WO2021164050A1 - Procédé et kit de détection rapide d'amplification isotherme à double colorant multi-cible - Google Patents

Procédé et kit de détection rapide d'amplification isotherme à double colorant multi-cible Download PDF

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WO2021164050A1
WO2021164050A1 PCT/CN2020/077400 CN2020077400W WO2021164050A1 WO 2021164050 A1 WO2021164050 A1 WO 2021164050A1 CN 2020077400 W CN2020077400 W CN 2020077400W WO 2021164050 A1 WO2021164050 A1 WO 2021164050A1
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isothermal amplification
target
primer
primers
seq
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徐堤
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北京天恩泽基因科技有限公司
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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
    • C12Q1/70—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
    • 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

Definitions

  • the invention relates to a biological detection reagent, in particular to a rapid detection method, a kit and a detection primer for multi-target double-dye isothermal amplification nucleic acid.
  • the SARS-CoV-2 virus has caused China to initiate first-level disease control and defense measures, and its rapid and accurate diagnosis is of great significance for the rapid diagnosis and prevention and control of the epidemic.
  • the core of the virus particle is composed of virus and protein and is an RNA virus with a lipid bilayer membrane on the outside.
  • SARS-CoV-2 GenBank ID: MN908947.3
  • MN908947.3 The whole genome sequence of SARS-CoV-2 (GenBank ID: MN908947.3) has been confirmed, laying the foundation for rapid molecular diagnosis research.
  • Molecular diagnosis targeting the four proteins of spike, RdRp, 3CLpro and PLpro may be applicable to rapid diagnosis of new coronavirus infection.
  • the conventional methods for detecting microbial infections have been successfully applied abroad.
  • immunological methods ELISA, etc.
  • these methods can perform specific detection, but have obvious disadvantages such as time-consuming, labor-intensive, low sensitivity and poor specificity.
  • the ATP method needs to consume a lot of more expensive reagents, and the cleaning and maintenance requirements of the instrument are particularly high.
  • customers often suspect the possibility of false negatives.
  • the MicroStar instrument is expensive and the operating cost is also very expensive. It usually takes more than 16 hours and cannot be used to detect unknown samples with high bacterial counts.
  • the whole process of using traditional biochemical detection methods requires at least 7-10 days, and the detection limit is low. ,Time-consuming.
  • the immunization method is relatively fast, but the preparation of monoclonal antibodies is difficult, cross-reaction is easy to occur, and the specificity is poor.
  • the PCR or fluorescent quantitative PCR method is fast, specific, and highly sensitive, but requires an expensive PCR machine.
  • There have been a small number of foreign literature reports using mRNA template to target iap gene to detect live Listeria monocytogenes virus by RT-PCR but membrane hybridization is required to display the detection results, which requires a long time, complicated technology, and high cost, and it is not easy to popularize and apply.
  • Fluorescence staining technology can detect live bacteria, but the technology is complicated and requires high-end equipment. Therefore, the establishment of a simple, sensitive, rapid, and specific genetic micro-shop detection method including pathogenic bacteria is currently an urgent technical problem to be solved.
  • Loop-mediated isothermal amplification gene technology (loop-mediated isothermal amplication, LAMP for short) is a new nucleic acid amplification technology developed by Notomi et al. in 2000. A set of two pairs of specific genes is designed for 6 regions of the target gene sequence to be tested. Primers, using strand displacement DNA polymerase (Bst DNA polymerase) can perform specific, efficient, and rapid nucleic acid amplification under isothermal conditions of about 65°C. The amplification results can directly detect the by-product magnesium pyrophosphate precipitation by naked eyes. To judge or detect its turbidity, it can also be stained with a double-stranded fluorescent dye, preferably SYBR Green I, which can be judged by the naked eye.
  • a double-stranded fluorescent dye preferably SYBR Green I
  • Chinese invention patents (application numbers: 200710030435.0, 200710030437.X, 200710132320.2, 200710026389.7, 200810052321.0, 200810015001.8, 200810093986.6) respectively disclose methods for detecting pathogens using loop-mediated isothermal amplification gene technology.
  • the existing loop-mediated isothermal amplification gene technology detection products in the industry still have certain false positives, which can only target a single target, and still cannot meet the sudden large number of detection needs in the detection of emergency epidemics and scientific research scenarios.
