WO2022110335A1 - Kit for rapidly detecting hepatitis b virus gene and detection method therefor - Google Patents
Kit for rapidly detecting hepatitis b virus gene and detection method therefor Download PDFInfo
- Publication number
- WO2022110335A1 WO2022110335A1 PCT/CN2020/136280 CN2020136280W WO2022110335A1 WO 2022110335 A1 WO2022110335 A1 WO 2022110335A1 CN 2020136280 W CN2020136280 W CN 2020136280W WO 2022110335 A1 WO2022110335 A1 WO 2022110335A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- kit
- add
- hepatitis
- primer
- recombinase polymerase
- Prior art date
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 12
- 102000018120 Recombinases Human genes 0.000 claims description 18
- 108010091086 Recombinases Proteins 0.000 claims description 18
- 239000013615 primer Substances 0.000 claims description 18
- 230000003321 amplification Effects 0.000 claims description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108020001019 DNA Primers Proteins 0.000 claims description 5
- 239000003155 DNA primer Substances 0.000 claims description 5
- 108020005004 Guide RNA Proteins 0.000 claims description 5
- 108020004518 RNA Probes Proteins 0.000 claims description 5
- 239000003391 RNA probe Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 4
- 239000011654 magnesium acetate Substances 0.000 claims description 4
- 229940069446 magnesium acetate Drugs 0.000 claims description 4
- 235000011285 magnesium acetate Nutrition 0.000 claims description 4
- 239000011541 reaction mixture Substances 0.000 claims description 4
- 238000012197 amplification kit Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000013642 negative control Substances 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 239000013641 positive control Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 238000003149 assay kit Methods 0.000 claims 1
- 208000002672 hepatitis B Diseases 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 208000035473 Communicable disease Diseases 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 108091033409 CRISPR Proteins 0.000 abstract description 2
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 2
- 238000010362 genome editing Methods 0.000 abstract description 2
- 239000002245 particle Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 101710082933 Single-strand DNA-binding protein Proteins 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 2
- 108020004638 Circular DNA Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 108091012434 DNA polymerase binding proteins Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000700739 Hepadnaviridae Species 0.000 description 1
- 206010019705 Hepatic pain Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 241000700732 Orthohepadnavirus Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000009352 congenital transmission Effects 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the field of clinical medical diagnosis, and relates to the diagnosis of viral hepatitis B, and the fields of infectious diseases, molecular biology, cell biology and the like. More specifically, the present invention relates to a nucleic acid amplification detection method of hepatitis B virus (Hepatitis B virus, HBV) gene in blood, and provides a kit for rapid detection of hepatitis B virus gene and a detection method thereof.
- Hepatitis B virus Hepatitis B virus, HBV
- Hepatitis B is an infectious disease mainly caused by liver lesions caused by hepatitis B virus (HBV) infection.
- HBV hepatitis B virus
- liver function damage, loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue are the main manifestations.
- Some patients may have jaundice and fever.
- Some hepatitis B patients can become chronic, and even develop into liver cirrhosis, and a few can develop into liver cancer. Mainly through blood and blood products, mother-to-child transmission and sexual contact.
- HBV belongs to the genus Ortho Hepadnavirus of the family Hepadnaviridae. Three kinds of particles with different shapes were observed in the serum of HBV infected patients under electron microscope. The first is a small spherical hollow particle with a diameter of 22nm, and the second is a tubular particle of 22nmx40-100nm. Both particles are enveloped components and do not contain viral nucleic acid. The third is spherical particles with a diameter of 42 nm, called Dane particles, which are composed of an envelope, a core-shell and a core.
- the envelope is composed of HBsAg; the nucleocapsid is composed of hepatitis B core antigen (HBcAg); the core contains part of circular double-stranded DNA and DNA polymerase.
- the HBV genome is a partially circular double-stranded DNA consisting of positive and negative strands, and the negative strand is a long chain. It is a closed circular DNA with a total length of about 3200 nucleotides.
- the positive strand is a short, semi-closed circular DNA, and the full length is 50%-75% of the negative strand.
- Fluorescence quantitative PCR is a nucleic acid quantitative detection technology that integrates PCR technology, fluorescence signal detection and data analysis. Fluorescence quantitative PCR uses specific probes, which can specifically identify target sequences, have dual control of primers and probes, and integrate fluorescent labeling technology, laser detection technology, and digital imaging technology, with high specificity and sensitivity. characteristics, high accuracy and low false positive rate.
