WO2023109032A1 - Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation - Google Patents
Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation Download PDFInfo
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- WO2023109032A1 WO2023109032A1 PCT/CN2022/097203 CN2022097203W WO2023109032A1 WO 2023109032 A1 WO2023109032 A1 WO 2023109032A1 CN 2022097203 W CN2022097203 W CN 2022097203W WO 2023109032 A1 WO2023109032 A1 WO 2023109032A1
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- nucleic acid
- probe
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- primer
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- 238000002360 preparation method Methods 0.000 title description 11
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 41
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- 238000007862 touchdown PCR Methods 0.000 claims description 5
- 239000012103 Alexa Fluor 488 Substances 0.000 claims description 2
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- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
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- 241000202934 Mycoplasma pneumoniae Species 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
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- 108060002716 Exonuclease Proteins 0.000 description 1
- 101900330348 Influenza A virus Matrix protein 1 Proteins 0.000 description 1
- 101150109178 M1 gene Proteins 0.000 description 1
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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
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the sequence of the 5' end region of the Target probe is reversely complementary to the loop region of the Beacon probe, and the Beacon probe is naturally coiled in a free state, because the reporter group (R) and the 5-terminal multiple (1-8 1)
- the distance between the G bases is short and there is no fluorescence (self-quenching molecular beacon probe).
- the end region is cut off and is in a free state.
- the temperature is lower than the Tm value, it can hybridize with the loop region of the Beacon probe, and under the action of the 5' ⁇ 3' polymerase activity of DNA polymerase, it can be extended and amplified.
- the inventor combined the design of the PCR program, through the design of the PCR program, combined with the use of the landing PCR program, and also proposed a multiple nucleic acid detection method, which can further reduce the number of non-specific nucleic acids in the PCR reaction. Heterosexual amplification improves detection sensitivity.
- a multiple nucleic acid detection method which can further reduce the number of non-specific nucleic acids in the PCR reaction. Heterosexual amplification improves detection sensitivity.
- the real-time fluorescent quantitative PCR instrument can be used for testing in a real-time fluorescent quantitative PCR instrument with a simpler reaction system and lower detection cost. Accurate and qualitative detection can be achieved for each target gene in at most 20 kinds of target nucleic acid sequences to be tested in the sample, and the specificity of detection can be guaranteed by interpreting the specific melting peak in the melting curve.
- Fig. 3 is the agarose gel electrophoresis figure of the influenza A virus PCR product that adopts different PCR programs to amplify in embodiment 2.
- the number n of continuous guanines at the 5' end of the 5' end region sequence of the above-mentioned Beacon probe is an integer of 3-5. It is further preferred that n is 4, so as to effectively provide the fluorescence quenching function.
- the above-mentioned diseases are infectious diseases caused by cross-infection caused by various pathogens.
- the aforementioned infectious diseases are respiratory tract infection, digestive tract infection, blood infection and/or urinary tract infection and the like. More preferably, it is used for the detection and determination of pathogens in respiratory tract infections with similar symptoms such as cough, which can be influenza viruses such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus , Mycoplasma pneumoniae, parainfluenza virus and other infections.
- influenza viruses such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus , Mycoplasma pneumoniae, parainfluenza virus and other infections.
- Primer preparation dissolved in TE, the concentration of each universal primer is the same, the concentration of each CLO primer is also the same, the final concentration of each universal primer is 10 times the final concentration of a single CLO primer, for example: the final concentration of a single CLO primer 1.6pmol/ ⁇ L, the final concentration of a single universal primer is 16pmol/ ⁇ L, and the mixture formed is labeled as HXD-T.
- Combination 1 and combination 2 were used to detect influenza A virus quality control products, and the amplification curve and melting curve of the detection results are shown in Figure 2 below.
- the kit contains a negative quality control and a positive quality control, and the negative and positive quality controls and the samples to be tested need to be processed synchronously.
- Positive quality control products consist of pseudoviruses containing influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference gene fragments; negative quality control
- the product consists of a pseudovirus containing internal reference gene fragments. Pseudoviruses were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. and prepared.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
La présente invention concerne un système de détection d'acides nucléiques multiples, comprenant un ensemble d'amorces d'amplification et un ensemble de sondes de détection pour une séquence d'acide nucléique cible. Le système de détection comprend une sonde cible modifiée par LNA et une sonde Beacon permettant d'obtenir un effet d'extinction de la fluorescence au moyen de 1 à 8 bases G consécutives. La présente invention concerne en outre un procédé de détection d'acides nucléiques multiples combiné au système de détection d'acides nucléiques multiples susmentionné et à un programme de PCR par essais. Le procédé permet en outre de réduire l'amplification non spécifique dans une réaction par PCR et d'améliorer la sensibilité de la détection. La présente invention permet de surmonter les limites de la technique traditionnelle de typage par PCR quantitative fluorescente en temps réel, de réaliser un typage multiple sur un seul tube au moyen de signaux spéciaux et d'une analyse de la courbe de fusion, et de détecter des acides nucléiques cibles dans un échantillon avec un système de réaction plus simple et des coûts de détection plus faibles.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111537083.4 | 2021-12-14 | ||
| CN202111537083.4A CN114134219A (zh) | 2021-12-14 | 2021-12-14 | 一种多重核酸检测系统及其制备方法与应用 |
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| Publication Number | Publication Date |
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| WO2023109032A1 true WO2023109032A1 (fr) | 2023-06-22 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/097203 WO2023109032A1 (fr) | 2021-12-14 | 2022-06-06 | Système de détection d'acides nucléiques multiples, son procédé de préparation et son utilisation |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114134219A (fr) |
| WO (1) | WO2023109032A1 (fr) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116694743A (zh) * | 2023-06-29 | 2023-09-05 | 山东迪曼生物科技有限公司 | 一种利用荧光探针检测多靶标基因序列的方法 |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134219A (zh) * | 2021-12-14 | 2022-03-04 | 广州市金圻睿生物科技有限责任公司 | 一种多重核酸检测系统及其制备方法与应用 |
| WO2023236037A1 (fr) * | 2022-06-07 | 2023-12-14 | 广州市金圻睿生物科技有限责任公司 | Kit de détection d'acide nucléique du hpv, son procédé de préparation et son utilisation |
| CN117802237A (zh) * | 2023-10-20 | 2024-04-02 | 艾普拜生物科技(苏州)有限公司 | 信标探针及其组合、用于检测flt3-itd基因突变的pcr扩增试剂及试剂盒 |
| CN117363767B (zh) * | 2023-12-07 | 2024-04-05 | 上海美吉生物医药科技有限公司 | 一种用于靶基因实时荧光pcr检测的探针组合、引物组、试剂盒及其应用 |
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| CN114134219A (zh) | 2022-03-04 |
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