WO2024259848A1 - 一种实时荧光环介导等温核酸扩增检测方法 - Google Patents

一种实时荧光环介导等温核酸扩增检测方法 Download PDF

Info

Publication number
WO2024259848A1
WO2024259848A1 PCT/CN2023/128187 CN2023128187W WO2024259848A1 WO 2024259848 A1 WO2024259848 A1 WO 2024259848A1 CN 2023128187 W CN2023128187 W CN 2023128187W WO 2024259848 A1 WO2024259848 A1 WO 2024259848A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
primer
acid amplification
amplification
labeled
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/128187
Other languages
English (en)
French (fr)
Inventor
吴坚
陈艳菊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University
Original Assignee
Zhejiang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University filed Critical Zhejiang University
Publication of WO2024259848A1 publication Critical patent/WO2024259848A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/63—Vibrio

Definitions

  • the present invention relates to a real-time nucleic acid amplification method, in particular to a real-time, single or multiple specific loop-mediated isothermal nucleic acid amplification primer and an amplification detection method thereof, which can be applied to the fields of gene detection, medical diagnosis, food safety, analytical chemistry, etc.
  • Nucleic acid amplification detection methods achieve the purpose of detection by amplifying target nucleic acid molecules. They have the characteristics of high sensitivity and are therefore widely used in food safety, disease diagnosis, environmental monitoring and other fields. Common nucleic acid amplification detection methods include polymerase chain reaction (PCR) and isothermal amplification reaction. Loop-mediated isothermal amplification (LAMP) is one of the most commonly used isothermal amplification reactions.
  • the LAMP reaction system contains outer primers (F3/B3), inner primers (FIP/BIP) and loop primers (LF/LB). The inner primers form their own loop structure, which enables amplification under constant temperature conditions (65°C) and can be used for on-site detection.
  • the amplified nucleic acid sample needs to be tested for amplification products to determine whether it is the target product.
  • Common detection methods can be divided into endpoint detection methods and real-time detection methods.
  • End-point detection methods detect amplification products after amplification, such as gel electrophoresis and turbidity methods. These detection methods are non-specific and it is difficult to identify false positive test results.
  • Real-time detection methods monitor the amplification process in real time by adding embedded dyes (such as SYBR Green, SYTO 9, etc.) or sequence-specific probes to the amplification system. Real-time amplification detection methods based on embedded dyes have poor specificity and are difficult to achieve multiple detection.
  • the real-time amplification detection method based on sequence-specific probes has high specificity. By adding sequence-specific probes modified with different fluorescent groups in different LAMP systems, real-time multiple detection can be achieved.
  • LAMP amplification some researchers introduced a sequence (c-FIP/c-BIP) that is complementary to the 5' end of the inner primer (FIP/BIP) in the LAMP amplification system, and modified the fluorescent group and quenching group at the 5' end of the inner primer and the 3' end of the inner primer complementary pairing sequence, respectively.
  • the fluorescent group is quenched.
  • the inner primer complementary pairing sequence is displaced, so that the real-time amplification signal is detected.
  • DARQ detection of amplification by release of quenching
  • Tm melting temperature
  • the current real-time loop-mediated nucleic acid amplification detection method based on sequence-specific probes still has certain limitations. Therefore, it is urgent to develop a simple, enzyme-free, rapid, and real-time multiplex loop-mediated isothermal nucleic acid amplification detection method.
  • the present invention provides a simple-to-design nucleic acid amplification primer, sequence and detection method, which can realize single or multiple real-time LAMP amplification detection, and has the characteristics of simple design, no need for additional enzymes, rapidity and real-time, etc.
  • the method of the present invention only requires Bst amplification enzyme, which is an advantage of the present invention.
  • Other methods may require other enzymes in addition to the amplification enzyme, so the present invention has the advantages of being simple, fast and not requiring additional enzymes.
  • one or more primers among the inner primer (FIP/BIP) and the loop primer (LF/LB) of the loop-mediated isothermal amplification system are used as labeled primers, and the labeled primers are labeled/modified with fluorescent groups and quenching groups, and the fluorescent groups and quenching groups are labeled/modified at any position of the labeled primer except the 3' end;
  • the labeled primer is used to perform loop-mediated isothermal nucleic acid amplification.
  • the fluorescence emitted by the fluorescent group is detected in real time, and the nucleic acid amplification situation is judged in real time based on the obtained fluorescence situation and the curve.
  • the fluorescence emitted by the fluorescent group is detected, and the nucleic acid amplification result is judged based on the obtained fluorescence situation and the curve.
  • the loop-mediated isothermal amplification system/method does not contain any functional enzyme that can cut, break, etc. the primer containing the fluorescent marker itself and the double-stranded chain generated by it.
  • the labeled primer has a 5'-terminal extension segment, and the fluorescent group and the quenching group are separately or completely labeled on the 5'-terminal extension segment.
  • the 5' end extension segment refers to a segment formed by extending the 5' end sequence according to conventional design.
  • the 5'-end extension segment can be any sequence of appropriate length containing thymine (T) bases, and it is not required that the extension segment itself or the extension segment and the connected primer form a secondary structure such as a stem-loop.
  • T thymine
  • the labeling/modification of the fluorescent group and the quenching group can be the following:
  • the fluorescent group and the quenching group are simultaneously modified in the middle of the labeled primer;
  • any one of the fluorescent group and the quenching group is modified in the middle of the labeled primer, and the other is modified at the 5' end of the labeled primer;
  • any one of the fluorescent group and the quenching group is modified at the 5'-terminal extension of the labeled primer, and the other is modified at the middle, 5'-terminal or 5'-terminal extension of the labeled primer.
  • the distance between the fluorescent group and the quenching group is between 9 and 35 bases.
  • the labeled primer binds to its complementary fragment and, under the action of a nucleic acid amplification enzyme having only a strand displacement function, extends along the target nucleic acid fragment to form a stable double strand.
  • the amplification primers of different target nucleic acid fragments are labeled/modified with fluorescent groups with different emission wavelengths to achieve real-time multiple nucleic acid detection.
  • the present invention provides a single or multiple real-time nucleic acid amplification detection system, in which fluorescent groups and quenching groups are modified on one or more primers of the amplification system.
  • the fluorescent group and the quenching group are modified on the primer sequence at the same time.
  • the two groups can be modified in the middle of the primer, or one of the groups can be modified at the 5' end of the primer; the 5' end of the primer can also be extended, and one or two groups can be modified on the extended sequence of the 5' end of the primer.