- the thermal cycling module and the fluorescence detection module are indispensable components of real-time quantitative PCR, so real-time quantitative PCR often requires expensive and bulky special instruments. This makes real-time PCR unsuitable for use in non-laboratory settings.
- Recombinase polymerase amplification (RPA) technology is an isothermal amplification technology that can be applied to low temperature (about 37°C).
- Enzymes, DNA polymerases with strand displacement activity, single strand DNA-binding proteins (SSB) and Mg2+, etc. use primer-recombinase complexes to scan DNA strands, thereby promoting the corresponding homologous sites on the DNA strands.
- the identification, binding, and exchange of dots synthesize complementary new strands through the combined action of DNA polymerase and SSB. As the reaction proceeds, the amplification product grows exponentially.
- RPA can be used as a portable, fast and cost-effective detection tool to achieve accurate on-site detection outside the laboratory.
- the invention provides a detection kit for hepatitis B virus with stronger sensitivity and specificity and a detection method thereof.
- the present invention provides primers for rapid detection of hepatitis B virus, the primers are recombinase polymerase amplification DNA primers:
- the primer is used for the preparation of a kit for rapid detection of hepatitis B virus.
- a kit for rapid detection of hepatitis B virus including: recombinase polymerase amplification system and Cas13 reaction system; the recombinase polymerase amplification system includes TwistAmp liquid recombinase polymerase amplification kit and recombinase polymerase Enzymatic amplification of DNA primers:
- the Cas13 reaction system includes recombinant CRISPR-Cas13a protein, single-stranded guide RNA and single-stranded RNA probe.
- the recombinase polymerase amplification system includes:
- the Cas13 reaction system includes:
- the method for detecting hepatitis B virus gene by the kit includes the following steps:
- Detection use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
- the RPA of the present application is combined with the gene editing system of CRISPR/Cas13a, which can detect hepatitis B virus in a portable, rapid and precise manner with good sensitivity and specificity.
- Fig. 1 Example 1 change curve of fluorescence intensity with time.
- Fig. 2 The effect of adding template concentration on fluorescence intensity in Example 1.
- TwistAmp liquid recombinase polymerase amplification kit including 20x core reaction mixture, 2x reaction buffer, 280mM magnesium acetate (MgOAc), 10x electron mixture, purchased from TwistDx Company.
- Trizma hydrochloric acid buffer-400mM Tris pH 7.4 (Sigma Aldrich).
- NxGen T7 RNA polymerase purchased from Lucigen Company.
- Detection use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Disclosed is a kit for rapidly detecting hepatitis B virus gene and a detection method therefor, which relate to the diagnosis of viral hepatitis B, and the fields of infectious diseases, molecular biology and cell biology. According to the kit and the detection method, the RPA is combined with a gene editing system of CRISPR/Cas13a, so that the hepatitis B virus can be conveniently, rapidly and accurately detected, and the kit and the detection method have good sensitivity and specificity.
Description
本发明属临床医学诊断领域,涉及乙型病毒性肝炎的诊断,以及感染病学、分子生物学和细胞生物学等领域。更具体地,本发明涉及血液中乙型肝炎病毒(Hepatitis B virus,HBV)基因的核酸扩增检测方法,提供了一种用于快速检测乙型肝炎病毒基因的试剂盒及其检测方法。The invention belongs to the field of clinical medical diagnosis, and relates to the diagnosis of viral hepatitis B, and the fields of infectious diseases, molecular biology, cell biology and the like. More specifically, the present invention relates to a nucleic acid amplification detection method of hepatitis B virus (Hepatitis B virus, HBV) gene in blood, and provides a kit for rapid detection of hepatitis B virus gene and a detection method thereof.
乙型肝炎是由乙型肝炎病毒(Hepatitis B virus,HBV)感染引起的以肝脏病变为主的一种传染病。临床上以肝功能损害、食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现,部分患者可有黄疸发热。部分乙肝患者可慢性化,甚至发展成肝硬化,少数可发展为肝癌。主要通过血和血制品、母婴传播及性接触传播。Hepatitis B is an infectious disease mainly caused by liver lesions caused by hepatitis B virus (HBV) infection. Clinically, liver function damage, loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue are the main manifestations. Some patients may have jaundice and fever. Some hepatitis B patients can become chronic, and even develop into liver cirrhosis, and a few can develop into liver cancer. Mainly through blood and blood products, mother-to-child transmission and sexual contact.
