/μL; 3) The probe used is Bun-RNHLF, that is, when the probe is designed at the position between F2 and F1c, the amplification of Bunia positive quality control and its serial dilutions is shown in Figure 6, and the amplification sensitivity is 10 -7 ng/μL; 4) When the probe used is Bun-RNHLB, that is, when the probe is designed at a position between B2 and B1c, the amplification of Bunia positive quality control and its serial dilutions is shown in Figure 7. The amplification sensitivity is 10 -8 ng/μL; 5) When the probe used is Bun-RNHP, that is, when the probe is designed at a position between F1c and B1c, the amplification of Bunia positive quality control products and their gradient dilutions As shown in Figure 8, the amplification sensitivity was 10 -9 ng/μL. This shows that the probe can be designed at any position within the target area, but the sensitivity of the design at each position is different in a test item.
实施例四 三重实时LAMP的检测结果Example 4 Detection results of triple real-time LAMP
1.检测引物与探针及阳性质控品如实施例一与实施例二中所述，只是将检测布尼亚病毒的探针(Bun-RNHP)又重新合成了一条标记有Cy5信号的探针，即布尼亚病毒的 检测探针标记为Cy5荧光报告基团、ACTB基因的检测探针标记为ROX荧光报告基团、寨卡病毒的检测探针标记为FAM荧光报告基团。1. Detection primers and probes and positive quality control materials are as described in Example 1 and Example 2, except that the Bunia virus detection probe (Bun-RNHP) is re-synthesized to a probe labeled with Cy5 signal. The needle, that is, the detection probe of Bunia virus is labeled with Cy5 fluorescent reporter, the detection probe of ACTB gene is labeled with ROX fluorescent reporter, and the detection probe of Zika virus is labeled FAM fluorescent reporter.
1.ACTB基因、寨卡病毒cRNA、布尼亚病毒cRNA三重反应体系如下表所示：1. The triple reaction system of ACTB gene, Zika virus cRNA, and Bunya virus cRNA is shown in the following table:
Figure PCTCN2020074925-appb-000009
Figure PCTCN2020074925-appb-000009
实时LAMP扩增反应程序为：90cycle:63℃,15s；63℃,30s(此步收集荧光)。适用机型：ABI 7500、SLAN。The real-time LAMP amplification reaction program is: 90cycle: 63°C, 15s; 63°C, 30s (collect fluorescence in this step). Applicable models: ABI 7500, SLAN.
2.实验结果2. Experimental results
将内参ACTB阳性质控品的10 4copies/μL梯度的模板，搭配寨卡病毒阳性质控品(ZK-NS5)与布尼亚病毒阳性质控品(Bun-S)的10 -9ng/μL模板的扩增结果如图9所示，三个模板都得到了较好的扩增；2)将内参ACTB阳性质控品的10 5copies/μL梯度的模板，搭配寨卡病毒阳性质控品(ZK-NS5)与布尼亚病毒阳性质控品(Bun-S)的10 -8ng/μL模板的扩增结果如图10所示，三个模板都得到了较好的扩增；3)将内参ACTB阳性质控品的10 6copies/μL梯度的模板，搭配寨卡病毒阳性质控品(ZK-NS5)与布尼亚病毒阳性质控品(Bun-S)的10 -7ng/μL模板的扩增结果如图11所示，三个模板都得到了较好的扩增，只是与前两个低浓度的扩增Ct值相比，有的靶标的Ct值反而变大，说明在模板浓度变高时，各模板之间的扩增受到了些许干扰，但是在三重LAMP中，每个靶标的检测 结果仍能达到单重时的检测灵敏度。 Combine the 10 4 copies/μL gradient template of ACTB positive quality control material with Zika virus positive quality control material (ZK-NS5) and Bunia virus positive quality control material (Bun-S) 10 -9 ng/ [mu] L template amplification results shown in Figure 9, three templates have been better amplification; 2) 10 5 copies of the internal control ACTB positive control materials / μL template gradient, Zika virus with positive controls The amplification results of 10 -8 ng/μL template of ZK-NS5 and Bunia virus positive quality control material (Bun-S) are shown in Figure 10. All three templates have been well amplified; 3) Combine the 10 6 copies/μL gradient template of ACTB positive quality control material with Zika virus positive quality control material (ZK-NS5) and Bunia virus positive quality control material (Bun-S) 10 -7 The amplification results of the ng/μL template are shown in Figure 11. All three templates have been amplified well, but compared with the first two low-concentration amplified Ct values, the Ct values of some targets have become larger. , Which shows that when the template concentration becomes higher, the amplification between the templates is slightly interfered, but in the triple LAMP, the detection result of each target can still reach the detection sensitivity of single-plex.
