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WO2021075912A1 - Ensemble d'amorces pour une réaction d'amplification multi-isotherme à haute sensibilité capable de cribler et de détecter simultanément mycobacterium tuberculosis et des mycobactéries non tuberculeuses - Google Patents

Ensemble d'amorces pour une réaction d'amplification multi-isotherme à haute sensibilité capable de cribler et de détecter simultanément mycobacterium tuberculosis et des mycobactéries non tuberculeuses Download PDF

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Publication number
WO2021075912A1
WO2021075912A1 PCT/KR2020/014175 KR2020014175W WO2021075912A1 WO 2021075912 A1 WO2021075912 A1 WO 2021075912A1 KR 2020014175 W KR2020014175 W KR 2020014175W WO 2021075912 A1 WO2021075912 A1 WO 2021075912A1
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mycobacterium tuberculosis
primer set
fluorescein
tuberculosis
seq
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Korean (ko)
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임채승
윤정
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Korea University Research and Business Foundation
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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
    • C12Q1/6876—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
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • 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

Definitions

  • the present invention relates to a primer set for multiple isothermal amplification reactions capable of simultaneously discriminating and detecting Mycobacterium tuberculosis (MTB) and Nontuberculous mycobacteria (NTM).
  • MTB Mycobacterium tuberculosis
  • NTM Nontuberculous mycobacteria
  • Tuberculosis is a droplet infectious disease caused by Mycobacterium tuberculosis (MTB) and is one of the leading causes of infectious disease deaths that have resulted in millions of deaths over the past centuries. It is also estimated to be 10 million new cases and about 2 million deaths each year.
  • MTB Mycobacterium tuberculosis
  • NTM Nontuberculous mycobacteria
  • M avium complex accounts for 60-80%
  • M. kansasii accounts for 15-20% in foreign countries
  • M. abscessus accounts for 20-30% in Korea
  • NTM is isolated from clinical specimens in Korea. And the number of patients diagnosed and treated for NTM lung disease is increasing significantly.
  • LAMP loop-mediated isothermal amplification reaction
  • the present inventors have completed the present invention by studying a method for simultaneously identifying MTB and NTM without an experienced inspector and expensive equipment using the LAMP method.
  • the technical problem to be achieved by the present invention is to provide a primer set for detecting Mycobacterium tuberculosis and a set of primers for detecting Mycobacterium tuberculosis and non-TB, and a primer set capable of discriminating and detecting Mycobacterium tuberculosis and an Mycobacterium tuberculosis, including each of the above primer sets, and It is to provide a kit and a method for discriminating and detecting Mycobacterium tuberculosis and Antituberculosis using the same.
  • the present invention provides a LAMP (Loop-mediated isothermal amplification) primer set for detecting Mycobacterium tuberculosis, including the primers of SEQ ID NOs: 1 to 6.
  • LAMP Loop-mediated isothermal amplification
  • the present invention provides a set of LAMP primers for detecting Mycobacterium tuberculosis and non-Tuberculosis Mycobacterium tuberculosis, comprising the primers of SEQ ID NOs: 7 to 12.
  • the present invention provides a primer set for differential detection of Mycobacterium tuberculosis and non-Mycobacterium tuberculosis, including primers of SEQ ID NOs: 1 to 6 and primers of SEQ ID NOs: 7 to 12.
  • the primer set may further include primers of SEQ ID NOs: 13 to 18.
  • the SEQ ID NO: 5; SEQ ID NO: 11; And/or the primer of SEQ ID NO: 18; may be labeled with a fluorescent material.
  • the fluorescent material may be labeled on thymine (T), which is the 15th base of SEQ ID NO: 5, and may be labeled on thymine, which is the 18th base of SEQ ID NO: 11, It can be labeled on the 18th base, thymine.
  • T thymine
  • thymine which is the 15th base of SEQ ID NO: 5
  • thymine which is the 18th base of SEQ ID NO: 11
  • the fluorescent material is FAM (6-carboxyfluorescein), Texas red, fluorescein, HEX (2',4',5',7'-tetrachloro- 6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein) isothiocyanate), oregon green, alexa fluor, JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine), Tetrachloro-Fluorescein (TET), tertramethylrodamine isothiocyanate (TRITC), 6-carboxytetramethyl-rhodamine (TAMRA), NED (N-(1-Naphthyl)
  • the SEQ ID NO: 5; SEQ ID NO: 11; And SEQ ID NO: 18 primers may be labeled with fluorescent materials emitting different wavelengths.
  • the present invention provides a kit for detecting Mycobacterium tuberculosis comprising the primers of SEQ ID NOs: 1 to 6.
  • the present invention provides a kit for detecting non-tuberculosis antibacterial bacteria comprising the primers of SEQ ID NOs: 7 to 12.
  • the present invention provides a kit for differential detection of Mycobacterium tuberculosis and non-Mycobacterium tuberculosis comprising a primer set of SEQ ID NOs: 1 to 18.
  • the kit may further include a reagent for performing an amplification reaction, and the reagent may include one or more selected from the group consisting of DNA polymerase, dNTPs, and buffers.