HBV属嗜肝DNA病毒科(Hepadnaviridae)正嗜肝DNA病毒属(Ortho Hepadnavirus)。HBV感染者血清中电镜下可见3种不同形态的颗粒。第一种为直径22nm的小球形空心颗粒,第二种为22nmx40-100nm的管状颗粒。这两种颗粒均为包膜成分,不含病毒的核酸。第三种为直径42nm的球形颗粒,称Dane颗粒,由包膜、核壳和核心组成。包膜由HBsAg组成;核壳由乙型肝炎核心抗原(HBcAg)组成;核心内有部分环状双链DNA和DNA聚合酶。HBV belongs to the genus Ortho Hepadnavirus of the family Hepadnaviridae. Three kinds of particles with different shapes were observed in the serum of HBV infected patients under electron microscope. The first is a small spherical hollow particle with a diameter of 22nm, and the second is a tubular particle of 22nmx40-100nm. Both particles are enveloped components and do not contain viral nucleic acid. The third is spherical particles with a diameter of 42 nm, called Dane particles, which are composed of an envelope, a core-shell and a core. The envelope is composed of HBsAg; the nucleocapsid is composed of hepatitis B core antigen (HBcAg); the core contains part of circular double-stranded DNA and DNA polymerase.
HBV基因组为部分环状双链DNA,由正链和负链组成,负链为长链。呈闭合型的环状DNA,全长约3200个核苷酸。正链为短链,呈半闭合型的环状DNA,全长为负链的50%-75%。The HBV genome is a partially circular double-stranded DNA consisting of positive and negative strands, and the negative strand is a long chain. It is a closed circular DNA with a total length of about 3200 nucleotides. The positive strand is a short, semi-closed circular DNA, and the full length is 50%-75% of the negative strand.
目前,常用HBV-DNA检测方法主要是荧光定量PCR法。荧光定量PCR法是一种集PCR技术、荧光信号检测和数据分析于一体的核酸定量检测技术。荧光定量PCR使用了特异性探针,能够对靶序列进行特异性的识别,具有引物探针双重控制,且综合了荧光标记技术、激光检测技术、数码显像技术,具有高特异性、高灵敏性,高准确性及低假阳性率等特点。其中热循环模块和荧光检测模块是荧光定量PCR不可缺少的组成部分,因此荧光定量PCR往往需要昂贵笨重的特殊仪器。这使得荧光定量PCR不适合在非实验室环境中使用。At present, the commonly used HBV-DNA detection method is mainly fluorescence quantitative PCR method. Fluorescence quantitative PCR is a nucleic acid quantitative detection technology that integrates PCR technology, fluorescence signal detection and data analysis. Fluorescence quantitative PCR uses specific probes, which can specifically identify target sequences, have dual control of primers and probes, and integrate fluorescent labeling technology, laser detection technology, and digital imaging technology, with high specificity and sensitivity. characteristics, high accuracy and low false positive rate. Among them, the thermal cycling module and the fluorescence detection module are indispensable components of real-time quantitative PCR, so real-time quantitative PCR often requires expensive and bulky special instruments. This makes real-time PCR unsuitable for use in non-laboratory settings.
重组酶聚合酶扩增技术(Recombinase polymerase amplification,RPA)技术是一种可应用于低温(37℃左右)的等温扩增技术,反应体系包括能结合单链核酸(寡核苷酸引物)的重组酶、具有链置换活性的DNA聚合酶、DNA单链结合蛋白(single strand DNA-binding protein,SSB)和Mg2+等,利用引物-重组酶复合物扫描DNA链,进而促进DNA链上对应同源位点的识别、结合、互换,通过DNA聚合酶和SSB共同作用合成互补新链,随着反应的进行,扩增产物以指数状态增长。RPA可作为一种便携、快速、性价比高的检测工具,实现实验室外的现场精确检测。Recombinase polymerase amplification (RPA) technology is an isothermal amplification technology that can be applied to low temperature (about 37°C). Enzymes, DNA polymerases with strand displacement activity, single strand DNA-binding proteins (SSB) and Mg2+, etc., use primer-recombinase complexes to scan DNA strands, thereby promoting the corresponding homologous sites on the DNA strands The identification, binding, and exchange of dots synthesize complementary new strands through the combined action of DNA polymerase and SSB. As the reaction proceeds, the amplification product grows exponentially. RPA can be used as a portable, fast and cost-effective detection tool to achieve accurate on-site detection outside the laboratory.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种灵敏度和特异性更强的检测乙型肝炎病毒的试剂盒及其检测方法。The invention provides a detection kit for hepatitis B virus with stronger sensitivity and specificity and a detection method thereof.