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。It should be understood that the disclosed invention is not limited to the specific methods, schemes and materials described, as these can be varied. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims.
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。Those skilled in the art will also recognize, or be able to confirm, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also included in the appended claims.

Claims (11)

  1. 单管检测一种或多种待测目标核酸序列的方法，其特征在于，所述方法包括以下步骤：A method for detecting one or more target nucleic acid sequences in a single tube, characterized in that the method comprises the following steps:
    步骤1：针对每种待测目标核酸序列设计特异性等温扩增引物和荧光探针RNHP，每种待测目标核酸序列荧光探针RNHP标记不同的荧光，使得不同待测目标核酸序列能够在不同荧光信号通道中区分；所述荧光探针RNHP包含至少1个RNA碱基，在所述RNA碱基左侧靠近所述探针5’端一侧的探针碱基上标记荧光基团，在所述RNA碱基靠近所述探针3’端一侧的探针碱基上标记淬灭基团；所述至少1个RNA碱基优选地为至少1个连续的RNA碱基，更优选地为1个RNA碱基；Step 1: Design specific isothermal amplification primers and fluorescent probe RNHP for each target nucleic acid sequence to be tested. The fluorescent probe RNHP for each target nucleic acid sequence to be tested is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be different The fluorescent signal channel is distinguished; the fluorescent probe RNHP contains at least one RNA base, and a fluorescent group is labeled on the probe base on the left side of the RNA base near the 5'end of the probe, and The probe base on the side of the RNA base close to the 3'end of the probe is labeled with a quenching group; the at least one RNA base is preferably at least one continuous RNA base, more preferably Is 1 RNA base;
    步骤2：在核糖核酸酶RNaseH存在下，在核酸聚合酶的作用下等温扩增每种待测目标核酸序列，每种荧光探针RNHP结合到相应的待测目标核酸序列上，形成探针-目标核酸杂交双链；所述核糖核酸酶RNaseH切割探针-目标核酸杂交双链中的RNA碱基，使得所述RNA碱基右侧的含有淬灭基团的探针片段游离出去，而在RNA碱基左侧的含有荧光基团的片段仍然保持形成杂交链且荧光基团发出荧光；和Step 2: In the presence of ribonuclease RNaseH, each target nucleic acid sequence to be tested is amplified isothermally under the action of nucleic acid polymerase, and each fluorescent probe RNHP binds to the corresponding target nucleic acid sequence to be tested to form a probe- The target nucleic acid hybrid double-stranded; the ribonuclease RNaseH cleaves the RNA base in the probe-target nucleic acid hybrid double-strand, so that the probe fragment containing the quencher group on the right side of the RNA base is freed, and the The fragment containing the fluorophore to the left of the RNA base still remains to form a hybrid chain and the fluorophore emits fluorescence; and
    步骤3：通过待测目标核酸序列形成的带不同荧光标记的杂交链产物，实现待测目标核酸序列的检测。Step 3: The detection of the target nucleic acid sequence to be tested is realized through the hybridization chain products with different fluorescent labels formed by the target nucleic acid sequence to be tested.
  2. 根据权利要求1所述的方法，其特征在于，所述荧光探针RNHP长度为16-45bp，所述荧光探针RNHP上标记的荧光基团和淬灭基团的碱基之间的距离为5-15bp；所述RNA碱基的左侧所述探针片段中G+C含量为40％-60％；和所述RNA碱基的右侧所述探针片段的长度为2-4bp。The method according to claim 1, wherein the length of the fluorescent probe RNHP is 16-45 bp, and the distance between the fluorescent group labeled on the fluorescent probe RNHP and the base of the quenching group is 5-15 bp; the G+C content in the probe fragment on the left side of the RNA base is 40%-60%; and the length of the probe fragment on the right side of the RNA base is 2-4 bp.