  • an object of the present invention is to provide a rapid isothermal amplification test for detecting the concentration of target nucleic acid in a biological sample.
  • Method the second purpose is to provide a kit for rapid detection of isothermal amplification
  • the third purpose is to provide a method for preparing the above-mentioned kit.
  • the fourth purpose of the present invention is to provide a detection method and reagents for the above-mentioned isothermal amplification.
  • the primer composition of the box is a rapid isothermal amplification test for detecting the concentration of target nucleic acid in a biological sample.
  • the isothermal amplification detection method, the kit, and the primer composition of the present invention have the characteristics of simplicity, sensitivity, rapidity, and good specificity, and can be widely used for emergency epidemic early warning, epidemic epidemiological investigation, clinical infection and epidemic-related food and aquatic products .
  • Environmental testing and medical and health, cosmetics pollution source testing, rapid public health epidemiology investigation, customs import and export quarantine and other fields can quickly target gene testing, such as the clinical testing of a new coronavirus infection in early 2020.
  • An isothermal amplification rapid detection method for detecting whether a target nucleic acid is contained in a sample is characterized in that the isothermal amplification rapid detection method includes the steps of: collecting a biological sample containing the target nucleic acid, extracting the target nucleic acid, and comparing the target nucleic acid with a positive control
  • the negative control and the isothermal amplification reaction system are added to the mixing tube of the isothermal amplification reaction system and the primer composition to perform the isothermal amplification reaction.
  • the color change in the reaction tube and the fluorescence scanning reading are both independently or combined to determine the test object sample by naked eyes at room temperature. Whether it contains target nucleic acid, among which,
  • the isothermal amplification reaction system includes a buffer composition, a visualization composition, and a polymerase that catalyzes the amplification reaction under isothermal conditions;
  • the normal temperature visualization composition includes a visible light chromogenic dye and a fluorescent signal generating compound, and the visible light chromogenic dye And the fluorescent signal generating compound are added to the amplification system at the same time before the isothermal amplification reaction; the visible light chromogenic dye and the fluorescent signal generating compound are used alone or in combination to detect the target nucleic acid containing the specimen to be tested.
  • the fluorescent signal generating compound includes a fluorescent dye and a fluorescent probe, or one selected from a fluorescent dye and a fluorescent probe; the positive control is a synthetic target nucleic acid with a known concentration, which can be amplified by a LAMP primer increase;
  • the isothermal amplification detection method is selected from loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (Nucleic acid sequence-based amplification, NASBA), and rolling circle amplification technology (Rolling circle amplification). , RCA), Single primer isothermal amplification (SPIA), Helicase-dependent isothermal DNA amplification (HDA), Recombinase Polymerase Amplification (RPA) or In Strand Displacement Amplification (SDA), or the isothermal amplification detection method is loop-mediated isothermal amplification.
  • LAMP loop-mediated isothermal amplification
  • Nucleic acid sequence-based amplification Nucleic acid sequence-based amplification
  • NASBA rolling circle amplification technology
  • Rolling circle amplification rolling circle amplification
  • RCA Single primer isothermal amplification
  • HDA Helicase-dependent isothermal DNA amplification
  • RPA Recombinase Polymerase Amplification
  • SDA In Strand
  • the macroscopic observation is selected from completing the above isothermal amplification reaction at room temperature.
  • visual observation is the determination of endpoint data.
  • the measurement endpoint data is to firstly look at the result with the naked eye to judge negative and positive, and then further fluorescently detect the reaction tube with insignificant color change, and the endpoint data (end point) is measured, and the fluorescence value is more than 10 times of the negative control and judged as positive.
  • visual observation is the measurement of real-time data.
  • the real-time data is measured directly on a fluorescent PCR instrument, and the real-time data is measured by reading the fluorescent reading data every time interval elapses, and judging whether the target nucleic acid contains the target nucleic acid or not the target pathogenic microorganism is infected by the negative control and the positive control.
  • the interval for reading fluorescence readings for measuring real-time data is 1 to 5 minutes. In some embodiments, the interval for reading fluorescence readings for measuring real-time data is preferably 1 to 3 minutes. In the method, the interval time for reading the fluorescence readings described in the measurement of real-time data is preferably 1 minute.
  • test tube by visually observing the visible light color identification, compared with the negative control tube, it is shown that the test tube has a clear dark color as positive, and the color without obvious change is negative; further, if the fluorescence signal is detected as the absolute value of the fluorescence signal exceeds 10000 is positive, and the fluorescence signal is lower than 5000 is negative.