本发明提供用于快速检测乙型肝炎病毒的引物,所述引物为重组酶聚合酶扩增DNA引物:The present invention provides primers for rapid detection of hepatitis B virus, the primers are recombinase polymerase amplification DNA primers:
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′Primer 1-5'-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3'
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′。Primer 2-5'-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3'.
所述引物用于制备快速检测乙型肝炎病毒的试剂盒的应用。The primer is used for the preparation of a kit for rapid detection of hepatitis B virus.
用于快速检测乙型肝炎病毒的试剂盒,包括:重组酶聚合酶扩增体系和Cas13反应体系;所述重组酶聚合酶扩增体系包括TwistAmp液态重组酶聚合酶扩增试剂盒和重组酶聚合酶扩增DNA引物:A kit for rapid detection of hepatitis B virus, including: recombinase polymerase amplification system and Cas13 reaction system; the recombinase polymerase amplification system includes TwistAmp liquid recombinase polymerase amplification kit and recombinase polymerase Enzymatic amplification of DNA primers:
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTT-CTG-3′Primer 1-5'-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTT-CTG-3'
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′Primer 2-5'-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3'
所述Cas13反应体系包括重组CRISPR-Cas13a蛋白、单链引导RNA和单链RNA探针。The Cas13 reaction system includes recombinant CRISPR-Cas13a protein, single-stranded guide RNA and single-stranded RNA probe.
单链引导RNA:Single-stranded guide RNA:
5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′
单链RNA探针:Single-stranded RNA probes:
5′-FAM-TUUUUUC-BHQ-1-3′。5'-FAM-TUUUUUC-BHQ-1-3'.
具体的,specific,
所述重组酶聚合酶扩增体系包括:The recombinase polymerase amplification system includes:
所述Cas13反应体系包括:The Cas13 reaction system includes:
试剂盒检测乙型肝炎病毒基因的方法,包括如下步骤:The method for detecting hepatitis B virus gene by the kit includes the following steps:
1)重组酶聚合酶扩增1) Recombinase polymerase amplification
(1)取HBV阳性对照稀释成3x10
5拷贝/μl,3x10
4拷贝/μl,3x10
3拷贝/μl,3x10
2拷贝/μl,3x10拷贝/μl,取1μl加入以下反应体系:
( 1 ) Dilute the HBV positive control to 3x105 copies/μl, 3x104 copies/μl, 3x103 copies/μl, 3x102 copies/μl, 3x10 copies/μl, take 1μl and add it to the following reaction system:
(2)加入1.25μl的20x核心反应混合液,混匀离心;(2) Add 1.25μl of 20x core reaction mixture, mix well and centrifuge;
(3)加入1μl模板(不同稀释度的质粒模板和阴性对照);(3) Add 1 μl of template (plasmid template of different dilution and negative control);
(4)在管盖上加入1.25μl乙酸镁(MgOAc,280mM);(4) Add 1.25 μl of magnesium acetate (MgOAc, 280 mM) to the cap of the tube;
(5)颠倒混匀,离心后39度孵育20min。(5) Invert and mix, and incubate at 39°C for 20min after centrifugation.
2)用Cas13蛋白检测HBV2) Detection of HBV with Cas13 protein
(1)取上述1ul反应产物加入Cas13反应体系,加入384孔透明圆底酶标板充分混合。设3个复孔:(1) Take 1 ul of the above reaction product and add it to the Cas13 reaction system, then add it to a 384-well transparent round-bottom microtiter plate and mix well. Set up 3 duplicate holes:
(2)反应时间30min。(2) The reaction time is 30 minutes.
(3)检测:用多功能酶标仪检测仪,检测480nm激发光520nm发射波长的荧光强度。(3) Detection: use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
本申请RPA与CRISPR/Cas13a的基因编辑系统相结合,可以便携、快速、精确地检测乙型肝炎病毒,具有很好的灵敏度和特异性。The RPA of the present application is combined with the gene editing system of CRISPR/Cas13a, which can detect hepatitis B virus in a portable, rapid and precise manner with good sensitivity and specificity.