  • the present invention comprises the steps of: (1) separating genomic DNA (gDNA) from a biological sample; (2) MTB primer set of SEQ ID NOs: 1 to 6 using the isolated gDNA as a template; And amplifying a target sequence by performing an isothermal amplification reaction using a primer set comprising: and an MTB and NTM primer set of SEQ ID NOs: 7 to 12; And (3) detecting the amplified product; It provides a method for differential detection and diagnosis of Mycobacterium tuberculosis and Mycobacterium tuberculosis, including.
  • the biological sample may be one or more selected from the group consisting of sputum, bronchial aspiration, and bronchial washing, and body fluid of a patient with respiratory disease. .
  • the primer set of step (2) may further include an internal control primer set of SEQ ID NOs: 13 to 18.
  • step (3) may be performed by one or more methods selected from the group consisting of DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement, but preferably fluorescence measurement It can be done by a method.
  • the method includes an MTB primer set after step (3); And if all the products amplified by the MTB and NTM primer set are detected, it is interpreted as MTB positive, and the product amplified by the MTB and NTM primer set is not detected, but the product amplified by the MTB and NTM primer set is detected as NTM positive. It may further include an interpreting step.
  • the method may include interpreting negative when an amplification product by an internal control primer set is detected after step (3) and an amplification product by another primer set is not detected. have.
  • the method may further include the step of quantifying MTB and/or NTM by measuring the detection time of step (3) after step (3).
  • the "quantification” may be to quantify the detection time of the fluorescent substance according to the DNA concentration compared to the standard curve when performing the amplification reaction using the primer set according to the present invention, and preferably, the amplification product of step (c) is fluorescent. By detecting using an index, the detection time of the fluorescent substance may be measured to quantify the DNA concentration.
  • the primer set of the present invention is based on real-time multiplex LAMP and shortens the time to identify the causative agent of lung disease than conventional PCR and real time PCR-based detection, and because it is based on LAMP, it is tested.
  • a simple, economical and lightweight device can be used compared to a conventional PCR device because it does not require temperature change.
  • the present invention is advantageous in terms of time and cost in that it can detect both MTB and NTM by performing multiplex tests and discriminate them at the same time, and is expected to be used as a molecular diagnostic test method that replaces the existing tuberculosis detection method.
  • Figure 1 is a tuberculosis GYRB gene sequence specific MTB primer set, 16S rRNA gene sequence-specific MTB and NTM primer set capable of amplifying both non-Tuberculosis Mycobacterium tuberculosis and Mycobacterium tuberculosis, and an internal control primer set to amplify human G6PD.
  • MTB tuberculosis GYRB gene sequence specific MTB primer set
  • 16S rRNA gene sequence-specific MTB and NTM primer set capable of amplifying both non-Tuberculosis Mycobacterium tuberculosis and Mycobacterium tuberculosis
  • an internal control primer set to amplify human G6PD.
  • FIG. 3 is a result of testing the presence or absence of a cross-reaction targeting actual MTB and NTM infected specimens in order to confirm whether the TB GYRB gene sequence-specific MTB primer set is specifically amplified for MTB and is non-specific for non-Tuberculosis mycobacterial NTM.
  • the present inventors have completed the present invention by studying a method for quickly and accurately identifying pathogens of patients with infectious lung disease in the field.
  • the present inventors produced a primer set specific for the gyrB gene of Mycobacterium tuberculosis (MTB) and a primer set specific for 16S rRNA of Mycobacterium tuberculosis (MTB) and 16S rRNA of Mycobacterium tuberculosis (MTB).
  • the LAMP-based primer set of the present invention does not require temperature change, so a simple and light amplification device can be used, and the time required for analysis can be drastically reduced.
  • the present invention can reduce the time to identify cells by performing multiplex LAMP including the gyrB -specific primer set and the 16S rRNA-specific primer set to simultaneously discriminate and detect Mycobacterium tuberculosis and non-Tuberculosis bacteria. .
  • a primer set specific to the G6PD gene is prepared and provided as an internal control primer set, thereby improving the reliability of the multiplex LAMP analysis result.
  • the present invention is a primer that acts specifically on MTB (MTB primer), 25 kinds of NTM and primers that simultaneously act on MTB (MTB and NTM primer), and a primer that can confirm amplification with internal control (Internal control primer) set.
  • the present invention provides a composition for detecting MTB bacteria comprising the primer set.
  • SEQ ID NO: 1-6 in Table 1 is a set of six primers for MTB (MTB primer)
  • SEQ ID NO: 7-12 is a set of six primers (MTB and NTM primers) for 25 kinds of NTM and MTB
  • sequence Numbers 13-18 are a set of 6 primers that can amplify the G6PD gene as an internal control.
  • the primer set of Table 1 and a composition comprising the same can be used for the detection of MTB and NTM bacteria using the Multiplex LAMP method.