  • SYBR Green I and EvaGreen I are used as fluorescent dyes, and the FAM channel is used to collect fluorescence data.
  • the concentration of the visible light dye is 0.01-2%.
  • the concentration of the fluorescent dye is 0.1-100 uM.
  • the concentration of the fluorescent probe is 0.1-100 uM.
  • reaction temperature is controlled at 50-70°C
  • reaction temperature is controlled at 55-65°C
  • reaction temperature is controlled at 60-65°C.
  • reaction time is 10-60 minutes.
  • reaction time is 20-60 minutes.
  • reaction time is 30-60 minutes.
  • the isothermal reaction system contains a buffer composition, an isothermal amplification polymerase, a visible light chromogenic dye, and a fluorescent dye.
  • the isothermal amplification polymerase is Bst DNA polymerase.
  • the isothermal amplification polymerase is an upgraded version of Bst DNA polymerase.
  • the isothermal amplification polymerase phi29 DNA Polymerase.
  • the buffer composition is LAMP mix
  • the reaction system of the LAMP mix for each 20 ⁇ L reaction system includes 5 ⁇ L reaction solution containing dual dyes, 1 ⁇ L Bst DNA polymerase, 2.5 ⁇ L primer mixture, and 2 ⁇ L template to be tested. Or positive control solution and 9.5 ⁇ L ddH 2 O.
  • the isothermal amplification reaction is loop-mediated isothermal amplification.
  • the reaction procedure of the isothermal amplification reaction is to react the prepared PCR tube at 60-65°C for 1 to 1.5 hours, and terminate the reaction at 80°C.
  • the rapid detection method for isothermal amplification of the present invention can be applied to pathogens including human pathogenic pathogens, fish and shellfish and other aquatic organisms, pathogens in agriculture and animal husbandry, plant pathogenic microorganisms, animal and plant genetically modified fields, and food safety fields. Rapid detection of specific genes, the test samples include clinical specimens for medical tests, genetically modified animal tissues, animal husbandry and veterinary animal specimens, genetically modified plant tissues, crop plants, rice flour, flour, soy products, rice products, complementary foods for infants and young children, canned food, potato And extruded foods, plant-based feeds, etc.
  • the isothermal amplification detection method can be applied to rapid genetic detection of human pathogenic pathogens.
  • the isothermal amplification detection method can be applied to rapid gene detection of pathogenic pathogens in aquatic products such as fish and shellfish.
  • the isothermal amplification detection method can be applied to rapid genetic detection of pathogens in agriculture, animal husbandry and aquaculture.
  • the isothermal amplification detection method can be applied to rapid genetic detection of plant pathogenic microorganisms.
  • the isothermal amplification detection method can be applied to the rapid detection of specific genes in the field of animal and plant transgenics.
  • the kit for the first purpose of the rapid detection method of isothermal amplification which includes an isothermal amplification reaction system, a primer composition, a positive control and a negative control
  • the isothermal amplification reaction system includes an isothermal amplification polymerase, a visualization combination
  • the visualization composition includes a visible light chromogenic dye and a fluorescent signal generating compound
  • the fluorescent signal generating compound includes a fluorescent chromogenic dye and a fluorescent probe or is selected from a fluorescent chromogenic dye and a fluorescent probe
  • the positive control solution is the target nucleic acid extraction solution to be tested.
  • the kit is a multi-target double-dye loop-mediated isothermal amplification (LAMP) kit
  • LAMP loop-mediated isothermal amplification
  • the loop-mediated isothermal amplification (LAMP) kit uses visible light at room temperature to develop color and fluorescence. Scanning readings are used alone or in combination to determine the positiveness of the target nucleic acid in the biological sample to be tested and the content of the nucleic acid content of the clinical specimen of the test subject.
  • the kit functions as follows (a) or (b):
  • the isothermal amplification polymerase is Bst DNA polymerase.
  • the Bst DNA polymerase contains 8-16 activity units per microliter.
  • the isothermal amplification polymerase is an upgraded version of Bst DNA polymerase.
  • the isothermal amplification polymerase phi29 DNA Polymerase.
  • the primer composition includes a LAMP primer composition directed against a target sequence of a biological origin.
  • the primer composition includes a LAMP primer composition targeting two target sequences of a biological origin.