  • the same or different fluorescent groups and corresponding quenching groups may be modified on the multiple primers to enhance the signal.
  • the primer When there is no amplification reaction, the primer is single-stranded DNA.
  • the "curled" state of single-stranded DNA makes the fluorescent group and the quenching group close in space, and the fluorescence signal is quenched; after the amplification reaction occurs, the primer modified with the fluorescent group and the quenching group binds to the target nucleic acid and is extended according to the target nucleic acid sequence to form double-stranded DNA. Since the rigidity of double-stranded DNA is greater than that of single-stranded DNA, the fluorescent group and the quenching group in the double-stranded DNA are far away, the fluorescence quenching effect is weakened, and the fluorescence signal generated in the system is enhanced.
  • nucleic acid amplification of the present invention after the primer modified with the fluorescent group and the quenching group is combined with the sequence of the target fragment, it will be extended from the 3' end.
  • This extension also increases the length of the primer labeled with the fluorescent group and the quenching group, and the base of the primer and the sequence of the target fragment are complementary to each other, so that a stable double-stranded chain can be naturally formed without the need for other means.
  • This also greatly reduces the possibility that the primer modified with the fluorescent group and the quenching group is replaced due to the amplification of other primers in the system, and can form a stable double-stranded chain, thereby realizing efficient, simple and convenient nucleic acid amplification detection.
  • the fluorescence signal enhancement brought about by the conversion of the single strand into a double strand in the present invention is weaker.
  • the currently common fluorescence amplification instrument such as the Thermo Fisher real-time fluorescence quantitative QuantStudio3PCR instrument, the enhancement of the real-time fluorescence signal brought about by the formation of the double strand during the amplification process is clearly visible, thus enabling efficient, simple and convenient nucleic acid amplification detection.
  • the primer When at least one of the fluorescent group and the quenching group is modified to the 5'-end extension of the nucleic acid amplification primer, the primer will become part of the product after the sequence of the target fragment is combined, which makes target detection easier, improves the fluorescence detection effect, and makes the detection results more accurate.
  • the present invention has the following advantages:
  • the present invention utilizes the universal LAMP amplification system. It only needs to select one or more primers based on the existing primers and modify them with fluorescent groups and quenching groups. There is no need to perform additional optimization of the primers and reaction system in order to achieve specific fluorescence detection, nor is there any need to add additional enzymes.
  • the fluorescent group and quenching group can be modified at the 5’ end of the primer and the middle region except the 3’ end.
  • the 5' end of the primer can be extended and one or both of the fluorescent group and the quenching group can be labeled on the extended segment to avoid the influence.
  • the 5' end of the primer can also be extended to introduce a T base for labeling.
  • the 5'-end extension segment of the primer can be any sequence of appropriate length containing thymine (T) bases, and it is not required that the extension segment itself or the extension segment and the connected primer form a secondary structure such as a stem-loop.
  • T thymine
  • the selection of any random sequence can avoid complementarity with the detection target sequence and avoid affecting the amplification reaction.
  • this method can theoretically be used to detect any amplification system.
  • FIG1 is a schematic diagram of the LAMP isothermal amplification fluorescence detection principle proposed by the present invention.
  • FIG. 2 is a diagram of the single-plex real-time LAMP amplification detection of 400 (a), 40 (b), 4 (c), and 0 copies/ ⁇ L (d) of Vibrio parahaemolyticus DNA proposed by the present invention.
  • FIG3 is a real-time LAMP reaction proposed by the present invention to detect 400 copies/ ⁇ L of Vibrio parahaemolyticus DNA;
  • FIG4 is a diagram of the single-plex real-time LAMP amplification detection of 600 (a), 60 (b), 6 (c), and 0 copies/ ⁇ L (d) of Salmonella DNA proposed by the present invention
  • FIG5 is a real-time LAMP reaction detection 4 (a), 0 copies/ ⁇ L (b) Vibrio parahaemolyticus DNA proposed by the present invention
  • FIG6 is a dual real-time LAMP amplification proposed by the present invention for simultaneous detection of Vibrio parahaemolyticus and Salmonella DNA.
  • the system includes:
  • the primers can be inner primers (FIP/BIP) or loop primers (LB/LF).
  • One of the primer sequences of the inner primer (FIP/BIP)/loop primer (LB/LF) can be modified with a fluorescent group and a quenching group according to the above requirements, and two or more primer sequences of the inner primer (FIP/BIP)/loop primer (LB/LF) can also be modified with a fluorescent group and a quenching group according to the above requirements.
  • the fluorescent group and quenching group can be modified at the 5' end and the middle region of the primer. If there is no base T in the middle region, the 5' end of the primer can be extended and the T base can be incorporated;
  • the 5' end of the primer can be extended and one or both of the fluorescent group and the quenching group can be labeled on the extended segment to avoid the influence.
  • the 5'-end extension segment of the primer can be any sequence of appropriate length containing thymine (T) bases, and it is not required that the extension segment itself or the extension segment and the connected primer form a secondary structure such as a stem-loop.
  • T thymine
  • the above primers and their extension segments can all use common dNTPs, and do not need to be modified with special groups such as locked nucleic acids. Although the use of modified groups such as locked nucleic acids can increase the melting point of the corresponding double strands, the design of the primers and subsequent amplification will have certain effects. In the present invention, it is only necessary to modify the normally designed LAMP primers with fluorescent groups and quenching groups.
  • the fluorescence quenching effect is poor and there will be a large background signal; when the distance between the fluorescent group and the quenching group is too close, even if a double strand is formed, there will be no good fluorescence signal enhancement.
  • the preferred distance between the two is between 9-35 bases.
  • primers required for the LAMP amplification system such as outer primers (F3/B3), inner primers (FIP/BIP), loop primers (LB/LF), etc., can be kept consistent with the primers in ordinary LAMP amplification if they are not modified with fluorescence.
  • reaction concentration of each primer concentration can also be adjusted according to actual needs.
  • Polymerase a polymerase with strand displacement activity, such as Bst DNA polymerase, Bst 2.0 DNA polymerase, etc.
  • Bst DNA polymerase a polymerase with strand displacement activity
  • Bst 2.0 DNA polymerase a polymerase with strand displacement activity
  • no additional tool enzyme is required to cut the fluorescently labeled primer or the double-stranded primer generated thereby.
  • Buffer Select an appropriate buffer according to the type of polymerase in the system.
