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WO2024098642A1 - Reagent for detecting monkeypox virus by means of constant-temperature amplification and method for detecting same - Google Patents

Reagent for detecting monkeypox virus by means of constant-temperature amplification and method for detecting same Download PDF

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Publication number
WO2024098642A1
WO2024098642A1 PCT/CN2023/085612 CN2023085612W WO2024098642A1 WO 2024098642 A1 WO2024098642 A1 WO 2024098642A1 CN 2023085612 W CN2023085612 W CN 2023085612W WO 2024098642 A1 WO2024098642 A1 WO 2024098642A1
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Prior art keywords
monkeypox virus
nucleic acid
reagent
isothermal amplification
mpv
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PCT/CN2023/085612
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French (fr)
Chinese (zh)
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王香玲
卓明辉
张�成
彭成彬
洪阅滨
段雪昆
王露
何燕华
江贤华
张国锋
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厦门宝太生物科技股份有限公司
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Publication of WO2024098642A1 publication Critical patent/WO2024098642A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804Nucleic acid analysis using immunogens
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a reagent for isothermal amplification detection of monkeypox virus and a detection method thereof, belonging to the technical field of virus detection.
  • Monkeypox virus is a double-stranded DNA virus that is a member of the genus Orthopoxvirus in the family Poxviridae. MPV causes disease in humans and many other animals and is considered a zoonosis, primarily transmitted to humans from animals such as rodents and primates. However, host animals that may harbor or transmit MPV are unknown.
  • Transmission of the virus can occur through contact with body fluids, wounds on the skin or internal mucosal surfaces, respiratory droplets, or contaminated objects. Consumption of inadequately cooked meat or other products from infected animals may also increase the risk of infection. Vertical transmission from mother to fetus through the placenta or close contact during or after birth has been documented. The prevalence of asymptomatic transmission is currently unknown. These modes of transmission place health workers, family members, or other close contacts at increased risk of infection.
  • monkeypox has been reassessed as an emerging public health threat because the frequency and geographic distribution of cases have increased, particularly in West Africa, where contact between susceptible people and infected animals is increasing due to factors such as deforestation and insecurity.
  • Increased human-to-human transmission of monkeypox may be attributed in part to the cessation of smallpox vaccination campaigns, as smallpox immunity provides some cross-protection against monkeypox.
  • climate change and increased population mobility are also potential factors contributing to increased human-to-human transmission.
  • the median age of cases has also increased in recent years. Plus, from children to young people.
  • PCR polymerase chain reaction
  • antigen and antibody serological tests may give false positive results due to serological cross-reactions between orthopoxviruses and people who have recently or previously received smallpox vaccination; the expertise and equipment required for PCR, electron microscopy, and immunohistochemistry limit their feasibility in rural or resource-poor areas without large laboratories; and these methods take a long time to detect, with test results available within 1 to 2 hours.
  • the present invention provides a reagent for detecting monkeypox virus by isothermal amplification and a detection method thereof, which can effectively solve the above-mentioned problems.
  • the present invention is achieved in that:
  • a method for isothermal amplification detection of monkeypox virus comprising the following steps:
  • S1 extracting monkeypox virus nucleic acid
  • the nucleic acid extraction reagents used include hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water;
  • step S2 designing primers and immunochromatographic probes, and applying enhanced recombinase-dependent amplification technology to amplify the monkeypox virus nucleic acid extracted in step S1; the 5' end of one of the primers is modified with biotin, The 5' end of the immunochromatographic probe is modified with FAM;
  • the ratios of hydrochloric acid, acetic acid, sodium chloride, Tween 20 and Tritonx-100 are 10-30 mM, 10-20 mM, 50-100 mM, 0.05%-1 v/v%, and 0.05%-1 v/v%.
  • the reagent used in the enhanced recombinase-dependent amplification technology is a lyophilized reagent
  • the lyophilized reagent includes the primer, the immunochromatographic probe, a lyophilized buffer and a lyophilized enzyme mixture.