  • the primer composition includes a LAMP primer composition targeting three target sequences of a biological origin.
  • the primer set of the target sequence is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the primer composition includes a target sequence for different biological sources.
  • the primer composition includes two target sequences directed against different biological sources.
  • the primer composition includes three target sequences for different biological sources.
  • the primer composition includes a LAMP primer composition targeting multiple target sequences from different biological sources, and the multiple sequences include target sequence A, target sequence B, and target sequence C.
  • the primer set A of the target sequence A is composed of outer primer A1, outer primer A2, inner primer A1 and inner primer A2 with a volume ratio of 1:1:4:4; in a preferred embodiment, the primer set B of the target sequence point B is composed of outer primer B1, outer primer B2, inner primer B1 and inner primer B2 with a volume ratio of 1:1:4:4; in a preferred embodiment, the The primer set C of the target sequence point C is composed of outer primer A1, outer primer C2, inner primer C1 and inner primer C2 with a volume ratio of 1:1:4:4.
  • Visible light coloring dyes are selected from FD&C blue #1, fast green FCF, erythrosine, allure red AC, tartrazine, sunset yellow FCF, indigo carmine, betaine, chlorophyll, caramel pigment, butterfly pea, fragrance Blue, ultramarine blue, peacock blue, calcein green, hydroxynaphthol blue, cobalt blue, phthalocyanine and Coomassie brilliant blue, or PCR grade green such as blue dye.
  • the visible light coloring dye is selected from one of peacock blue, malachite green, calcein green, hydroxynaphthol blue, and Prussian blue.
  • the visible light chromogenic dye is selected from peacock blue, malachite green, calcein green, hydroxynaphthol blue, and Prussian blue.
  • the visible light chromogenic dye is Prussian blue.
  • the visible light dye is calcein green.
  • the visible light dye is hydroxynaphthol blue.
  • Nucleic acid fluorescent dyes include but are not limited to SYBR series dyes, UltraPower nucleic acid dyes, GelRed&GelGreen, propidium iodide (PI), DAPI, Hoechst 33342, thiazole orange, and acridine orange.
  • SYBR Green I has the specific selectivity of double-stranded DNA. YBR Green II and SYBR Green I are just the opposite. It is a highly sensitive dye for detecting RNA or single-stranded DNA. SYBR Gold dye can be used to detect double-stranded DNA and single-stranded DNA. DNA and RNA, SYBR Safe.
  • the fluorescent dye is SYBR Green I, and the FAM channel is used to collect fluorescence data; in some embodiments, the fluorescent dye is EvaGreen I, and the FAM channel is used to collect fluorescence data.
  • Fluorescent probes are nucleic acid probes artificially synthesized according to the sequence of the target site.
  • the probe labeled with FAM collects fluorescence data in the FAM channel during detection.
  • the buffer composition of the reaction solution system is a LAMP mix
  • the dUTP-UNG contained in the LAMP mix can prevent cross-fouling.
  • the LAMP mix consists of 10 ⁇ Thermopol reaction buffer with a volume ratio of 5:2:1:2, 7.5-12.5mM dNTP, 100-200mM MgSO4 and 25-37.5M betaine; the 10 ⁇ Thermopol contains 200mM pH8.8 trihydroxymethylaminomethane hydrochloride, 100mM potassium chloride, 100mM ammonium sulfate, 20mM magnesium sulfate and a volume percentage concentration of 1% Triton X-100.
  • the positive control solution is the target nucleic acid extraction solution to be tested. In one embodiment, the positive control
  • the solution is the new coronavirus SARS-CoV-2 genomic RNA.
  • the positive control is the new coronavirus SARS-CoV-2 genomic RNA.
  • the positive control is the porA gene nucleic acid of human pathogenic Neisseria gonorrhoeae.
  • the positive control is bovine cytB gene.
  • the positive control is a pilin gene extract of Aeromonas hydrophila.
  • the positive control is an exogenous gene of transgenic sugar beet H7-1 strain.
  • the sample is a clinical specimen of sputum.
  • the sample is a clinical specimen of nasal wash.
  • the sample is a clinical specimen of a throat swab.
  • the clinical specimen sample is venous blood.
  • the said is a clinical specimen of urine. In one embodiment, the said is a stool clinical specimen.
  • the said is a clinical specimen of cerebrospinal fluid.