  • Magnesium sulfate The content of magnesium sulfate in the system can be 2 mM, or greater than 2 mM.
  • Amplification enhancers such as betaine, trehalose, etc. can be added to the amplification system.
  • Target molecule It can be RNA or DNA. If the target molecule is RNA, reverse transcriptase can be added to convert it into DNA.
  • the primers modified with the fluorescent group and the quenching group bind to the target fragment. Since there is no modification of any group at its 3' end, the primer can be extended from the 3' end after binding to the target sequence to form a double-stranded DNA.
  • the long double-stranded chain generated in this way helps the fluorescent-labeled primer to bind to the target chain and avoid falling off.
  • the double-stranded DNA sequence has greater rigidity, so that the modified fluorescent group and the quenching group are kept away from each other, and the fluorescence signal is enhanced.
  • the labeled primer here participates in amplification normally, so although the Bst amplification enzyme in LAMP has strand displacement activity, it will not displace the labeled primer alone in subsequent amplification.
  • the effect of the labeled primer on amplification efficiency is also very small.
  • the concentrations of different target primer sets in the system can be adjusted normally as needed, and different fluorescent groups should be modified on different primer sets to achieve multiplex detection.
  • the LAMP amplification detection process under the scheme of the present invention is shown in FIG1 , and is specifically as follows:
  • the detection method of the present invention is illustrated here by taking fluorescent labeling on FIP as an example.
  • F is a fluorescent group
  • Q is a corresponding quenching group, wherein the fluorescent group is labeled at the 5' end of the FIP primer, and the quenching group is labeled in the middle of the primer.
  • the labeling can also be performed on the BIP primer or the loop primer.
  • the specific amplification process of LAMP is the same as the general LAMP amplification method.
  • the F2 fragment in FIP binds and extends with the F2c in the target nucleic acid.
  • the outer primer F3 it is displaced by the chain displacement activity of the Bst enzyme.
  • the displaced single strand is amplified by BIP as a template. In this way, a double strand containing a fluorescent marker group is formed.
  • the double strand is more rigid, so the distance between the fluorescent group and the quenching group is increased, the quenching effect is weakened, and the fluorescence signal is increased.
  • double chains containing fluorescent groups will be continuously formed, so that the LAMP amplification process can be detected by real-time detection of fluorescent signals.
  • the fluorescent signal can also be detected after the amplification is completed.
  • This embodiment provides a real-time single-plex LAMP amplification detection method, in which a fluorescent group and a quenching group (FQ-VP-LB) are modified on the loop primer LB, and the spacing between them is 9 bases.
  • the specific sequence information is shown in Table 1.
  • the extracted Vibrio parahaemolyticus O3:K6 DNA was used as the detection object.
  • the LAMP amplification system included: 1.6 ⁇ M FIP, 1.6 ⁇ M BIP, 0.4 ⁇ M LF, 0.4 ⁇ M FQ-LB, 0.2 ⁇ M F3, 0.2 ⁇ M B3, 1 ⁇ isothermal amplification buffer, 0.8 M betaine, 1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA polymerase, 2 mM magnesium sulfate, and target DNA.
  • the fluorescent signal in addition to labeling the fluorescent signal in LB, the fluorescent signal can also be further labeled on LF, so that the detection system will have a stronger real-time fluorescent signal.
  • the detection object is still Vibrio parahaemolyticus, and the target concentration is 400 copies/ ⁇ L.
  • the same fluorescent and quenching groups are also modified on LF. Compared with the fluorescent signal in Figure 2 where only the fluorescent group is modified on LB, it can be seen that the fluorescent signal of modifying the two loop primers is stronger.
  • This example provides a real-time single-plex LAMP amplification detection method, in which the 5' end of the loop primer LF is extended (the bold bases are the extended sequence), and the fluorescent group and the quenching group (FQ-St-LF) are modified on the extended LF, with a spacing of 9 bases.
  • the specific sequence information is shown in Table 2.
  • the extracted Salmonella typhimurium DNA was used as the detection object.
  • the LAMP amplification system included: 1.6 ⁇ M FIP, 1.6 ⁇ M FIP, 0.4 ⁇ M FQ-LF, 0.4 ⁇ M LB, 0.2 ⁇ M F3, 0.2 ⁇ M B3, 1 ⁇ isothermal amplification buffer, 0.8 M betaine, 1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA polymerase, 2 mM magnesium sulfate, and target DNA.
  • a series of nucleic acid samples with different concentrations were added to the LAMP amplification system, and fluorescence curves were collected in real time at 65°C. The detection results are shown in Figure 4 below.
  • the reaction can detect target DNA as low as 6 copies/ ⁇ L, and the amplification curve of the lowest concentration sample can also reach the fluorescence plateau within 35 minutes, indicating that the reaction speed is relatively fast. There is no amplification curve for the sample with 0 copies/ ⁇ L, indicating that there is no nonspecific amplification in the reaction.
  • This example provides a real-time single-plex LAMP amplification detection method, in which the 5' end of the inner primer BIP is extended (the bold bases are the extended sequence), and the fluorescent group and the quenching group (FQ-VP-BIP) are modified on the extended BIP, with a spacing of 35 bases.
  • the specific sequence information is shown in Table 3.
  • the extracted Vibrio parahaemolyticus O3:K6 DNA was used as the detection object.
  • the LAMP amplification system included: 1.6 ⁇ M FIP, 1.6 ⁇ M FQ-VP-BIP, 0.4 ⁇ M LF, 0.4 ⁇ M LB, 0.2 ⁇ M F3, 0.2 ⁇ M B3, 1 ⁇ isothermal amplification buffer, 0.8 M betaine, 1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA polymerase, 2 mM magnesium sulfate, and target DNA.
  • This embodiment provides a real-time dual LAMP amplification detection method, which simultaneously detects the extracted Vibrio parahaemolyticus O3:K6 DNA and Salmonella typhimurium DNA.
  • the sequences used in the two amplification reactions are shown in Tables 1 and 2 in the above embodiments.
  • the LAMP amplification system comprises: 1 ⁇ isothermal amplification buffer, 0.8 M betaine, 1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA polymerase, 2 mM magnesium sulfate, and two target DNAs.
  • the total primer concentration of the dual LAMP amplification system is: 1.6 ⁇ M FIP, 1.6 ⁇ M BIP, 0.4 ⁇ M LF, 0.4 ⁇ M LB, 0.2 ⁇ M F3, 0.2 ⁇ M B3.
  • concentration of the Vibrio parahaemolyticus LAMP primer group accounts for 30%
  • concentration of the Salmonella LAMP primer group accounts for 70%.
  • the fluorescence of the TexasRed channel and the HEX channel were collected in real time at 65°C using the Thermo Fisher Scientific real-time fluorescence quantitative QuantStudio3PCR instrument. The test results are shown in Figure 6.
  • the reaction can simultaneously detect 40 copies/ ⁇ L of Vibrio parahaemolyticus O3:K6 DNA and 60 copies/ ⁇ L of Salmonella typhimurium DNA, and the detection signals of the two fluorescence channels do not interfere with each other.
  • the gene sequence involved in the present invention is as follows:
  • TTATCCGTCAGCGTTGTGAAGCA (5’ end modified with TexasRed; middle bold T base modified with BHQ2)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明提供了一种实时荧光环介导等温核酸扩增检测方法。在通用环介导等温扩增体系的基础上，在内引物（FIP/BIP）、环引物（LF/LB）中的一条或多条进行荧光基团和淬灭基团的标记；荧光基团和淬灭基团标记在所述引物除3'端的任何位置；利用所述引物进行核酸扩增，针对其中的荧光基团和淬灭基团之间相互作用产生的荧光进行检测，实现核酸扩增检测。