  • the primers include MPV-eRDA-F and MPV-eRDA-R, and their base sequences are respectively shown in SEQ ID NO:2-SEQ ID NO:3;
  • the immunochromatography probe includes MPV-eRDA-P, and its base sequences are respectively shown in SEQ ID NO:4.
  • the lyophilization buffer comprises Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate.
  • the freeze-dried enzyme mixture includes creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo).
  • a nucleic acid extraction reagent comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, Triton x-100 and water.
  • a nucleic acid extraction method comprises adding a sample to the nucleic acid extraction reagent, stirring the sample and then allowing the sample to stand.
  • a pair of primers for isothermal amplification detection of monkeypox virus including MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO:2-SEQ ID NO:3, respectively.
  • a freeze-dried reagent for isothermal amplification detection of monkeypox virus comprises primers for enhanced recombinase-dependent amplification, immunochromatographic probes, freeze-dried buffer and freeze-dried enzyme mixture.
  • the rapid nucleic acid extraction reagent used in the method for detecting monkeypox virus by isothermal amplification of the present invention is a hands-free lysis solution, and there is no cumbersome operation of the conventional magnetic bead extraction method, such as lysis magnetic absorption, washing, drying, elution and the like. Only one step is needed for extracting nucleic acid from the sample, that is, adding the sample to be tested into the nucleic acid extraction reagent, stirring and standing to complete the extraction of the sample nucleic acid.
  • the method has the advantages of simple operation, high efficiency and rapidity.
  • the primers designed for the method for detecting monkeypox virus by warm amplification of the present invention have good specificity and high sensitivity.
  • the detection reagent is a freeze-dried reagent, which has the characteristics of easy storage, easy transportation, good stability, etc.
  • the detection time of the sample by the detection reagent is short, that is, the sample detection can be completed within 25 minutes, which is of great significance for the screening of imported diseases at ports and meets the current needs of health and epidemic prevention.
  • FIG. 1 is a comparison diagram of the nucleic acid extraction reagent provided in an embodiment of the present invention and the Tiangen magnetic bead extraction reagent.
  • FIG. 2 is an appearance diagram of a freeze-dried reagent for isothermal amplification detection of monkeypox virus nucleic acid provided in an embodiment of the present invention.
  • FIG3 is a performance test diagram of a freeze-dried reagent for isothermal amplification detection of monkeypox virus nucleic acid provided by an embodiment of the present invention.
  • FIG. 4 is a sensitivity test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided in an embodiment of the present invention.
  • FIG. 5 is a reaction temperature test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided by an embodiment of the present invention.
  • FIG6 is a specificity test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided in an embodiment of the present invention.
  • the present invention provides a method for isothermal amplification detection of monkeypox virus, comprising the following steps:
  • S1 extracting monkeypox virus nucleic acid
  • the nucleic acid extraction reagents used include hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water;
  • step S2 designing primers and immunochromatographic probes, and applying enhanced recombinase-dependent amplification technology to amplify the monkeypox virus nucleic acid extracted in step S1; the 5' end of one of the primers is modified with biotin, and the 5' end of the immunochromatographic probe is modified with FAM;
  • the immunochromatography analysis is to drop the monkeypox virus nucleic acid solution amplified in step S2 onto the immunochromatography test paper, react for a period of time, and if the T line on the immunochromatography test paper shows a red band, it is determined that there is monkeypox virus, otherwise, there is no monkeypox virus.
  • the nucleic acid extraction reagent used in this method includes hydrochloric acid and acetic acid as protein denaturants, Tween 20 as a surfactant, and Tritonx-100 as a nucleic acid protective agent and nucleic acid binding agent.
  • This nucleic acid extraction reagent abandons the traditional method of using guanidine salt and SDS to lyse and release nucleic acids, but uses hydrochloric acid and acetic acid to lyse cell denatured proteins to release nucleic acids, and uses Tritonx-100 to bind and protect nucleic acids to prevent nucleic acids from being degraded.