图1实施例1随时间荧光强度变化曲线。Fig. 1 Example 1 change curve of fluorescence intensity with time.
图2实施例1加入模板浓度对荧光强度的影响。Fig. 2 The effect of adding template concentration on fluorescence intensity in Example 1.
(一)材料(1) Materials
(1)重组酶聚合酶扩增DNA引物:(1) Recombinase polymerase amplifies DNA primers:
引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′Primer 1-5'-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3'
引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′Primer 2-5'-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3'
上述引物均于上海生工生物工程技术服务有限公司合成。The above primers were synthesized in Shanghai Sangon Bioengineering Technology Service Co., Ltd.
(2)单链引导RNA:(2) Single-stranded guide RNA:
5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′,5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′,
于上海生工生物工程技术服务有限公司合成。Synthesized at Shanghai Sangon Bioengineering Technology Service Co., Ltd.
(3)单链RNA探针:5′-FAM-TUUUUUC-BHQ-1-3′,(3) Single-stranded RNA probe: 5′-FAM-TUUUUUC-BHQ-1-3′,
于上海生工生物工程技术服务有限公司合成。Synthesized at Shanghai Sangon Bioengineering Technology Service Co., Ltd.
(4)TwistAmp液态重组酶聚合酶扩增试剂盒,包含20x核心反应混合液、2倍反应缓冲液、280mM乙酸镁(MgOAc)、10x电子混合液,购于TwistDx公司。(4) TwistAmp liquid recombinase polymerase amplification kit, including 20x core reaction mixture, 2x reaction buffer, 280mM magnesium acetate (MgOAc), 10x electron mixture, purchased from TwistDx Company.
(5)Trizma盐酸缓冲液-400mM Tris pH 7.4(Sigma Aldrich)。(5) Trizma hydrochloric acid buffer-400mM Tris pH 7.4 (Sigma Aldrich).
(6)重组CRISPR-Cas13a蛋白,购于上海惠诚生物科技有限公司。(6) Recombinant CRISPR-Cas13a protein, purchased from Shanghai Huicheng Biotechnology Co., Ltd.
(7)RNase酶抑制剂,购于上海碧云天生物技术有限公司。(7) RNase enzyme inhibitor, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
(8)NxGen T7 RNA聚合酶,购于Lucigen公司。(8) NxGen T7 RNA polymerase, purchased from Lucigen Company.
(9)10mM NTP混合液,购于上海生工生物工程技术服务有限公司。(9) 10mM NTP mixture, purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
(10)氯化镁溶液(120mM),用六合氯化镁加ddH
2O配成。
(10) Magnesium chloride solution (120mM), prepared with hexahydrate magnesium chloride and ddH 2 O.
(二)方法:(2) Method:
1.重组酶聚合酶扩增1. Recombinase Polymerase Amplification
(1)取HBV阳性对照稀释成3x10
5拷贝/μl,3x10
4拷贝/μl,3x10
3拷贝/μl,3x10
2拷贝/μl,3x10拷贝/μl,取1μl加入以下反应体系:
( 1 ) Dilute the HBV positive control to 3x105 copies/μl, 3x104 copies/μl, 3x103 copies/μl, 3x102 copies/μl, 3x10 copies/μl, take 1μl and add it to the following reaction system:
(2)加入1.25μl的20x核心反应混合液,混匀离心;(2) Add 1.25μl of 20x core reaction mixture, mix well and centrifuge;
(3)加入1μl模板(不同稀释度的质粒模板和阴性对照);(3) Add 1 μl of template (plasmid template of different dilution and negative control);
(4)在管盖上加入1.25μl乙酸镁(MgOAc,280mM);(4) Add 1.25 μl of magnesium acetate (MgOAc, 280 mM) to the cap of the tube;
(5)颠倒混匀,离心后39度孵育20min。(5) Invert and mix, and incubate at 39°C for 20min after centrifugation.
2.用Cas13蛋白检测HBV2. Detection of HBV with Cas13 protein
(1)取上述1ul反应产物加入Cas13反应体系,加入384孔透明圆底酶标板充分混合。设3个复孔:(1) Take 1 ul of the above reaction product and add it to the Cas13 reaction system, then add it to a 384-well transparent round-bottom microtiter plate and mix well. Set up 3 duplicate holes:
(2)反应时间30min。(2) The reaction time is 30 minutes.