Description

一种实时荧光环介导等温核酸扩增检测方法 技术领域
本发明涉及了一种实时核酸扩增方法，尤其是涉及了一种实时、单一或多重的特异性环介导等温核酸扩增引物及其扩增检测方法，可应用于基因检测、医学诊断、食品安全、分析化学等领域。
 背景技术
核酸扩增检测方法通过对目标核酸分子进行扩增达到检测的目的，具有灵敏度高的特点，因此被广泛应用于食品安全、疾病诊断、环境监测等领域。常见的核酸扩增检测方法有聚合酶链式反应（polymerase chain reaction， PCR）和恒温扩增反应。环介导等温扩增反应（loop-mediated isothermal amplification， LAMP）是最常用的恒温扩增反应之一。LAMP反应体系中包含外引物（F3/B3）、内引物（FIP/BIP）和环引物（LF/LB），通过内引物形成自身环结构，使其在恒温条件（65°C）下就能实现扩增，可用于现场检测。
扩增后的核酸样本需要通过扩增产物检测来判定其是否为目标产物，常见的检测方法可分为终点检测法和实时检测法。
终点检测法在扩增结束后对扩增产物进行检测，例如凝胶电泳法、浊度法等，这些检测方法是非特异性的，难以辨别假阳性的检测结果。实时检测法通过在扩增体系中加入嵌入式染料（如SYBR Green, SYTO 9等）或者序列特异性探针对扩增过程进行实时监测。基于嵌入式染料的实时扩增检测方法特异性差，难以实现多重检测。
基于序列特异性探针的实时扩增检测方法特异性较高，通过在不同的LAMP体系中加入修饰不同荧光基团的序列特异性探针，即可实现实时多重检测。以LAMP扩增为例，有研究者在LAMP扩增体系中引入一条和内引物（FIP/BIP）5’端互补配对的序列(c-FIP/c-BIP)，分别在内引物的5’端和内引物互补配对序列的3’端修饰荧光基团和猝灭基团，其形成双链复合物时荧光基团被猝灭，扩增过程中，内引物互补配对序列被置换下来，使得实时扩增的信号被检测，该方法被称为DARQ（detection of amplification by release of quenching, DARQ）。DARQ方法可实现实时LAMP检测，但内引物互补配对序列的引入明显降低了LAMP反应的速度，且在LAMP扩增前，需要通过高温变性-退火步骤使内引物和内引物互补配对序列形成双链复合物，这使得反应过程复杂化。也有研究者在内引物（FIP/BIP）或者环引物(LF/LB)的5’端添加一段特异性的内酶切序列，在内切序列的两端修饰荧光基团和猝灭基团，使得LAMP扩增产物可以被特定的内切酶切割，从而达到实时检测的目的，但此方法需要在扩增体系中引入一种新的酶，这增加了反应体系的复杂性。
有研究者在扩增体系加入一段分子信标，使得分子信标和扩增片段上的序列特异性识别时打开茎环结构，产生荧光信号，但此方法要求分子信标序列和目标结合序列区域有合适Tm值（熔解温度，temperature of melting，Tm），这使得此方法存在一定的局限性。
综上所述，当前基于序列特异性探针的实时环介导核酸扩增检测方法仍存在一定的局限性。因此，急需发展一种设计简便、无需额外酶、快速、实时的多重环介导等温核酸扩增检测方法。
 发明内容
为解决上述问题和背景技术的缺陷，基于LAMP技术，本发明提供了一种设计简便的核酸扩增引物、序列和检测方法，可实现单一或者多重实时LAMP扩增检测，具有设计简便、无需额外酶、快速、实时的特点等。
本发明方法只需要Bst扩增酶，这是本发明的优势。而其他的方法除了扩增酶，可能需要别的酶，因此本发明具有简便快捷、无需额外酶的优点。
本发明技术方案和情况是：
首先在通用环介导等温扩增体系/方法基础上，以环介导等温扩增体系的内引物（FIP/BIP）、环引物（LF/LB）中的一条或多条引物作为标记引物，在标记引物上进行荧光基团和猝灭基团的标记/修饰，且荧光基团和猝灭基团标记/修饰在所述标记引物除3’端的任何位置；
然后利用所述标记引物进行环介导等温核酸扩增，在核酸扩增过程中，对荧光基团所发出的荧光进行实时检测，根据所得到的荧光情况以及曲线对核酸扩增情况进行实时判断。或者在核酸扩增后，对荧光基团所发出的荧光进行检测，根据所得到的荧光情况以及曲线对核酸扩增结果进行判断。
所述环介导等温扩增体系/方法中不含有任何能对含有荧光标记的引物本身及其生成的双链进行切割、断裂等的功能酶。
所述标记引物具有5’端延长段，所述荧光基团和猝灭基团分别或全部标记在所述5’端延长段。
所述的5’端延长段是指5’端的序列按照常规设计进行延长设计而形成的一段。
所述5’端延长段选用包含有胸腺嘧啶（T）碱基的合适长度的任意序列，并且不要求延长段本身或延长段和所连接引物形成茎环等二级结构。
具体而言，所述荧光基团和猝灭基团的标记/修饰可以是以下几种：
所述荧光基团和猝灭基团同时修饰在标记引物的中间；
或者荧光基团和猝灭基团中的任意其中一个修饰在标记引物的中间、另一个修饰在标记引物的5’端；
或者荧光基团和猝灭基团中的任意其中一个修饰在标记引物的5’端延长段、另一个修饰在标记引物的中间、5’端或者5’端延长段。
所选标记引物上，所述荧光基团和所述猝灭基团之间的间距在9-35个碱基之间。
在进行环介导等温核酸扩增时，当存在目标核酸片段时，标记引物和其互补片段结合，并在仅具有链置换功能的核酸扩增酶作用下，沿目标核酸片段延伸形成稳定双链。
在进行环介导等温核酸扩增时，当存在多个目标核酸片段时，对不同目标核酸片段的扩增引物标记/修饰上不同发射波长的荧光基团，实现实时多重核酸检测。
由此，本发明提供一种可实现单一或者多重实时核酸扩增检测体系，在扩增体系的一个或多个引物上修饰荧光基团和猝灭基团。
荧光基团和猝灭基团同时修饰在引物的序列上，既可以把这两个基团修饰在引物的中间，也可以把其中一个基团修饰在引物的5’端；也可以将引物的5’端进行延长，将一个或两个基团修饰在引物5’端延长的序列上。
在核酸扩增时存在多个引物，也可以在多个引物上修饰相同或不同的荧光基团及对应猝灭基团，增强信号。
在无扩增反应时，该引物为单链DNA，“蜷缩”状态的单链DNA使得荧光基团和猝灭基团在空间上较近，荧光信号被猝灭；发生扩增反应后，修饰有荧光基团和猝灭基团的引物与目标核酸结合，并根据目标核酸序列进行延伸形成双链DNA，由于双链DNA的刚性比单链大，双链中的荧光基团和猝灭基团远离，荧光猝灭效应减弱，使得体系中产生的荧光信号增强。
在本发明核酸扩增时，修饰有荧光基团和猝灭基团的所述引物和目标片段的序列结合后，将从3’端开始延伸，这一延伸，也使得标记有荧光基团和猝灭基团的引物的长度增加，并且引物的碱基和目标片段的序列相互互补，从而可以自然形成稳定的双链，而不需要其他手段，这也大大降低了修饰荧光基团和猝灭基团的引物由于体系中其他引物的扩增而被替换下来的可能性，能够形成稳定的双链，实现了高效率、简单便捷的核酸扩增检测。
并且本发明相比于采用四氢呋喃、内切酶等现有技术的其他手段将带有荧光基团和猝灭基团的序列切割，从而使得两者完全分离的情况，本发明中只是由于单链变成双链所带来的荧光信号增强要弱一些，但是采用目前常见的荧光扩增仪，如赛默飞实时荧光定量QuantStudio3PCR仪等，扩增过程中由于形成双链所带来的实时荧光信号的增强是清晰可见的，因此能够带来高效率、简单便捷的核酸扩增检测。
当荧光基团和猝灭基团其中至少一个修饰到酸扩增引物的5’端延长段时，引物和目标片段的序列结合后，将使得核酸扩增引物变成产物的一部分，这样目标检测更简易，提升了荧光检测效果，使得检测结果更准确。
相比于现在的技术，本发明具有如下优势：
1）实用性强。本发明利用通用LAMP扩增体系，只需要在现有引物的基础上，选择其中一个引物或多个引物，将其修饰上荧光基团和猝灭基团即可。不需要为了实现特异性荧光检测而对引物及反应体系进行额外的优化，也不需要增加额外的酶。
2）序列依赖性低。荧光基团和猝灭基团可修饰在引物的5’端和除3’端外的中间区域。
若所选引物上的碱基序列对荧光基团的信号有影响，可将引物5’端进行延长，并将荧光基团和猝灭基团中的一个或全部都标记在延长段，从而避免影响。
若所选引物中间区域没有胸腺嘧啶T，也可将引物5’端进行延长，引入T碱基，进行标记。
引物的5’端延长段选用包含有胸腺嘧啶（T）碱基的合适长度的任意序列，并且不要求延长段本身或延长段和所连接引物形成茎环等二级结构。
任意随机序列的选取可以避免其和检测目标序列的互补，避免对扩增反应的影响。
因此该方法理论上可用于任何扩增体系的检测。
3）操作简便：本发明所提出的扩增检测方法的检测步骤与普通扩增反应步骤一样，无需额外的操作步骤，节约时间。
4）节约成本：本发明所提出的扩增检测方法无需额外酶制剂的参与。
5）特异性高：可以避免内，外引物二聚体等导致的非特异性扩增。
 附图说明