  • the nucleic acid residues lysed by this method have a positive effect on The inhibitory effect of subsequent nucleic acid amplification reagents is very small, which is conducive to the subsequent detection of nucleic acids.
  • the use of this nucleic acid extraction reagent does not require the cumbersome operations of conventional magnetic bead extraction methods, such as lysis magnetic absorption, washing, drying, elution and other steps.
  • the nucleic acid extraction of the sample only requires one step, that is, adding the sample to be tested to the nucleic acid extraction reagent, stirring and standing to complete the extraction of the sample nucleic acid. It has the advantages of simple operation, high efficiency and speed.
  • the present invention uses enhanced recombinase dependent amplification (eRDA) technology for the first time to detect monkeypox virus.
  • eRDA enhanced recombinase dependent amplification
  • This technology is based on the nucleic acid sequence of the target gene of the monkeypox virus, and designs specific eRDA primers. Under constant temperature conditions, the primers use the recombinase chain displacement function to efficiently and specifically bind to the template complementary pairing and realize rapid amplification of the template under the action of DNA polymerase.
  • the immunochromatographic probe specifically binds to the amplified template under the chain displacement function of the recombinase, and is specifically cut from the THF site of the probe under the action of the nuclease, so that the probe 3'OH is released and becomes a specific primer, which reacts with the primer modified with biotin to produce a complex containing FAM-biotin, which can specifically bind to the T line on the immunochromatographic test strip to display a red band. If no specific amplification occurs, the FAM-biotin complex will not be formed, and the red band will not be displayed on the T line, thereby completing the detection.
  • the principle of this detection has low instrument requirements, and only a simple constant temperature heater is required, which is cheaper than the fluorescent quantitative PCR instrument required for qPCR.
  • the result detection is displayed on the flow chromatography test strip, so the result can be intuitively judged, and the result visualization is high.
  • the embodiment of the present invention uses a nucleic acid extraction reagent to extract nucleic acid, and the extraction takes only 4 to 6 minutes.
  • the technical means is the enhanced recombinant constant temperature amplification technology eRDA, which can be completed in only 8 to 12 minutes.
  • the result is interpreted by the flow chromatography test strip, and the result can be obtained in 8 to 12 minutes. That is, the entire test only takes 20 to 30 minutes to complete the test from sample input to result output, which is faster than ordinary qPCR (3 to 4 hours to get the result) and LAMP technology (90 minutes to get the result), and is particularly suitable for entry-exit inspection and quarantine inspection work requirements.
  • the detection sensitivity of the embodiment of the present invention is high, and the detection limit can be up to 25 copies/rxn.
  • the method of the embodiment of the present invention is mainly used for the detection of monkeypox virus for the purpose of laboratory scientific research.
  • the ratio of hydrochloric acid, acetic acid, sodium chloride, Tween 20, and Tritonx-100 is 10-30 mM, 10-20 mM, 50-100 mM, 0.05%-1 v/v%, and 0.05%-1 v/v%. Within this range, the nucleic acid extraction efficiency is the highest.
  • the reagent used in the enhanced recombinase-dependent amplification technology is a lyophilized reagent, which includes the primer, the immunochromatographic probe, a lyophilized buffer and a lyophilized enzyme mixture.
  • the lyophilized reagent has the advantages of easy storage, easy transportation and good stability, thereby improving the detection efficiency.
  • the primers include MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO: 2-SEQ ID NO: 3 respectively;
  • the immunochromatographic probe includes MPV-eRDA-P, whose base sequences are shown in SEQ ID NO: 4 respectively.
  • This primer is designed based on the gene fragment of the standard of monkeypox virus, suitable for enhanced recombinase dependent amplification (enhanced recombinase dependent amplification, eRDA) of the gene of monkeypox virus, and has good primer specificity and high sensitivity.
  • the lyophilization buffer comprises Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate.