  • the configuration of the multiplex LAMP primer set for differential detection of MTB and NTM of the present invention is shown in Table 2 below, and the reaction time and temperature are shown in Table 3 below.
  • the fluorescent substance attached to the primer set is not limited as long as it emits fluorescence, but non-limiting examples thereof include FAM (6-carboxyfluorescein), Texas red, fluorescein, HEX (2', 4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetra Methyl rhodamine, fluorescein isothiocyanate (FITC), oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein) ), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-r
  • the present invention attaches a fluorescent material to thymine, which is the 15th base in the forward loop primer (FLP) consisting of the base of SEQ ID NO: 5 of the MTB primer set, and the sequence number of the MTB and NTM primer sets.
  • FLP forward loop primer
  • thymine which is the 15th base in the forward loop primer (FLP) consisting of the base of SEQ ID NO: 5 of the MTB primer set, and the sequence number of the MTB and NTM primer sets.
  • FLP forward loop primer
  • BLP reverse loop primer
  • the position of the thymine to which the fluorescent substance is attached is located in the last 4 nucleotide sequences at the 3'end of the primer oligo sequence, G or C is located in front of the thymine, and bases such as GG GC CC are located in the two terminal bases of the thymine, so that the fluorescence of thymine is expressed. Is designed to optimize.
  • the biological sample may include cells or tissues of various parts of the body including sputum, bronchial aspiration, bronchial washing, body fluid, and the like.
  • the "loop-mediated isothermal amplification (LAMP)" is a method of performing a reaction under isothermal conditions using a polymerase such as Bst or Gsp that does not require a PCR cycle unlike the existing PCR method, Basically, using four primers, amplification of a specific region of a gene by synthesizing both ends in a loop structure to infinity, and a method of confirming the amplification by using a fluorescent detection reagent or precipitation reaction of the amplified DNA product.
  • a reagent for detecting fluorescence may be used, but the present invention is not limited thereto.
  • the "real-time multiple isothermal amplification reaction” is a method of confirming the presence or absence of amplification in real time, and can simultaneously detect various genes, preferably simultaneously detecting MTB and/or NTM in a sample in real time, and It can be determined, but is not limited thereto.
  • the primer set of the present invention is preferably used for isothermal amplification (LAMP; Loop-mediated isothermal amplification), and the isothermal amplification reaction may be a one-step LAMP for amplifying target DNA by LAMP, It may be a real-time LAMP (real-time LAMP), or a combination thereof.
  • the term "primer” is a template under appropriate conditions (for example, 4 different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer solution. -Refers to a single-stranded oligonucleotide that can act as a starting point for DNA synthesis.
  • the appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template.
  • the primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template.
  • the primer of the present invention can be chemically synthesized using a method known in the art, such as, for example, a phosphoramidite solid support method. In addition, it can be modified by methylation, capping, or the like by a known method.
  • the primer of the present invention may contain a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (e.g. horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., 32P), fluorescent molecules, chemical groups (e.g., biotin), etc. have.
  • inner primer refers to a single-stranded oligonucleotide that can act as a starting point for synthesizing a new DNA chain by binding to a template DNA.
  • outer primer refers to a single-stranded oligonucleotide that binds to the template DNA from the outside of the position where the inner primer binds to the template DNA. After the chain is elongated, a strand displacement occurs due to the binding of the external primer and the template DNA, and the first formed chain is separated.
  • the "loop primer” means that the initial stem loop structure chain formed by binding the inner primer and the outer primer to the template DNA increases the number of generations of the loop structure. It refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis, allowing the reaction to be accelerated.
  • the base numbering for specifying the base to which the fluorescent substance is attached in the ring primer is performed in the same order as 3'to 5'in the same manner as the method of confirming the base sequence of the oligonucleotide.
  • DNA Nucleic acid (DNA) of MTB bacteria and NTM extracted from sputum and bronchial aspirate of tuberculosis patients and non-tuberculosis mycobacterium infected patients were obtained at Korea University Guro Hospital, and the infection of each sample was clinically confirmed at the hospital.
  • the present invention relates to a primer set and the like capable of simultaneously discriminating between Mycobacterium tuberculosis and Mycobacterium tuberculosis, and it is possible to detect MTB and NTM quickly and accurately without heavy and complex PCR equipment by the present invention, and to differentiate and diagnose them. Is expected to be used as a molecular diagnostic test that replaces the existing tuberculosis detection method.