图1是本发明提出的LAMP等温扩增荧光检测原理图；
图2是本发明提出的单重实时LAMP扩增检测400（a），40（b），4（c），0 copies/µL（d）的副溶血性弧菌DNA。
图3是本发明提出的实时LAMP反应检测400 copies/µL副溶血性弧菌DNA；
图4是本发明提出的单重实时LAMP扩增检测600（a），60（b），6（c），0 copies/µL（d）的沙门氏菌DNA；
图5是本发明提出的实时LAMP反应检测4（a），0 copies/µL（b）副溶血性弧菌DNA；
图6是本发明提出的双重实时LAMP扩增同时检测副溶血性弧菌和沙门氏菌DNA。a) 40 copies/µL 副溶血性弧菌DNA；b) 60 copies/µL 沙门氏菌DNA；c) 0 copies/µL 沙门氏菌DNA; d) 0 copies/µL副溶血性弧菌DNA。
 具体实施方式
下面结合具体的实施例和附图来对本发明做进一步地解释。下列实施例仅用于说明本发明，并不用来限定本发明的实施范围。
具体实施以LAMP扩增为例，所述体系包括：
1）在正常LAMP扩增体系中，选取需要修饰荧光基团和猝灭基团的一条或多条引物进行修饰。
修饰荧光基团和猝灭基团的引物：该引物可为内引物（FIP/BIP），也可以为环引物（LB/LF）。
可以在内引物（FIP/BIP）/环引物（LB/LF）其中之一引物序列按照上述要求修饰上荧光基团和猝灭基团，也在内引物（FIP/BIP）/环引物（LB/LF）的两个或多个引物序列均按照上述要求修饰上荧光基团和猝灭基团。
荧光基团和猝灭基团可修饰在引物的5’端和中间区域。若中间区域没有碱基T，可将引物5’端进行延长，掺入T碱基；
若所选引物上的碱基序列对荧光基团的信号有影响，也可以可将引物5’端进行延长，并将荧光基团和猝灭基团中的一个或全部都标记在延长段，从而避免影响。
引物的5’端延长段选用包含有胸腺嘧啶（T）碱基的合适长度的任意序列，并且不要求延长段本身或延长段和所连接引物形成茎环等二级结构。
上述引物及其延伸段均采用普通dNTP即可，无需利用锁核酸等特殊基团修饰。利用锁核酸等修饰基团，虽然可以提高相应双链的熔点，但引物的设计、后续的扩增等会带来一定的影响。本发明中，只需对正常设计的LAMP引物进行荧光基团和猝灭集团的修饰即可。
在引物上标记时，当荧光基团和猝灭基团之间的间距过大时，荧光的猝灭效应较差，会有较大的本底信号；而当荧光基团和猝灭基团之间的间距过近时，即使形成双链也无法有较好的荧光信号增强。优选的两者之间的间距为9-35个碱基之间。
2）其他引物：
LAMP扩增体系需要的其他引物，如外引物（F3/B3）、内引物（FIP/BIP）、环引物（LB/LF）等，若没有选择进行荧光修饰，则与普通LAMP扩增中的引物保持一致即可。
在多重扩增中也可以根据实际需要对各引物浓度的反应浓度进行调整。
这里充分体现出了本发明的优势，也即除了在需要修饰的引物上进行荧光及猝灭基团的标记外，其他和普通的LAMP扩增反应完全一样，即不需要增加额外的酶，也不会对已确定的LAMP反应体系产生任何影响，不存在会抑制扩增速度等问题。
3）聚合酶：具有链置换活性的聚合酶，例如Bst DNA聚合酶、Bst 2.0 DNA聚合酶等。在本发明中，不需要任何额外的工具酶来对荧光标记的引物或由其生成的双链进行切割。
4）缓冲液：根据体系中的聚合酶类型选择合适的缓冲液。
5）硫酸镁：体系中硫酸镁的含量可为2 mM,或者大于2 mM。
6）脱氧核苷酸：可以选用dNTPs，也可以选择dNTPs/dUTPs。
7）扩增增强剂：可在扩增体系中加入扩增增强剂，如甜菜碱、海藻糖等。
8）靶标分子：可以为RNA，也可以为DNA。若靶标分子为RNA，则可加入逆转录酶将其转化为DNA。
当核酸扩增体系溶液中存在靶标分子时，发生正常的LAMP扩增，修饰荧光基团和猝灭基团的引物和目标片段结合。由于其3’端没有修饰有任何基团，所以引物和目标序列结合后可以从3’端开始进行延伸，形成双链DNA。这样生成的长双链，有助于带有荧光标记引物和目标链结合，避免脱落。相比于单链，双链DNA序列有更大的刚性从而使得修饰的荧光基团和猝灭基团相互远离，荧光信号增强。
同时，这里的标记引物正常参与扩增，所以LAMP中的Bst扩增酶虽然有链置换活性，但在后续扩增中，也不会把标记引物单独置换下来。标记引物对扩增效率的影响也很小。
若进行多重扩增检测时，可根据需要，正常调整体系中的不同靶标引物组浓度之间的浓度，且不同引物组上要修饰不同的荧光基团，以实现多重检测。
这样，无需常用的嵌入式荧光染料，就可以实现扩增过程的实时荧光检测。通过在不同靶标的引物上修饰不同的荧光基团，就可以实现单管内的多重检测。
本发明方案下的LAMP扩增检测过程如图1所示，具体如下：
正常设计LAMP扩增的引物后，这里以在FIP上进行荧光标记为例进行本发明检测方法的图示说明。图中F为荧光基团，Q为相应的猝灭基团，其中荧光基团标记在FIP引物的5’端，猝灭基团标记在引物的中间。在这里只是在FIP上进行了标记，在实际中，也可以在BIP引物，或是环引物上进行标记。
利用了标记引物后，LAMP的具体扩增过程和通用的LAMP扩增方式一样。首先，FIP中的F2片段和目标核酸中的F2c结合并延伸。然后，外引物F3的作用下，在Bst酶的链置换活性作用下被置换下来。置换下来的单链被BIP作为模板，进行扩增。这样就形成了包含有荧光标记基团的双链。相比于卷曲的单链，双链刚性较强，因此增大了荧光基团和猝灭基团之间的距离，猝灭作用减弱，从而荧光信号增大。
在后续的正常LAMP扩增过程中，将不断形成这样含有荧光基团的双链，从而可以通过实时检测荧光信号，就可以实现LAMP扩增过程的检测。也可以在扩增结束后，对荧光信号进行检测。
本发明的实施例如下：
实施例1：
本实施例提供一种实时单重LAMP扩增检测方法，在环引物LB上修饰荧光基团和猝灭基团（FQ-VP-LB），其间距为9个碱基，具体序列信息如表1所示。
以提取的副溶血性弧菌 Vibrio parahaemolyticus O3:K6 DNA为检测对象，该LAMP扩增体系包含：1.6 µM FIP, 1.6 µM BIP, 0.4 µM LF, 0.4 µM FQ-LB, 0.2 µM F3, 0.2 µM B3, 1× isothermal amplification buffer, 0.8 M 甜菜碱，1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA聚合酶，2 mM 硫酸镁，靶标DNA。
将一系列浓度的核酸样本加入LAMP扩增体系中，利用赛默飞实时荧光定量QuantStudio3 PCR仪65°C实时采集TexasRed通道的荧光。检测结果如图2所示，该反应能检测低至4 copies/µL的目标DNA，且最低浓度DNA样本的扩增曲线在40分钟内也可以达到荧光平台期，说明该反应速度较快。0 copies/µL的样本无扩增曲线，说明该反应无非特异性扩增。
在上述反应体系中，除了LB中标记荧光信号，也可以进一步在LF上标记荧光信号，这样检测体系会有更强的实时荧光信号图3中，检测对象仍为副溶血弧菌，靶标浓度为400 copies/µL，除了LB上修饰了荧光和猝灭基团外，在LF上也修饰了同样的荧光和猝灭基团。相比于图2中只在LB上修饰了荧光基团的荧光信号，可以看到修饰2条环引物的荧光信号更强。
表1 副溶血性弧菌LAMP扩增序列
名称 序列（5’到3’）
VP-F3 ATCGCACCAGCTACTCGA
VP-B3 CGGCGAAGAACGTAATGTCT
VP-FIP CCACCAGTAGCCGTCAATGGTGAAGATGATCCAGCGACCGAT
VP-BIP ACACCAACACGTCGCAAAACGCGTTCTCGTTCGCCAAAT
VP-LB TTATCCGTCAGCGTTGTGAAGCA
FQ-VP-LB 5’ TexasRed-TTATCCGTCAGCG/iBHQ2dT/TGTGAAGCA
VP-LF TCGTTTTTTGCCCATTCCCA
实施例2：
本实施例提供一种实时单重LAMP扩增检测方法，将环引物LF的5’端延长（加粗碱基为延长序列），并在延长的LF上修饰荧光基团和猝灭基团（FQ- St-LF），其间距为9个碱基，具体序列信息如表2所示。