  3. 根据权利要求1所述的方法，其特征在于，所述待测目标核酸序列为DNA序列，所述核酸聚合酶为Bst聚合酶，或所述待测目标核酸序列为RNA序列，所述核酸聚合酶为Bst聚合酶与AMV逆转录酶；和所述核糖核酸酶RNaseH是耐热核糖核酸酶RNaseH，所述耐热核糖核酸酶RNaseH优选地为RNaseH2。The method of claim 1, wherein the target nucleic acid sequence to be tested is a DNA sequence, the nucleic acid polymerase is Bst polymerase, or the target nucleic acid sequence to be tested is an RNA sequence, and the nucleic acid polymerase is The enzymes are Bst polymerase and AMV reverse transcriptase; and the ribonuclease RNaseH is a thermostable ribonuclease RNaseH, and the thermostable ribonuclease RNaseH is preferably RNaseH2.
  4. 根据权利要求1所述的方法，其特征在于，所述等温扩增为环介导等温扩增、重组酶聚合酶等温扩增、单引物等温扩增、依赖解旋酶的等温扩增、链替代扩增、交叉引物扩增技术、核酸依赖性扩增检测技术或切刻内切酶核酸等温扩增。The method of claim 1, wherein the isothermal amplification is loop-mediated isothermal amplification, recombinase polymerase isothermal amplification, single-primer isothermal amplification, helicase-dependent isothermal amplification, chain Alternative amplification, cross-primer amplification technology, nucleic acid-dependent amplification detection technology or nicking endonuclease nucleic acid isothermal amplification.
  5. 单管检测一种或多种待测目标核酸序列的试剂盒，其特在于，所述试剂盒包括：核糖核酸酶RNaseH，核酸聚合酶，和针对每种待测目标核酸序列设计特异性等温扩增引物和荧光探针RNHP，每种待测目标核酸序列荧光探针RNHP标记不同的荧光，使得不同待测目标核酸序列能够在不同荧光信号通道中区分；所述荧光探针RNHP包含至少1个RNA碱基，在所述RNA碱基左侧靠近所述探针5’端一侧的探针碱基上标记荧光基团，在所述RNA碱基靠近所述探针3’端一侧的探针碱基上标记淬灭基团；所述至少1 个RNA碱基优选地为至少1个连续的RNA碱基，更优选地为1个RNA碱基。A kit for detecting one or more target nucleic acid sequences to be tested in a single tube, characterized in that the kit includes: ribonuclease RNaseH, nucleic acid polymerase, and a specific isothermal expansion designed for each target nucleic acid sequence to be tested Primer enhancer and fluorescent probe RNHP, each target nucleic acid sequence to be tested fluorescent probe RNHP is labeled with different fluorescence, so that different target nucleic acid sequences to be tested can be distinguished in different fluorescent signal channels; the fluorescent probe RNHP includes at least one RNA base, a fluorescent group is labeled on the probe base on the left side of the RNA base near the 5'end of the probe, and the RNA base is marked on the probe base near the 3'end of the probe The probe base is labeled with a quencher group; the at least one RNA base is preferably at least one continuous RNA base, more preferably one RNA base.
  6. 根据权利要求5所述的试剂盒，其特征在于，所述荧光探针RNHP长度为16-45bp，所述荧光探针RNHP上标记的荧光基团和淬灭基团的碱基之间的距离为5-15bp；所述RNA碱基的左侧所述探针片段中G+C含量为40％-60％；和所述RNA碱基的右侧所述探针片段的长度为2-4bp。The kit according to claim 5, wherein the length of the fluorescent probe RNHP is 16-45 bp, and the distance between the fluorescent group labeled on the fluorescent probe RNHP and the base of the quenching group The length of the probe fragment on the left side of the RNA base is 40%-60%; and the length of the probe fragment on the right side of the RNA base is 2-4 bp. .
  7. 根据权利要求5所述的试剂盒，其特征在于，所述待测目标核酸序列为DNA序列，所述核酸聚合酶为Bst聚合酶，或所述待测目标核酸序列为RNA序列，所述核酸聚合酶为Bst聚合酶与AMV逆转录酶；和所述核糖核酸酶RNaseH是耐热核糖核酸酶RNaseH，所述耐热核糖核酸酶RNaseH优选地为RNaseH2。The kit according to claim 5, wherein the target nucleic acid sequence to be tested is a DNA sequence, the nucleic acid polymerase is Bst polymerase, or the target nucleic acid sequence to be tested is an RNA sequence, and the nucleic acid The polymerase is Bst polymerase and AMV reverse transcriptase; and the ribonuclease RNaseH is a thermostable ribonuclease RNaseH, and the thermostable ribonuclease RNaseH is preferably RNaseH2.
