WO2021151395A1 - Procédé de détection multiple d'une séquence nucléotidique cible sur la base d'une courbe de fusion obtenue par une sonde oligonucléotidique à double marquage, et kit associé - Google Patents

Procédé de détection multiple d'une séquence nucléotidique cible sur la base d'une courbe de fusion obtenue par une sonde oligonucléotidique à double marquage, et kit associé Download PDF

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WO2021151395A1
WO2021151395A1 PCT/CN2021/074527 CN2021074527W WO2021151395A1 WO 2021151395 A1 WO2021151395 A1 WO 2021151395A1 CN 2021074527 W CN2021074527 W CN 2021074527W WO 2021151395 A1 WO2021151395 A1 WO 2021151395A1
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concentration
primer
labeled oligonucleotide
target
dual
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PCT/CN2021/074527
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English (en)
Chinese (zh)
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许向华
张玲华
周中人
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上海快灵生物科技有限公司
上海妙灵生物工程有限公司
上海快灵生物工程有限公司
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Publication of WO2021151395A1 publication Critical patent/WO2021151395A1/fr

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • 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 requirement for symmetric PCR primers requires that the concentration difference of the primer pair does not exceed 50%.
  • the concentration of symmetric PCR primers varies little, and the product is mainly double-stranded.
  • the focus of detection is on the amplification curve, and set the corresponding fluorescence channel to have only one target sequence.
  • the present invention simultaneously has multiple amplification and performs melting curve analysis on asymmetrically amplified single-stranded products; compared with melting curve analysis, the results are obtained from the amplification curve, which takes much shorter time.
  • the melting temperature Tm values of the plurality of dual-labeled fluorescent probes labeled with the same fluorescent group differ from each other by no less than 2°C; more preferably, the plurality of said dual-labeled fluorescent probes labeled with the same fluorescent group differ from each other by no less than 2°C; The melting temperature Tm values of the dual-labeled fluorescent probes differ from each other by no less than 4°C.
  • Figure 17 is the amplification curve of Example 5 with the primer ratio of FIPV0.4+Bar1:20+FELV1:7+FIV1:7.
  • Figure 17-1 is the amplification curve of channel 1 (amplification curve of asymmetric amplification).
  • 17-2 is channel 2 (amplification curve of symmetrical amplification);
  • the Ct value of the amplification curve of the test sample of the asymmetric amplification probe fluorescence channel is less than 30 and there is a characteristic peak with a TM value of 75 ⁇ 1°C, which is judged to be positive for swine fever virus; the test sample has no Ct value or no obvious Amplification curve without a characteristic peak with a TM value of 75 ⁇ 1°C is judged to be negative for swine fever virus; the tested sample is 30 ⁇ Ct ⁇ 34 and an obvious amplification curve appears with a characteristic peak with a TM value of 75 ⁇ 1°C , Judged as suspicious; suggest re-sampling to extract nucleic acid, and then judge the result after amplification.
  • the preliminary results of the PCR for polymodal swine disease in this example are shown in Fig. 3 and Fig. 4.
  • the first channel detects porcine epidemic diarrhea virus (PEDV)-VAR variant strain-S protein gene, (PEDV)-CLA classic strain-S protein gene and rotavirus (PRoV)-VP6 gene, probe TM is 69°C respectively , 73°C, 63°C;
  • the second channel detects the transmissible gastroenteritis virus (TGEV)-M protein gene and the porcine D-coronavirus (PDCoV)-N gene, the probe TM is 58°C and 67°C, respectively.
  • the Tm values of the primers are all around 60°C, and the Tm of the probe for at least one detection target in the two channels is higher than the Tm of the primer for the detection target.
  • the best primer ratio is FIPV0.4+Bar1:20+FELV1:8+FIV1:8, and the detection limit is 2 channels of FIPV-10 copies, 1 channel of FIV-Bar-100 copies, and FELV-1000 copies.
  • the results of amplification curves and melting curves with different primer ratios are as follows.
  • the 5-item detection system for cat respiratory tract is equipped with asymmetric PCR-melting curve internal standard, including 5 sets of primers and probes. Among them, the ratio of the primers for detecting RNA is 1:1, and the melting curve and amplification curve of each target are tested by plasmid. The sensitivity is up to 1 copy. The detection of FHV and FCV nucleic acids in Miao Sanduo cat rhinotracheitis vaccine samples has good results.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (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