  • the preparation method of the isothermal amplification detection kit to achieve the second objective of the present invention isothermal amplification detection kit.
  • the isothermal amplification is loop-mediated isothermal amplification (LAMP);
  • the primer composition includes a LAMP primer composition targeting one target sequence, two target sequences, three target sequences, or multiple target sequences from a biological source, and the multiple target sequences include a target sequence. Sequence A and target sequence B and target sequence C;
  • the primer composition includes a LAMP primer composition for one target sequence, two target sequences, three target sequences, or multiple target sequences from different biological sources, and the multiple sequences include the target sequence A And target sequence B and target sequence C.
  • the target sequence A primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the target sequence B primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the target sequence C primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers;
  • a primer composition used for the rapid detection of the isothermal amplification technology of the first aspect and the isothermal amplification detection kit of the second aspect wherein:
  • the isothermal amplification is loop-mediated isothermal amplification (LAMP);
  • the primer composition includes a LAMP primer composition for one target sequence, two target sequences, three target sequences, or multiple target sequences from a biological source, and the multiple target sequences include target sequence A and target sequence B And target sequence C; or the primer composition includes a LAMP primer composition for one target sequence, two target sequences, three target sequences, or multiple target sequences from different biological sources, and the multiple sequences include target sequence A And target sequence B and target sequence C, where
  • the target sequence A primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the target sequence B primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the target sequence C primer set is composed of two pairs of primers, a pair of primers are outer primers, and a pair of primers are inner primers.
  • the organism is SARS-CoV-2
  • the primer set for the target A target sequence A is a pair of outer primers (F3A and B3A), and a pair of primers are inner primers (FIPA and BIPA)
  • the sequence is shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively,
  • the primer set for the point target sequence B is a pair of primers (F3B and B3B), a pair of primers (FIPB and BIPB), the sequence is as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 are shown,
  • the primer set of the target sequence C has a pair of outer primers (F3C and B3C), and a pair of inner primers (FIPC and BIPC).
  • the sequences are as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 9, respectively. 11 and SEQ ID No. 12.
  • the biological African swine fever virus includes a LAMP primer composition against African swine fever virus
  • the LAMP primer composition of the African swine fever virus includes a target sequence D as an external primer (F3D and B3D), a pair of primers (FIPD and BIPD), the sequence is as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No.18 is shown.
  • the loop-mediated isothermal amplification technology (loop-mediated isothermal amplication, referred to as LAMP) of the present invention can quickly detect the genomic nucleic acid of a sample infected with pathogenic microorganisms, such as the RNA of the novel coronavirus SARS-CoV-2 and the DNA of the swine fever virus.
  • the method is to use isothermal amplification reaction polymerase such as thermostable Bst DNA and two pairs of special inner and outer primers designed according to the target gene sequence to specifically identify six independent regions on the target sequence, and initiate a cyclic strand displacement reaction.
  • the target DNA region initiates the synthesis of the complementary strand, and as a result, the complementary sequence on the same strand repeatedly forms a stem-circle DNA mixture with many loops of cauliflower structure.
  • LAMP reaction loop-mediated isothermal amplification reaction
  • the LAMP reaction is completed within 10 to 60 minutes at a constant temperature (about 65°C).
  • This relatively mild temperature condition and no temperature cycle simplify the required equipment and overcome the inherent shortcomings of traditional PCR, such as long detection time, easy contamination, and high detection cost.
  • this detection method has low requirements on the technical quality of the detection personnel, and the actual operation is extremely simple and does not require special reagents and equipment, which is conducive to the establishment of a low-cost rapid screening system.
  • the LAMP method is a simple, rapid and highly specific gene amplification method. Comparing constant temperature gene amplification technology with PCR technology (including fluorescence real-time quantitative PCR technology), it can be found that this technology is equivalent to or superior to PCR technology in terms of sensitivity, specificity and detection range, and does not rely on any specialization. High-throughput rapid detection on site can be achieved with the latest equipment and equipment, and the detection cost is much lower than the fluorescence quantitative PCR technology. At present, there are no kits for sale in this area in China. The current national standards mainly focus on microbial isolation, culture and morphological identification, combined with biochemical analysis and serological identification.
  • the preliminary identification requires 2-3 days, and the completion of the identification report requires 10-15 days; the gene of the present invention is used
  • the diagnostic kit test only takes 0.5 hours.