(3)检测:用多功能酶标仪检测仪,检测480nm激发光520nm发射波长的荧光强度。(3) Detection: use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
(三)结果(3) Results
随着加入模板样本浓度的下降,孔内荧光强度逐渐下降,到30分钟时实验组相对于阴性组差异达到较为显著的程度(见图1)。重复进行实验共计3次,统计30分钟时各浓度梯度的荧光强度,可见样本浓度为30拷贝/μl时与对照组仍有明显差异。With the decrease of the concentration of the added template sample, the fluorescence intensity in the well gradually decreased, and the difference between the experimental group and the negative group reached a more significant degree at 30 minutes (see Figure 1). The experiment was repeated for a total of 3 times, and the fluorescence intensity of each concentration gradient was counted at 30 minutes. It can be seen that there is still a significant difference between the sample concentration and the control group when the concentration is 30 copies/μl.
Claims (6)
- 用于快速检测乙型肝炎病毒的引物,所述引物为重组酶聚合酶扩增DNA引物:Primers for rapid detection of hepatitis B virus, the primers are recombinase polymerase amplification DNA primers:引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′Primer 1-5'-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3'引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′。Primer 2-5'-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3'.
- 权利要求1所述引物用于制备快速检测乙型肝炎病毒的试剂盒的应用。The application of the primer of claim 1 for preparing a kit for rapid detection of hepatitis B virus.
- 一种用于快速检测乙型肝炎病毒的试剂盒,包括:重组酶聚合酶扩增体系和Cas13反应体系;A kit for rapid detection of hepatitis B virus, comprising: a recombinase polymerase amplification system and a Cas13 reaction system;所述重组酶聚合酶扩增体系包括TwistAmp液态重组酶聚合酶扩增试剂盒和重组酶聚合酶扩增DNA引物:The recombinase polymerase amplification system includes a TwistAmp liquid recombinase polymerase amplification kit and a recombinase polymerase amplification DNA primer:引物1-5′-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3′Primer 1-5'-GAAATTAATACGACTCACTATAGGGGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTG-3'引物2-5′-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3′Primer 2-5'-TCCCGTGCTGGTTGTTGAGGATCCTGGAATTAGAG-3'所述Cas13反应体系包括重组CRISPR-Cas13a蛋白、单链引导RNA和单链RNA探针单链引导RNA:The Cas13 reaction system includes recombinant CRISPR-Cas13a protein, single-stranded guide RNA and single-stranded RNA probe single-stranded guide RNA:5′-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3′单链RNA探针:5'-ACUACCCCAAAAACGAAGGGGACUAAAACGACAAACGGGCAACAUACCUUGAUAGUC-3' single-stranded RNA probe:5′-FAM-TUUUUUC-BHQ-1-3′。5'-FAM-TUUUUUC-BHQ-1-3'.
- 权利要求3-5任一所述试剂盒检测乙型肝炎病毒基因的方法,包括如下步骤:The method for detecting hepatitis B virus gene of any one of the described test kits of claims 3-5, comprising the steps:1)重组酶聚合酶扩增1) Recombinase polymerase amplification(1)取HBV阳性对照稀释成3x10 5拷贝/μl,3x10 4拷贝/μl,3x10 3拷贝/μl,3x10 2拷贝/μl,3x10拷贝/μl,取1μl加入以下反应体系: ( 1 ) Dilute the HBV positive control to 3x105 copies/μl, 3x104 copies/μl, 3x103 copies/μl, 3x102 copies/μl, 3x10 copies/μl, take 1μl and add it to the following reaction system:(2)加入1.25μl的20x核心反应混合液,混匀离心;(2) Add 1.25μl of 20x core reaction mixture, mix well and centrifuge;(3)加入1μl模板(不同稀释度的质粒模板和阴性对照);(3) Add 1 μl of template (plasmid template of different dilution and negative control);(4)在管盖上加入1.25μl乙酸镁(MgOAc,280mM);(4) Add 1.25 μl of magnesium acetate (MgOAc, 280 mM) to the cap of the tube;(5)颠倒混匀,离心后39度孵育20min。(5) Invert and mix, and incubate at 39°C for 20min after centrifugation.2)用Cas13蛋白检测HBV2) Detection of HBV with Cas13 protein(1)取上述1ul反应产物加入Cas13反应体系,加入384孔透明圆底酶标板充分混合;设3个复孔:(1) Take 1 ul of the above reaction product and add it to the Cas13 reaction system, add a 384-well transparent round-bottom microtiter plate and mix well; set up 3 duplicate wells:(2)反应时间30min;(2) reaction time 30min;(3)检测:用多功能酶标仪检测仪,检测480nm激发光520nm发射波长的荧光强度。(3) Detection: use a multifunctional microplate reader to detect the fluorescence intensity of the excitation light at 480 nm and the emission wavelength of 520 nm.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011385303.1 | 2020-11-30 | ||