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Abstract

La présente invention concerne un ensemble d'amorces pour une réaction d'amplification multi-isotherme capable de cribler et de détecter simultanément Mycobacterium tuberculosis (MTB) et des mycobactéries non tuberculeuses (MNT) et analogues.
PCT/KR2020/014175 2019-10-18 2020-10-16 Ensemble d'amorces pour une réaction d'amplification multi-isotherme à haute sensibilité capable de cribler et de détecter simultanément mycobacterium tuberculosis et des mycobactéries non tuberculeuses Ceased WO2021075912A1 (fr)

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KR10-2019-0129647 2019-10-18
KR1020190129647A KR102274011B1 (ko) 2019-10-18 2019-10-18 결핵균 및 비결핵항산균을 동시에 감별하여 검출할 수 있는 고감도 다중 등온증폭반응용 프라이머 세트

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CN119020516A (zh) * 2024-10-30 2024-11-26 中国医学科学院病原生物学研究所 一种活性结核分枝杆菌的定量检测方法及其应用

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KR102428279B1 (ko) * 2021-04-02 2022-09-28 주식회사 이노제닉스 치료 효율 모니터링을 위한 16S rRNA 기반 결핵 분자진단 방법 및 그 응용
KR102692361B1 (ko) * 2021-12-14 2024-08-07 고려대학교 산학협력단 형광 프로브를 이용한 칸디다 판 및 칸디다 알비칸스 검출용 동시다중 등온증폭반응 프라이머 세트

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