  • the ratios of Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate in the lyophilization buffer are 100-150 mM, 10-14 mM, 2%-5% (w/v), 12%-16% (w/v), 1%-10% (w/v), 30-50 ⁇ M, 10 mM, 300-420 mM, and 30-40 mM.
  • the freeze-dried enzyme mixture includes creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo).
  • the ratios of creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo) in the freeze-dried enzyme mixture are 130-180 ng/ ⁇ L, 200-250 ng/ ⁇ L, 25-60 ng/ ⁇ L, 500-750 ng/ ⁇ L, 100-200 ng/ ⁇ L, and 20-50 ng/ ⁇ L.
  • the temperature of the amplification reaction of the enhanced recombinase-dependent amplification technology is 40-44°C.
  • a nucleic acid extraction reagent comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water.
  • a nucleic acid extraction method comprises adding a sample to the nucleic acid extraction reagent, stirring and then standing.
  • the standing time is 4 to 6 minutes.
  • a pair of primers for isothermal amplification detection of monkeypox virus including MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO:2-SEQ ID NO:3, respectively.
  • a freeze-dried reagent for isothermal amplification detection of monkeypox virus comprises primers for enhanced recombinase-dependent amplification, immunochromatographic probes, freeze-dried buffer and freeze-dried enzyme mixture.
  • the following raw materials 20mM HCL, 15mM acetic acid, 80mM NaCl, 0.85% by volume of Tween20, 0.65% by volume of Tritonx-100 and purified water are mixed evenly to obtain a rapid nucleic acid extraction reagent, which is dispensed into extraction tubes at a volume of 500 ⁇ L per person and sealed.
  • the gene fragment of the standard product of artificially synthesized monkeypox virus, the base sequence of the gene fragment is:
  • the gene fragment was cloned into the PGEM-T easy vector to obtain a recombinant plasmid vector (target gene vector).
  • the recombinant plasmid was extracted with the nucleic acid extraction reagent prepared in Example 1 of the present invention and the Tiangen magnetic bead extraction reagent at the same time.
  • the extracted DNA was diluted and then detected with the monkeypox virus nucleic acid detection kit (PCR-fluorescence method) of Shengxiang Biotechnology Co., Ltd.
  • the detection results are shown in Figure 1. The results show that the extraction efficiency of the nucleic acid rapid extraction reagent is slightly lower than that of the Tiangen magnetic bead extraction reagent, but within the 1Ct value, that is, the efficiency of the two extraction methods is not much different.
  • the prepared reagents were dispensed into eight tubes at 40 ⁇ L per person, opened and placed in a freeze dryer for freeze drying.
  • the freeze-dried reagents were capped and sealed in an environment with a humidity below 30% and a temperature below 25°C, and the reagent cards were sealed, and the sealed reagents were stored at 2-8°C.
  • primer F/R/probe design of primer F/R/probe is as follows:
  • MPV-eRDA-F Biotin-5-AAGACTTATTATCCTCTCTCATTGATTTTTCGC (SEQ ID NO: 2), in which the 5'-end modification group is biotin;
  • MPV-eRDA-R AACGTAGTACTATGGTGTACAGTTCCGAC (SEQ ID NO:3);
  • the sequence of the immunochromatographic probe is as follows:
  • the appearance of the lyophilized reagent was observed.
  • the lyophilized reagent had a smooth surface, adhered to the tube wall and was not easy to fall off.
  • the result is shown in Figure 2, that is, the lyophilized reagent met the requirements.
  • the lyophilized reagent was reconstituted with 40 ⁇ L of 100 cpsies and 50 cpsies DNA templates (target gene vectors prepared in Example 1), respectively, and the reagent was turned upside down 10 times and centrifuged on a handheld centrifuge. The centrifuged reagent was amplified at 42°C for 10 minutes. After the amplification, a chromatography test was performed. From the results in Figure 3, it can be seen that the lyophilized reagent of the present invention has good performance.