La présente invention concerne un procédé de détection multiple d'une séquence nucléotidique cible sur la base d'une courbe de fusion obtenue par une sonde oligonucléotidique à double marquage, et un kit de détection associé. Selon la présente invention, une sonde oligonucléotidique à double marquage est utilisée pour l'analyse de courbe de fusion d'un produit d'amplification PCR multiple, et la détection est effectuée à l'aide d'une courbe d'amplification PCR obtenue par la sonde oligonucléotidique à double marquage et à l'aide d'une courbe de fusion d'un hybride à double brin d'un produit d'acide nucléique simple brin et de la sonde.
PCT/CN2021/074527 2020-01-30 2021-01-30 Procédé de détection multiple d'une séquence nucléotidique cible sur la base d'une courbe de fusion obtenue par une sonde oligonucléotidique à double marquage, et kit associé WO2021151395A1 (fr)

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CN202010077586 2020-01-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699033A (zh) * 2021-08-10 2021-11-26 上海交通大学 一种基于熔解曲线的多重数字核酸分析装置和分析方法
CN114317699A (zh) * 2021-12-23 2022-04-12 郑州华之源医学检验实验室有限公司 一种基于熔解曲线正负峰形分析的多重pcr检测方法及应用
CN114672595A (zh) * 2022-04-08 2022-06-28 山东傲农种猪有限公司 一种猪病毒性腹泻检测引物组合、检测试剂盒及应用
CN114807437A (zh) * 2022-04-06 2022-07-29 南京农业大学 一种用于检测猪流行性腹泻病毒和猪轮状病毒的四重荧光定量pcr检测试剂盒及其应用

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CN112921122A (zh) * 2021-03-26 2021-06-08 广西大学 猫常见病毒多重pcr快速检测试剂盒及其引物组
CN113684256B (zh) * 2021-08-30 2024-05-14 中国药科大学 基于绿色溶剂及可编程寡核苷酸探针的多重定位检测多种靶标的方法
CN114177963A (zh) * 2022-01-13 2022-03-15 深圳市刚竹医疗科技有限公司 核酸分析装置
CN114675038B (zh) * 2022-04-25 2023-02-10 陕西科技大学 一种蛋白质含量的热荧光分析检测方法及试剂盒
CN117343992A (zh) * 2022-06-29 2024-01-05 广州市基准医疗有限责任公司 一种多基因合并荧光通道检测的方法
CN116121241A (zh) * 2023-02-01 2023-05-16 广州臻富科技有限公司 探针、检测试剂盒及靶核酸甲基化的检测方法

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CN103635593A (zh) * 2011-05-04 2014-03-12 生物概念股份有限公司 用于检测核酸序列变体的方法
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CN102171366B (zh) * 2008-07-31 2014-11-26 奥西泰克有限公司 多重扩增与检测
CN107475446B (zh) * 2017-08-24 2020-12-11 复旦大学附属儿科医院 一种同时检测多种呼吸道病毒的多重pcr检测方法及其探针组和试剂盒

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CN101871007A (zh) * 2010-05-07 2010-10-27 无锡锐奇基因生物科技有限公司 用标记探针进行检测和熔解曲线分析的方法
CN103635593A (zh) * 2011-05-04 2014-03-12 生物概念股份有限公司 用于检测核酸序列变体的方法
CN105087763A (zh) * 2014-05-16 2015-11-25 北京万泰生物药业股份有限公司 实时荧光pcr和探针法融解曲线结合的多重核酸检测方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699033A (zh) * 2021-08-10 2021-11-26 上海交通大学 一种基于熔解曲线的多重数字核酸分析装置和分析方法
CN114317699A (zh) * 2021-12-23 2022-04-12 郑州华之源医学检验实验室有限公司 一种基于熔解曲线正负峰形分析的多重pcr检测方法及应用
CN114317699B (zh) * 2021-12-23 2023-01-10 郑州华之源医学检验实验室有限公司 一种基于熔解曲线正负峰形分析的多重pcr检测方法及应用
CN114807437A (zh) * 2022-04-06 2022-07-29 南京农业大学 一种用于检测猪流行性腹泻病毒和猪轮状病毒的四重荧光定量pcr检测试剂盒及其应用
CN114672595A (zh) * 2022-04-08 2022-06-28 山东傲农种猪有限公司 一种猪病毒性腹泻检测引物组合、检测试剂盒及应用
CN114672595B (zh) * 2022-04-08 2023-09-19 山东傲农种猪有限公司 一种猪病毒性腹泻检测引物组合、检测试剂盒及应用

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