CN202011385303.1A CN112359146A (en) | 2020-11-30 | 2020-11-30 | Kit for rapidly detecting hepatitis B virus gene and detection method thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022110335A1 true WO2022110335A1 (en) | 2022-06-02 |
Family
ID=74535851
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/136280 WO2022110335A1 (en) | 2020-11-30 | 2020-12-14 | Kit for rapidly detecting hepatitis b virus gene and detection method therefor |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN112359146A (en) |
WO (1) | WO2022110335A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113150127B (en) * | 2021-05-10 | 2021-12-21 | 北京祥瑞生物制品有限公司 | Nucleic acid antibody kit for rapidly detecting virus |
CN113684317B (en) * | 2021-09-09 | 2023-09-26 | 贵州中医药大学第二附属医院 | Ultra-sensitive rapid detection and identification system for B type and C type hepatitis B virus based on CRISPR-Cas12B |
CN117402719A (en) * | 2023-10-19 | 2024-01-16 | 上海交通大学医学院附属仁济医院 | Method and kit for detecting circular RNA |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1378553A (en) * | 1998-12-23 | 2002-11-06 | 博伊斯汤普生植物研究所 | Expression of immunogenic hepatitis B surface antigens in transgenic plants |
US20020177127A1 (en) * | 2000-10-23 | 2002-11-28 | Yang Yeasing Y. | Compositions and methods for detecting human immunodeficiency virus 2 (HIV-2) |
KR20120044953A (en) * | 2012-03-19 | 2012-05-08 | 주식회사 맥스바이오텍 | Primemr and method for detecting hepatitis b virus, and detection kit thereof |
CN107604096A (en) * | 2017-10-13 | 2018-01-19 | 杭州迪安医学检验中心有限公司 | A kind of nucleotide sequence and kit for hepatitis type B virus detection |
CN109055499A (en) * | 2018-08-30 | 2018-12-21 | 杭州杰毅麦特医疗器械有限公司 | isothermal nucleic acid detection method and kit based on CRISPR-Cas |
CN110066865A (en) * | 2019-04-30 | 2019-07-30 | 常州桐树生物科技有限公司 | A kind of detection method and probe of the nucleic acid specific fragment based on CRISPR-Cas13a |
CN110343785A (en) * | 2019-08-05 | 2019-10-18 | 首都医科大学附属北京佑安医院 | Based on PCR-CRISPR-cas13a detects the kit of hepatitis b virus covalence closed cyclic DNA |
CN111041084A (en) * | 2018-10-12 | 2020-04-21 | 中国科学院上海生命科学研究院 | Detection kit for small fat Willi syndrome and use method thereof |
CN111778288A (en) * | 2020-07-17 | 2020-10-16 | 广州华腾生物医药科技有限公司 | Method, composition and application for constructing HBV transgenic mouse model |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111108220A (en) * | 2017-03-15 | 2020-05-05 | 博德研究所 | CRISPR-Effector System-based diagnostics for Virus detection |
WO2020124050A1 (en) * | 2018-12-13 | 2020-06-18 | The Broad Institute, Inc. | Tiled assays using crispr-cas based detection |
CN111748647A (en) * | 2019-03-28 | 2020-10-09 | 中国医科大学 | RPA primer, reagent and kit for detecting hepatitis B virus and application thereof |
CN111593138A (en) * | 2019-09-20 | 2020-08-28 | 山东省农业科学院家禽研究所 | Duck hepatitis B virus recombinant polymerase isothermal amplification detection method |
-
2020
- 2020-11-30 CN CN202011385303.1A patent/CN112359146A/en active Pending
- 2020-12-14 WO PCT/CN2020/136280 patent/WO2022110335A1/en active Application Filing
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1378553A (en) * | 1998-12-23 | 2002-11-06 | 博伊斯汤普生植物研究所 | Expression of immunogenic hepatitis B surface antigens in transgenic plants |
US20020177127A1 (en) * | 2000-10-23 | 2002-11-28 | Yang Yeasing Y. | Compositions and methods for detecting human immunodeficiency virus 2 (HIV-2) |
KR20120044953A (en) * | 2012-03-19 | 2012-05-08 | 주식회사 맥스바이오텍 | Primemr and method for detecting hepatitis b virus, and detection kit thereof |
CN107604096A (en) * | 2017-10-13 | 2018-01-19 | 杭州迪安医学检验中心有限公司 | A kind of nucleotide sequence and kit for hepatitis type B virus detection |