以提取的沙门氏菌 Salmonella typhimurium DNA为检测对象，该LAMP扩增体系包含：1.6 µM FIP, 1.6 µM FIP, 0.4 µM FQ-LF, 0.4 µM LB, 0.2 µM F3, 0.2 µM B3, 1× isothermal amplification buffer, 0.8 M 甜菜碱，1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA聚合酶，2 mM 硫酸镁，靶标DNA。将一系列浓度的核酸样本加入LAMP扩增体系中，65°C实时采集荧光曲线。检测结果如下图4所示，该反应能检测低至6 copies/µL的目标DNA，且最低浓度样本的扩增曲线在35分钟内也可以达到荧光平台期，说明该反应速度较快。0 copies/µL的样本无扩增曲线，说明该反应无非特异性扩增。
表2 沙门氏菌LAMP扩增序列
名称 序列（5’到3’）
St-F3 CGGCCCGATTTTCTCTGG
St-B3 CGGCAATAGCGTCACCTT
St-FIP GCGCGGCATCCGCATCAATATGCCCGGTAAACAGATGAGT
St-BIP GCGAACGGCGAAGCGTACTGTCGCACCGTCAAAGGAAC
FQ- St-LF 5-HEX- TGCAATGATAiBHQ1dTGGCCTTCAAATCGGCATCAAT
St-LF GGCCTTCAAATCGGCATCAAT
St-LB GAAAGGGAAAGCCAGCTTTACG
St-F3 CGGCCCGATTTTCTCTGG
实施例3：
本实施例提供一种实时单重LAMP扩增检测方法，将内引物BIP 5’端延长（加粗碱基为延长序列），并在延长的BIP上修饰荧光基团和猝灭基团（FQ-VP-BIP），其间距为35个碱基，具体序列信息如表3所示。
以提取的副溶血性弧菌Vibrio parahaemolyticus O3:K6 DNA为检测对象，该LAMP扩增体系包含：1.6 µM FIP, 1.6 µM FQ-VP-BIP, 0.4 µM LF, 0.4 µM LB, 0.2 µM F3, 0.2 µM B3, 1× isothermal amplification buffer, 0.8 M 甜菜碱，1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA聚合酶，2 mM 硫酸镁，靶标DNA。
将不同浓度的核酸样本加入LAMP扩增体系中，利用赛默飞实时荧光定量QuantStudio3PCR仪65°C实时采集TexasRed通道的荧光。检测结果如图5所示，该反应能检测低至4 copies/µL的目标DNA，且在40分钟内也可以达到荧光平台期，说明该反应速度较快。0 copies/µL的样本无扩增曲线，说明该反应无非特异性扩增。
表3 副溶血性弧菌LAMP扩增序列
名称 序列（5’到3’）
VP-F3 ATCGCACCAGCTACTCGA
VP-B3 CGGCGAAGAACGTAATGTCT
VP-FIP CCACCAGTAGCCGTCAATGGTGAAGATGATCCAGCGACCGAT
FQ-VP-BIP 5’TexasRed- TGCAATGACACCAACACGTCGCAAAACGCGTTCTCG/iBHQ2dT/TCGCCAAAT
VP-BIP ACACCAACACGTCGCAAAACGCGTTCTCGTTCGCCAAAT
VP-LB TTATCCGTCAGCGTTGTGAAGCA
VP-LF TCGTTTTTTGCCCATTCCCA
实施例4：
本实施例提供一种实时双重LAMP扩增检测方法，同时检测提取的副溶血性弧菌Vibrio parahaemolyticus O3:K6 DNA和沙门氏菌Salmonella typhimurium DNA，两扩增反应用到的序列如前述实施例中的表1和表2所示。该LAMP扩增体系包含： 1× isothermal amplification buffer, 0.8 M 甜菜碱，1.4 mM dNTPs, 8U Bst 2.0 WarmStart DNA聚合酶，2 mM 硫酸镁，两种靶标DNA。该双重LAMP扩增体系引物总浓度为：1.6 µM FIP, 1.6 µM BIP, 0.4 µM LF, 0.4 µM LB, 0.2 µM F3, 0.2 µM B3。其中副溶血性弧菌LAMP引物组浓度占30%，沙门氏菌LAMP引物组浓度占70%。利用赛默飞实时荧光定量QuantStudio3PCR仪65°C实时采集TexasRed通道和HEX通道的荧光。检测结果如图6所示，该反应能同时检测40 copies/µL的Vibrio parahaemolyticus O3:K6 DNA 和60 copies/µL Salmonella typhimurium DNA，两荧光通道检测信号无相互干扰。
 序列表自由内容
本发明涉及的基因序列如下：
SEQ ID No.1；
名称：VP-F3引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
ATCGCACCAGCTACTCGA
SEQ ID No.2；
名称：VP-B3引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
CGGCGAAGAACGTAATGTCT
SEQ ID No.3；
名称：VP-FIP引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
CCACCAGTAGCCGTCAATGGTGAAGATGATCCAGCGACCGAT
SEQ ID No.4；
名称：VP-BIP引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
ACACCAACACGTCGCAAAACGCGTTCTCGTTCGCCAAAT
SEQ ID No.5；
名称：VP-LB引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
TTATCCGTCAGCGTTGTGAAGCA
SEQ ID No.6；
名称：FQ-VP-LB引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
TTATCCGTCAGCGTTGTGAAGCA (5’端修饰TexasRed; 中间加粗T碱基修饰BHQ2)
SEQ ID No.7；
名称：VP-LF引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
TCGTTTTTTGCCCATTCCCA
SEQ ID No.8；
名称：St-F3引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
CGGCCCGATTTTCTCTGG
SEQ ID No.9；
名称：St-B3引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
CGGCAATAGCGTCACCTT
SEQ ID No.10；
名称：St-FIP引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
GCGCGGCATCCGCATCAATATGCCCGGTAAACAGATGAGT
SEQ ID No.11；
名称：St-BIP引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
GCGAACGGCGAAGCGTACTGTCGCACCGTCAAAGGAAC
SEQ ID No.12；
名称：FQ- St-LF引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
TGCAATGATATGGCCTTCAAATCGGCATCAAT（5’端修饰HEX；中间加粗T碱基修饰BHQ1）
SEQ ID No.13；
名称：St-LF引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
GGCCTTCAAATCGGCATCAAT
SEQ ID No.14；
名称：St-LB引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
GAAAGGGAAAGCCAGCTTTACG
SEQ ID No.15；
名称：St-F3引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
CGGCCCGATTTTCTCTGG
SEQ ID No.16；
名称：FQ-VP-BIP引物基因序列
生物体来源：人工序列(Artificial Sequence)/ synthetic construct
TGCAATGACACCAACACGTCGCAAAACGCGTTCTCGTTCGCCAAAT（5’修饰TexasRed，中间加粗碱基T修饰BHQ2）。