  • visible light and fluorescent dyes are added to the reaction solution of the present invention, and the identification result is more intuitive and clear.
  • swine vesicular disease virus SVDV
  • porcine circovirus type 2 PCV2
  • classical swine fever virus CSFV
  • porcine reproductive and respiratory syndrome virus PRRSV
  • porcine parvovirus PV
  • Newcastle disease virus NDV
  • H5N1 avian influenza virus H5N1avian influenza virus
  • H9 avian influenza virus H9avian influenza virus
  • duck plagues virus DMV
  • canine distemper virus canine distemper virus
  • canine parvovirus canine parvovirus
  • bovine leukemia virus BLV
  • sheep Infectious impetigo virus orf virus
  • trypanosomes Babesiagibsoni
  • Theileriaequi Babesiacaballi
  • Theileriauilenbergi UTRu6
  • Results are available in 10-30 minutes (depending on virus concentration). Reverse transcription and LAMP are completed in one step, which is more than 1 hour faster than RT-PCR.
  • the reaction can be done in a water bath or metal bath. Firstly, check the result with the naked eye to judge negative and positive, and then take the reaction tube with no obvious color change to measure the fluorescence. At this time, the measured end point is the fluorescence. If the value exceeds 10 times of the negative control, it is considered positive. (If SYBR Green I and EvaGreen I fluorescent dyes are used, the FAM channel is used to collect fluorescence data), only a constant temperature water bath of more than 1,000 yuan is needed, and a fluorescent PCR instrument of hundreds of thousands is not required, which is suitable for basic units.
  • the reaction can also be performed directly on a fluorescent PCR instrument, and the data is read every minute (if SYBR Green I and EvaGreen I fluorescent dyes are used, the fluorescence data is collected by the FAM channel), so the measurement is real time data.
  • RNA positive control and external reference to monitor extraction and amplification operations.
  • the specificity is higher than RT-PCR, because four primers are used instead of two.
  • the sensitivity is higher than that of RT-PCR, up to 10 copies/reaction, so the false negative rate is lower.
  • Visible light color development, fluorescent color development, and probe detection are synergistic, which is suitable for a large range of sample nucleic acid content.
  • Figure 1 A group of SARS-CoV-2 samples and then add the LAMP amplification primer set N3 of target site A to the LAMP amplification reaction system to visualize the color development of the isothermal amplification reaction of different concentrations of samples at room temperature
  • the negative control water reacts with normal human DNA and low-concentration 10E2 samples and 10E3 concentration samples.
  • the normal temperature color visualization observation result is negative.
  • the samples with 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by normal temperature color visualization.
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • A, B, C, and D fluorescence readings below 5000 are negative, and E, F, G, and H detections above 10,000 are positive.
  • Figure 4 A group of SARS-CoV-2 samples and then add the LAMP amplification primer set N1+2 of target sites B and C to the LAMP amplification reaction system to visualize the color development of the isothermal amplification reaction of different concentrations of samples at room temperature.
  • the negative control water reacts with normal human DNA and low-concentration 10E2 samples.
  • the result of the visual observation of normal temperature color development is negative, and the samples with concentrations of 10E3, 10E4, 10E5, 10E6 and 10E7 can be visually detected by normal temperature color development.
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • A, B, and C detect fluorescence readings below 5000 are considered negative, D, E, F, G, and H detect fluorescence readings above 10,000 are considered positive.
  • Figure 7 A group of SARS-CoV-2 samples and then add the LAMP amplification primer set N1+3 of target sites A and B to the LAMP amplification reaction system to visualize the color development of the isothermal amplification reaction of different concentrations of samples at room temperature.
  • the negative control water reacts with normal human DNA and low-concentration 10E2 samples and 10E3 concentration samples.
  • the normal temperature color visualization observation result is negative.
  • the samples with 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by normal temperature color visualization.
  • FIG. 8 Agarose gel electrophoresis results of a group of SARS-CoV-2 samples and the LAMP amplification reaction system of target sites A and B with the LAMP amplification primer set N1+3 on the isothermal amplification reaction products of samples of different concentrations .
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • Figure 9 A group of SARS-CoV-2 samples added with target sites A and B LAMP amplification primer set N1+3 LAMP amplification reaction system for the fluorescence coloring results of isothermal amplification reaction products of different concentrations of samples,
  • A, B, C, and D with fluorescence readings below 5000 are negative.