  8. 根据权利要求5所述的试剂盒，其特征在于，所述试剂盒是检测寨卡病毒的环介导等温扩增检测试剂盒，所述试剂盒利用检测寨卡病毒核酸的引物探针搭配检测内参的引物探针形成的二重实时环介导等温扩增来完成，所述试剂盒内检测寨卡病毒核酸所用的环介导等温扩增引物分别是SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6，所用的RNHP探针是SEQ ID NO：7；所述试剂盒内检测作为内参的人类ACTB基因所用的环介导等温扩增引物分别是SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13，所用的RNHP探针是SEQ ID NO：14；两种RNHP探针分别标记不同的荧光，各个序列如下：The kit according to claim 5, wherein the kit is a loop-mediated isothermal amplification detection kit for detecting Zika virus, and the kit uses primers and probes for detecting Zika virus nucleic acid in combination with detection The double real-time loop-mediated isothermal amplification formed by the primer probe of the internal reference is completed. The loop-mediated isothermal amplification primers used in the detection of Zika virus nucleic acid in the kit are SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, the RNHP probe used is SEQ ID NO: 7; the human ACTB gene as an internal reference is detected in the kit The loop-mediated isothermal amplification primers used are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the used RNHP The probe is SEQ ID NO: 14; the two RNHP probes are labeled with different fluorescence, and the respective sequences are as follows:
    引物序列号Primer sequence number 引物序列Primer sequence SEQ ID NO：1SEQ ID NO: 1 TGACCAGGGCCTCCTTTTGTGGGGGCTGGAGTTACTACGTTGACCAGGGCCTCCTTTTGTGGGGGCTGGAGTTACTACGT SEQ ID NO：2SEQ ID NO: 2 TGAAGAACCCGTGTTGGTGCAGCCGCCATATGAAAGACGTTGAAGAACCCGTGTTGGTGCAGCCGCCATATGAAAGACGT SEQ ID NO：3SEQ ID NO: 3 TTGAACTTTGCGGATGGTGGTTGAACTTTGCGGATGGTGG SEQ ID NO：4SEQ ID NO: 4 AAGCTATGGGTGGAACATAGTCCAAGCTATGGGTGGAACATAGTCC SEQ ID NO：5SEQ ID NO: 5 TGATCTTGGATGTGGCAGAGTGATCTTGGATGTGGCAGAG SEQ ID NO：6SEQ ID NO: 6 CCTATGTCACACAGCAACGTCCTATGTCACACAGCAACGT SEQ ID NO：7SEQ ID NO: 7 TGTTGGTGCAAAGC TATgGGT TGTTGGTGCAAAGC T ATgGGT SEQ ID NO：8SEQ ID NO: 8 GCGGATGTCCACGTCACACTTCCTGTGGCATCCACGAAGCGGATGTCCACGTCACACTTCCTGTGGCATCCACGAA SEQ ID NO：9SEQ ID NO: 9 AACACAGTGCTGTCTGGCGGTGCCAGGGCAGTGATCTCAACACAGTGCTGTCTGGCGGTGCCAGGGCAGTGATCTC SEQ ID NO：10SEQ ID NO: 10 TCATGATGGAGTTGAAGGTAGTTCATGATGGAGTTGAAGGTAGT SEQ ID NO：11SEQ ID NO: 11 CACCACCATGTACCCTGGCACCACCATGTACCCTGG SEQ ID NO：12SEQ ID NO: 12 TTCCCTCTCAGGCATGGATTCCCTCTCAGGCATGGA SEQ ID NO：13SEQ ID NO: 13 AGGAAAGACACCCACCTTGAAGGAAAGACACCCACCTTGA SEQ ID NO：14SEQ ID NO: 14 CACCACCATGTACCC TGGCaTTG CACCACCATGTACCC T GGCaTTG
  9. 根据权利要求8所述的试剂盒，其特征在于，所述试剂盒还包括阳性质控品和阴性空白对照品；所述阳性质控品为内参基因片段的标准质粒和寨卡病毒cRNA。The kit according to claim 8, wherein the kit further comprises a positive quality control substance and a negative blank control substance; the positive quality control substance is a standard plasmid of internal reference gene fragments and Zika virus cRNA.