Claims (7)

  1. 一种实时荧光环介导等温核酸扩增检测方法，其特征如下：
    首先在环介导等温扩增体系基础上，以内引物、环引物中的一条或多条引物作为标记引物，在标记引物上进行荧光基团和猝灭基团的标记/修饰，且荧光基团和猝灭基团标记/修饰在所述标记引物除3’端的任何位置；
    然后利用所述标记引物进行核酸扩增，在核酸扩增过程中，对荧光基团所发出的荧光进行实时检测，根据所得到的荧光情况对核酸扩增情况进行判断。
  2. 根据权利要求1所述的核酸扩增方法，其特征在于：
    所述环介导等温扩增体系中不含有任何能对含有荧光标记的引物本身及其生成的双链进行切割、断裂等的功能酶。
  3. 根据权利要求1所述的核酸扩增方法，其特征在于：
    所述标记引物具有5’端延长段，所述荧光基团和猝灭基团分别或全部标记在所述5’端延长段。
  4. 根据权利要求3所述的核酸扩增方法，其特征在于：
    所述5’端延长段选用包含有胸腺嘧啶（T）碱基的任意序列，并且不要求延长段本身或延长段和所连接引物形成茎环等二级结构。
  5. 根据权利要求1-4任一所述的核酸扩增方法，其特征在于：
    所选标记引物上，所述荧光基团和所述猝灭基团之间的间距在9-35个碱基之间。
  6. 根据权利要求1-4任一所述的核酸扩增方法，其特征在于：
    在进行核酸扩增时，当存在目标核酸片段时，标记引物和其互补片段结合，并在具有链置换功能的核酸扩增酶作用下，沿目标核酸片段延伸形成双链。
  7. 根据权利要求1-4任一所述的核酸扩增方法，其特征在于：
    在进行核酸扩增时，当存在多个目标核酸片段时，对不同目标核酸片段的扩增引物标记/修饰上不同发射波长的荧光基团及对应的猝灭基团，实现实时多重核酸检测。
PCT/CN2023/128187 2023-06-21 2023-10-31 一种实时荧光环介导等温核酸扩增检测方法 Ceased WO2024259848A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310740901.3 2023-06-21
CN202310740901.3A CN116875664A (zh) 2023-06-21 2023-06-21 一种实时荧光环介导等温核酸扩增检测方法