  • the method for isothermal amplification detection of monkeypox virus is as follows:
  • the extraction tube Open the nozzle of the extraction tube, put the collected swab containing the sample to be tested (the sample includes the lesion rash, acne surface or exudate, etc.) into the extraction tube and stir for 15 circles, and discard the swab; or add 5 ⁇ L of liquid sample such as whole blood or serum, cover the nozzle cover of the extraction tube on the extraction tube after adding the sample, mix well, and stand at room temperature for 5 minutes.
  • the extraction tube is filled with the nucleic acid extraction reagent prepared in Example 1 (extraction reagent 500 ⁇ L/tube/person).
  • Sample loading Add 2 drops (40 ⁇ L) of the sample extracted from the upper speed to the MPV freeze-dried reagent prepared in Example 2 (1 test is 1 reaction well), and shake up and down 10 times.
  • Step 4 Result detection:
  • the chromatography test strip results are analyzed. In the chromatography results, if the C line has a band, if the T line has no band, there is no monkeypox virus; if the T line has a band, there is monkeypox virus.
  • the PGEM-T easy plasmid (the target gene vector prepared in Example 1) containing the target gene fragment was cloned as a positive sample standard DNA template for sensitivity test: a positive template of known concentration was taken and diluted with TE gradient to 10cpsies/rxn, 25cpsies/rxn, 50cpsies/rxn, and 100cpsies/rxn. 40 ⁇ L of each concentration gradient was added to the system, and each concentration was repeated 2 times. The test was performed according to the method of Example 3, and the test results are shown in Figure 4. As can be seen from the results, the method of the embodiment of the present invention can stably detect plasmid DNA of 25cpsies/rxn.
  • the PGEM-T easy plasmid (the target gene vector prepared in Example 1) containing the cloned target gene fragment was used as the positive sample standard DNA template.
  • 40 ⁇ L of 100 cpsies/rxn DNA template was tested according to the method of Example 3. The test temperature was set to 37°C, 42°C, and 45°C, the reaction time was 10 min, and an immunochromatography test was performed after the reaction. Two replicates were performed at each temperature. The test results are shown in Figure 5. The results show that the efficiency is not much different at the reaction temperature of 37-45°C, but the chromaticity is most stable at 42°C overall.
  • the PGEM-T easy plasmid (target gene vector prepared in Example 1) containing the target gene fragment cloned therein was used as a positive sample standard and tested and analyzed with varicella-zoster virus, rubella virus, herpes simplex virus, vaccinia virus, vaccinia virus, Streptococcus pyogenes, etc. according to the method of Example 3 of the present invention, and the results are shown in Figure 6. It can be seen that only the T line of the positive sample standard is colored, and the T lines of the other viruses are not colored. The results show that the method of the embodiment of the present invention has good detection specificity.

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Abstract

The present invention provides a reagent for detecting a monkeypox virus by means of constant-temperature amplification and a method for detecting same. The method comprises the following steps: S1, extracting a nucleic acid of the monkey poxvirus, wherein a nucleic acid extraction reagent used comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, TritonX-100, and water; S2, designing primers and an immunochromatography probe, and amplifying the nucleic acid of the monkeypox virus extracted in the step S1 with an enhanced recombinase-dependent amplification technology, wherein the 5' terminus of one of the primers modifies a biotin, and the 5' terminus of the immunochromatography probe modifies FAM; and S3, analyzing an immunochromatography result and determining whether the monkeypox virus exists or not.

Description

一种恒温扩增检测猴痘病毒的试剂及其检测方法A reagent for isothermal amplification detection of monkeypox virus and a detection method thereof 技术领域Technical Field
本发明涉及一种恒温扩增检测猴痘病毒的试剂及其检测方法,属于病毒检测技术领域。The invention relates to a reagent for isothermal amplification detection of monkeypox virus and a detection method thereof, belonging to the technical field of virus detection.