CN109055499A (en) * | 2018-08-30 | 2018-12-21 | 杭州杰毅麦特医疗器械有限公司 | isothermal nucleic acid detection method and kit based on CRISPR-Cas |
CN111041084A (en) * | 2018-10-12 | 2020-04-21 | 中国科学院上海生命科学研究院 | Detection kit for small fat Willi syndrome and use method thereof |
CN110066865A (en) * | 2019-04-30 | 2019-07-30 | 常州桐树生物科技有限公司 | A kind of detection method and probe of the nucleic acid specific fragment based on CRISPR-Cas13a |
CN110343785A (en) * | 2019-08-05 | 2019-10-18 | 首都医科大学附属北京佑安医院 | Based on PCR-CRISPR-cas13a detects the kit of hepatitis b virus covalence closed cyclic DNA |
CN111778288A (en) * | 2020-07-17 | 2020-10-16 | 广州华腾生物医药科技有限公司 | Method, composition and application for constructing HBV transgenic mouse model |
Non-Patent Citations (1)
Title |
---|
CHEN, SHAN, XIE XUE-PING, ZOU CHUN-MEI, QI BING-JIE, ZHOU XIE-MIN, GUO-HUA: " Detection methods and devices of visual detection in closed tube recombinase polymerase amplification of hepatitis B virus", JOURNAL OF MEDICAL POSTGRADUATES, vol. 33, no. 5, 1 May 2020 (2020-05-01), pages 515 - 520, XP055933985 * |
Also Published As
Publication number | Publication date |
---|---|
CN112359146A (en) | 2021-02-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Kang et al. | Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example | |
WO2022110335A1 (en) | Kit for rapidly detecting hepatitis b virus gene and detection method therefor | |
CN110551846B (en) | Cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
CN111549176B (en) | LAMP primer group and kit for detecting SARS-CoV-2 | |
CN108486234A (en) | A kind of method and its application of CRISPR partings PCR | |
JPH03133379A (en) | Extraction of nucleic acid without using protein decomposing enzyme and pcr augmenting method | |
Josko | Molecular virology in the clinical laboratory | |
CN107022651B (en) | Kit for rapidly detecting hepatitis C virus nucleic acid and detection method thereof | |
CN108676920A (en) | It is a kind of quickly to detect mouse norovirus primer, kit and its RT-RPA methods | |
WO2010102460A1 (en) | A method and kit for quantitative and qualitative detection of genetic material of pathogenic microorganisms | |
CN112725531B (en) | Hepatitis B virus rapid detection system combining MCDA with biosensor | |
US20210147910A1 (en) | Hybrid multi-step nucleic acid amplification | |
US20070281295A1 (en) | Detection of human papillomavirus E6 mRNA | |
Wang et al. | Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection | |
CN105018647B (en) | A kind of kit and its detection method based on the accurate quantitative typing detection HPV16/18 of digital pcr | |
US20240150856A1 (en) | Multiplexed nucleic acid detection kit for human papillomavirus (hpv) typing, and detection method | |
Shi et al. | A real time quantitative PCR-based method for the detection and quantification of simian virus 40 | |
CA3067868A1 (en) | Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction | |
WO2023207909A1 (en) | Crispr-based nucleic acid detection kit and use thereof | |
CN111719015A (en) | Human immunodeficiency virus HIV-1 detection kit | |
CN115820818A (en) | One-step nucleic acid detection method and application thereof | |
CN113234866B (en) | Detection kit for synchronously detecting pathogens of multiple blood circulation systems and detection method thereof | |
KR101503039B1 (en) | Diagnostic primer for the heatitis c virus, probe, kit including same, and method for diagnosing the hepatitis c virus using the kit | |
CN113801966B (en) | Fluorescent quantitative PCR method and kit for detecting novel coronavirus subgenomic | |
Zhao | From single cell gene-based diagnostics to diagnostic genomics: current applications and future perspectives |
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: 20963223 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: 20963223 Country of ref document: EP Kind code of ref document: A1 |