  • D, E, F, G, and H with fluorescence readings of more than 10,000 are considered positive.
  • Figure 10 A group of SARS-CoV-2 samples and then add the LAMP amplification primer set N2+3 of target sites A and C to the LAMP amplification reaction system to visualize the color development of the isothermal amplification reaction of different concentrations of samples at room temperature.
  • FIG. 12 Fluorescence coloring results of a group of SARS-CoV-2 samples and the LAMP amplification reaction system of target sites A and C with the LAMP amplification primer set N2+3 on the isothermal amplification reaction products of samples of different concentrations.
  • A, and B do not detect fluorescence readings below 5000 as negative, and C, D, E, F, G and H detect fluorescence readings above 10,000 as positive.
  • Figure 13 A group of SARS-CoV-2 samples and then add the LAMP amplification primer set N1+2+3 of target sites A
  • Figure 14 Agar for a group of SARS-CoV-2 samples and then add LAMP amplification primer set N1+2+3 at target sites A
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • Figure 15 Fluorescence of the LAMP amplification reaction system of a set of SARS-CoV-2 samples and the LAMP amplification primer set N1+2+3 at target sites A
  • a primer set for target A target sequence A A pair of primers are outer primers (F3A and B3A), and a pair of primers are inner primers (FIPA and BIPA).
  • the sequences are as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4 are shown,
  • the primer set for the point target sequence B is a pair of primers (F3B and B3B), a pair of primers (FIPB and BIPB), the sequence is as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 are shown,
  • the primer set of the target sequence C has a pair of outer primers (F3C and B3C), and a pair of inner primers (FIPC and BIPC).
  • the sequences are as SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 9, respectively. 11 and SEQ ID No. 12.
  • the primer composition includes a LAMP primer composition for African swine fever virus, the LAMP primer composition of the African swine fever virus includes target sequence D as outer primers (F3D and B3D), and a pair of inner primers (FIPD and B3D).
  • BIPD BIPD
  • the sequence is shown as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16.
  • the gene diagnosis kit provided by the present invention is composed of a primer set, a positive control, a negative control, and a reaction solution containing dual dyes and dUTP and UNG enzymes.
  • primer composition including 3 sets of primers for 3 target sequences, each set has outer primer 1, outer primer 2, inner primer 1 and inner primer with a volume ratio of 1:1:4:4 2.
  • Composition in which the sequences of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 in primer composition 1, such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4 As shown, the sequences of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 in primer composition 2 are shown in SEQ ID No. 51, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8.
  • the sequences of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 in primer composition 1 are shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12.
  • the reaction solution is LAMP mix, which consists of 5:2:1:2 10 ⁇ Thermopol reaction buffer, 7.5-12.5mM dNTP, 100-200mM MgSO4 and 25-37.5M betaine; among them, 10 ⁇ Thermopol reaction buffer contains 200mM pH8.8 trihydroxymethylaminomethane hydrochloride, 100mM potassium chloride, 100mM ammonium sulfate, 20mM magnesium sulfate and a volume percentage concentration of 1% Triton X-100;
  • Bst DNA polymerase is Bst DNA polymerase, which contains 8 active units per microliter
  • the visible light dye is Prussian blue, and the fluorescent dye is SYBR Green I;
  • the positive control solution is the RNA extract of the new coronavirus SARS-CoV-2.
  • 20-30 ⁇ M primer composition includes 1 set of primers for 1 target sequence
  • each set of composition has 20-30 ⁇ M outer primer 1, outer primer 2, inner primer with a volume ratio of 1:1:4:4 1 and inner primer 2, wherein the sequences of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 in primer composition 1 are as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 18 are shown, the positive control is swine fever virus DNA extract, and the rest is the same as in Example 2.
  • Example 4 A set of LAMP primer set detects SARS-CoV-2 genomic RNA single target N3 isothermal amplification and normal temperature color detection method
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant of the viral RNA genome can be used as the template RNA to be used according to the conventional method.
  • the negative control water reacts with normal human DNA and low-concentration 10E2 samples and 10E3 concentration samples.
  • the normal temperature color visualization observation result is negative.
  • the samples with 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by normal temperature color visualization.
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • Example 4 Two sets of LAMP primer sets detect dual target isothermal amplification and normal temperature color detection method
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant can be used as the template RNA to be used.
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the samples with 10E3, 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by agarose gel electrophoresis.