Publications (1)

Publication Number Publication Date
WO2024259848A1 true WO2024259848A1 (zh) 2024-12-26

Family

ID=88270626

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/128187 Ceased WO2024259848A1 (zh) 2023-06-21 2023-10-31 一种实时荧光环介导等温核酸扩增检测方法

Country Status (2)

Country Link
CN (1) CN116875664A (zh)
WO (1) WO2024259848A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116254353A (zh) * 2022-11-24 2023-06-13 浙江大学杭州国际科创中心 一种检测副溶血弧菌的lamp引物、试剂盒及其检测方法
CN116875664A (zh) * 2023-06-21 2023-10-13 浙江大学 一种实时荧光环介导等温核酸扩增检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104962607A (zh) * 2015-04-07 2015-10-07 中国疾病预防控制中心传染病预防控制所 一种单一或多重目的基因片段恒温扩增的检测方法
US20200248241A1 (en) * 2017-08-18 2020-08-06 Lgc Genomics Limited Methods and kits for detection of nucleic acid molecules
CN116179655A (zh) * 2021-11-29 2023-05-30 北京迅识科技有限公司 一种实时荧光定量检测探针
CN116875664A (zh) * 2023-06-21 2023-10-13 浙江大学 一种实时荧光环介导等温核酸扩增检测方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024642A1 (en) * 2010-08-20 2012-02-23 Life Technologies Corporation Quantitative real-time pcr assay using fret dual-labeled primers
CN105506121A (zh) * 2016-01-10 2016-04-20 中国疾病预防控制中心传染病预防控制所 用于副溶血性弧菌和创伤弧菌检测的一组核苷酸序列
CN109750091B (zh) * 2019-03-13 2023-02-03 江苏宏微特斯医药科技有限公司 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
CN110358815B (zh) * 2019-07-18 2023-05-23 江苏宏微特斯医药科技有限公司 一种同时检测多个靶标核酸的方法及其试剂盒
WO2023071788A1 (zh) * 2021-10-27 2023-05-04 上海翔琼生物技术有限公司 一种用于检测核酸的荧光pcr方法
EP4446438A4 (en) * 2021-12-10 2025-10-08 Seegene Inc LABELED LOOP PRIMER COMPRISING A SINGLE-STRANDED INTERACTIVE DOUBLE LABELING SYSTEM, AND NUCLEIC ACID ISOTHERMAL AMPLIFICATION METHOD USING SAME

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104962607A (zh) * 2015-04-07 2015-10-07 中国疾病预防控制中心传染病预防控制所 一种单一或多重目的基因片段恒温扩增的检测方法
US20200248241A1 (en) * 2017-08-18 2020-08-06 Lgc Genomics Limited Methods and kits for detection of nucleic acid molecules
CN116179655A (zh) * 2021-11-29 2023-05-30 北京迅识科技有限公司 一种实时荧光定量检测探针
CN116875664A (zh) * 2023-06-21 2023-10-13 浙江大学 一种实时荧光环介导等温核酸扩增检测方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN, J. ET AL.: "Anti-primer quenching-based real-time PCR for simplex or multiplex DNA quantification and single-nucleotide polymorphism genotyping", NATURE PROTOCOLS, vol. 2, no. 1, 22 February 2007 (2007-02-22), XP055132325, DOI: 10.1038/nprot.2007.11 *

Also Published As

Publication number Publication date
CN116875664A (zh) 2023-10-13

Similar Documents

Publication Publication Date Title
EP4077722B1 (en) Methods of detecting an analyte
EP3880843B1 (en) Method for detection of rna
CN1468251B (zh) 一种用于均相检测核酸的特异性双链探针及其实施方法
JP3843215B2 (ja) 高い特異性を有するプライマー、増幅法およびキット
US20130178378A1 (en) Multiplex digital pcr
CN102071251B (zh) 一种pcr检测核酸的方法
US20020197611A1 (en) Method for real-time detection and quantification of nucleic acid sequences using fluorescent primers
JP7634551B2 (ja) 標的核酸を検出するためのループプライマー及びループ・デ・ループ方法
CA2847697A1 (en) Nucleic acid enzyme substrates
CN110195099B (zh) 一种多靶标基因并行检测组合探针及其试剂盒的应用
US20250283158A1 (en) Beacon-mediated exponential amplification reaction (bear) using a single enzyme and primer
Li et al. Efficient dual-amplification system for G-quadruplex-based non-enzymatic fluorescence detection of microRNA
Liang et al. A novel fluorescence method based on loop-mediated isothermal amplification and universal molecular beacon in Mycobacterium tuberculosis detection
CN116875664A (zh) 一种实时荧光环介导等温核酸扩增检测方法
CN111742057A (zh) 核酸扩增或与其相关的改进
CN108048533B (zh) 基于三通连接-核酸分子线路换能器的分子检测方法
CN114507706A (zh) 基于酶dna修复级联驱动荧光团编码/去编码的生物传感器及其在端粒酶检测中的应用
KR20230063085A (ko) 분할된 t7 프로모터를 이용한 등온 단일 반응용 프로브 세트 및 이의 용도
CN117625768A (zh) 一种通用数字pcr检测体系及其应用
CN111979302A (zh) 一种检测转录因子的荧光生物传感器及其检测方法和应用
Zeng et al. Strand Displacement Amplification for Multiplex
KR101785687B1 (ko) 다중 증폭 이중 시그널 증폭에 의한 타겟 핵산 서열의 검출 방법
Han et al. Optimization and performance evaluation of double-stranded probe in real-time PCR
KR20230063086A (ko) 분할된 t7 프로모터를 이용한 중증 급성 호흡기 증후군 코로나바이러스 2 검출 및/또는 이의 돌연변이 검출용 등온 단일 반응 프로브 세트 및 이의 용도
CN113308462B (zh) 一种核酸分子内扩增的探针及其检测方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23942115

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23942115

Country of ref document: EP

Kind code of ref document: A1