  10. 根据权利要求5所述的试剂盒，其特征在于，所述试剂盒是检测布尼亚病毒的环介导等温扩增检测试剂盒，所述试剂盒利用检测布尼亚病毒核酸的引物探针搭配检测内 参的引物探针形成的二重实时环介导等温扩增来完成，所述试剂盒内检测布尼亚病毒核酸所用的环介导等温扩增引物分别是SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，所用的RNHP是SEQ ID NO：21；所述试剂盒内检测作为内参的人类ACTB基因所用的环介导等温扩增引物分别是SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13，所用的RNHP探针是SEQ ID NO：14；两种RNHP探针分别标记不同的荧光，The kit according to claim 5, wherein the kit is a loop-mediated isothermal amplification detection kit for detecting Bunia virus, and the kit uses a primer probe for detecting Bunia virus nucleic acid The double real-time loop-mediated isothermal amplification formed by the primer probe with the detection internal reference is completed. The loop-mediated isothermal amplification primers used in the detection of Bunia virus nucleic acid in the kit are SEQ ID NO: 15 and SEQ. ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, the RNHP used is SEQ ID NO: 21; the kit detects human ACTB as an internal reference The loop-mediated isothermal amplification primers used in the gene are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and the used The RNHP probe is SEQ ID NO: 14; the two RNHP probes are labeled with different fluorescence,
    引物序列号Primer sequence number 引物序列Primer sequence SEQ ID NO：15SEQ ID NO: 15 CGGAGCCAGCAAGACAGAAGTTGACAGAGTTCACAGCAGCATCGGAGCCAGCAAGACAGAAGTTGACAGAGTTCACAGCAGCAT SEQ ID NO：16SEQ ID NO: 16 AAGCCTCCATCAGGGTCTTGGTTCGGGTCCCTGATTCCAACAAGCCTCCATCAGGGTCTTGGTTCGGGTCCCTGATTCCAAC SEQ ID NO：17SEQ ID NO: 17 CTCCTTCAGGGATCCTCTCCCTCCTTCAGGGATCCTCTCC SEQ ID NO：18SEQ ID NO: 18 CGTGGCTTCAGATACCCCTGCGTGGCTTCAGATACCCCTG SEQ ID NO：19SEQ ID NO: 19 CCGAACATCATTGGGGAAGACCGAACATCATTGGGGAAGA SEQ ID NO：20SEQ ID NO: 20 CCCTGAGATGATGTGCATGGCCCTGAGATGATGTGCATGG SEQ ID NO：21SEQ ID NO: 21 GGTGAAGGCATCTTGCCA TAaAGA GGTGAAGGCATCTTGCCA T AaAGA SEQ ID NO：8SEQ ID NO: 8 GCGGATGTCCACGTCACACTTCCTGTGGCATCCACGAAGCGGATGTCCACGTCACACTTCCTGTGGCATCCACGAA SEQ ID NO：9SEQ ID NO: 9 AACACAGTGCTGTCTGGCGGTGCCAGGGCAGTGATCTCAACACAGTGCTGTCTGGCGGTGCCAGGGCAGTGATCTC SEQ ID NO：10SEQ ID NO: 10 TCATGATGGAGTTGAAGGTAGTTCATGATGGAGTTGAAGGTAGT SEQ ID NO：11SEQ ID NO: 11 CACCACCATGTACCCTGGCACCACCATGTACCCTGG SEQ ID NO：12SEQ ID NO: 12 TTCCCTCTCAGGCATGGATTCCCTCTCAGGCATGGA SEQ ID NO：13SEQ ID NO: 13 AGGAAAGACACCCACCTTGAAGGAAAGACACCCACCTTGA SEQ ID NO：14SEQ ID NO: 14 CACCACCATGTACCC TGGCaTTG CACCACCATGTACCC T GGCaTTG
  11. 根据权利要求10所述的试剂盒，其特征在于，所述试剂盒还包括阳性质控品和阴性空白对照品；所述阳性质控品为内参基因片段的标准质粒和布尼亚病毒cRNA。The kit according to claim 10, wherein the kit further comprises a positive quality control substance and a negative blank control substance; the positive quality control substance is a standard plasmid of internal reference gene fragments and Bunya virus cRNA.
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