  • samples A, B, and C did not detect the sharp and sharp increase in copy number, and D, E, F, G, and H detected the sharp and sharp increase in copy number.
  • samples A, B, C, D , E, F and G correspond to samples 9-16 respectively.
  • Example 5 Two sets of LAMP primer sets detect dual target isothermal amplification and normal temperature color detection method
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant can be used as the template RNA to be used.
  • the negative control water reacts with normal human DNA and low-concentration 10E2 samples and 10E3 concentration samples.
  • the normal temperature color visualization observation result is negative.
  • the samples with 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by normal temperature color visualization.
  • the negative control water, normal human DNA and low concentration 10E2 sample agarose gel electrophoresis results are negative, and the samples with 10E3, 10E4, 10E5, 10E6 and 10E7 concentrations can be detected by agarose gel electrophoresis.
  • samples A, B, C and D fluorescence readings below 5000 are negative, E, F, G, and H fluorescence readings above 10,000 are positive.
  • samples A, B, C, D, E, F, and G correspond to samples respectively 17-24.
  • Example 6 Two sets of LAMP primer sets detect dual target isothermal amplification and normal temperature color detection method
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant can be used as the template RNA to be used.
  • Example 7 Three sets of LAMP primer sets detect dual target isothermal amplification and normal temperature color detection method
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant can be used as the template RNA to be used.
  • the negative control water, normal human DNA and low-concentration 10E2 sample agarose gel electrophoresis results are negative, and the 10E3, 10E4, 10E5, 10E6 and 10E7 concentration samples can be detected by agarose gel electrophoresis.
  • samples A, B, C, D, E, F And G correspond to samples 33-40, respectively.
  • the absolute reading is the fluorescence reading of each tube after the amplification is completed. If the negative control is not amplified, the dye will not fluoresce, so the reading is very low, only more than 1,000, while the amplified one has high fluorescence. We use this to judge whether to expand.
  • Example 8 Three sets of LAMP primer sets with and without UNG added and no UNG added Isothermal amplification and normal temperature color detection method for dual target detection
  • Tested samples tested samples such as nasal wash for the slowing down of new coronavirus infection.
  • the supernatant can be used as the template RNA to be used.
  • the 7-13 group of samples used the newly configured LAMP mix, and the reaction was taken out every 10 minutes to observe the color change
  • the N1+N2+N3 primers are the most sensitive and can detect 600 copies.
  • Results are available in 10-30 minutes (depending on virus concentration), RT and LAMP are completed in one step, which is better than RTPCR
  • the contained dUTP-UNG can prevent cross contamination.
  • the specificity is higher than RT-PCR, because four primers are used instead of two.
  • the sensitivity is higher than RT-PCR, up to 10 copies/reaction, so the false negative rate is lower.
  • Example 9 Method for isothermal amplification of dual targets of two sets of LAMP primer sets and normal temperature color development to detect swine fever virus infection
  • Samples to be tested samples of nasal wash from pigs infected with swine fever virus.
  • Pretreatment of the tested sample extract the virus genome according to the conventional method:
  • the primers are shown in SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 18.

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Abstract

L'invention concerne un procédé de détection permettant de détecter rapidement un gène cible en utilisant une technologie d'amplification isotherme à double colorant multi-cible, un kit et un groupe d'amorces à utiliser. Lorsque le procédé de détection pour la détection rapide d'un gène cible à l'aide d'une technologie d'amplification isotherme à double colorant multi-cible et d'un mélange d'amorces du kit sont utilisés, un témoin positif et un témoin négatif sont soumis à une réaction d'amplification isotherme dans un instrument d'amplification par PCR et un changement de couleur dans un tube de réaction est observé à l'oeil nu à température ambiante combiné à une lecture de balayage par fluorescence de façon à déterminer si un objet de détection contient un acide nucléique cible. L'instrument de détection présente les avantages suivants : simplicité de configuration, simplicité d'utilisation, adaptation à des scénarios d'échantillons de virus à forte et à faible concentration, sensibilité élevée, forte spécificité, résultats pouvant être déterminés à l'œil nu, faible coût, adaptation à la détection d'échantillons à faible et à forte concentration, possibilité de détection de base et sur site.
PCT/CN2020/077400 2020-02-21 2020-03-02 Procédé et kit de détection rapide d'amplification isotherme à double colorant multi-cible WO2021164050A1 (fr)

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