背景技术Background technique
猴痘病毒(MPV)是一种双链DNA病毒,是痘病毒科正痘病毒属的一员。猴痘病毒在人类和许多其他动物中引起疾病,被认为是一种人畜共患病,主要从啮齿动物和灵长类动物等动物传播给人类。然而,可能携带或传播猴痘病毒的寄主动物尚不清楚。Monkeypox virus (MPV) is a double-stranded DNA virus that is a member of the genus Orthopoxvirus in the family Poxviridae. MPV causes disease in humans and many other animals and is considered a zoonosis, primarily transmitted to humans from animals such as rodents and primates. However, host animals that may harbor or transmit MPV are unknown.
病毒的传播可通过接触体液、皮肤或内部粘膜表面的伤口、呼吸道飞沫或受污染的物体而发生。食用来自受感染动物的未充分煮熟的肉类或其他产品也可能增加感染风险。已记录通过胎盘或出生期间或出生后的密切接触从母亲到胎儿的垂直传播。目前尚不清楚无症状传播的流行率。这些传播方式使卫生工作者、家庭成员或其他密切接触者的感染风险增加。Transmission of the virus can occur through contact with body fluids, wounds on the skin or internal mucosal surfaces, respiratory droplets, or contaminated objects. Consumption of inadequately cooked meat or other products from infected animals may also increase the risk of infection. Vertical transmission from mother to fetus through the placenta or close contact during or after birth has been documented. The prevalence of asymptomatic transmission is currently unknown. These modes of transmission place health workers, family members, or other close contacts at increased risk of infection.
近年来,猴痘已被重新评估为一种新兴的公共卫生威胁,因为病例的频率和地理分布有所增加,特别是在西非,由于森林砍伐和不安全等因素,易感人群与受感染动物之间的接触正在增加。人与人之间的猴痘传播增加可能部分归因于天花疫苗接种活动的停止,因为天花免疫提供了一些针对猴痘的交叉保护。气候变化和人口流动性增加也是导致人际传播增加的潜在因素。近年来,病例的中位年龄也有所增 加,从儿童到年轻人。In recent years, monkeypox has been reassessed as an emerging public health threat because the frequency and geographic distribution of cases have increased, particularly in West Africa, where contact between susceptible people and infected animals is increasing due to factors such as deforestation and insecurity. Increased human-to-human transmission of monkeypox may be attributed in part to the cessation of smallpox vaccination campaigns, as smallpox immunity provides some cross-protection against monkeypox. Climate change and increased population mobility are also potential factors contributing to increased human-to-human transmission. The median age of cases has also increased in recent years. Plus, from children to young people.
由于猴痘和水痘的临床症状相似,医疗保健提供者往往难以仅根据临床症状诊断病例。通常根据临床表现和疾病进展进行推测性诊断。测试方法有:病毒DNA的聚合酶链式反应(PCR)检测、抗原和抗体血清检测、电子显微镜和免疫组织化学检测等。Because the clinical symptoms of monkeypox and varicella are similar, it is often difficult for healthcare providers to diagnose cases based on clinical symptoms alone. Diagnosis is usually presumptive based on clinical presentation and disease progression. Testing methods include polymerase chain reaction (PCR) testing for viral DNA, serum testing for antigens and antibodies, electron microscopy, and immunohistochemistry.
技术问题technical problem
然而,这些方法存在以下缺点:抗原和抗体血清检测会由于正痘病毒之间的血清学交叉反应以及最近或以前接种过天花疫苗的人可能出现假阳性结果;PCR、电子显微镜和免疫组织化学检测所需的专业知识和设备,限制了它们在没有大型实验室的农村或资源匮乏地区的可行性;同时这些方法检测时间长,在1~2h内才能得到检测结果。However, these methods have the following disadvantages: antigen and antibody serological tests may give false positive results due to serological cross-reactions between orthopoxviruses and people who have recently or previously received smallpox vaccination; the expertise and equipment required for PCR, electron microscopy, and immunohistochemistry limit their feasibility in rural or resource-poor areas without large laboratories; and these methods take a long time to detect, with test results available within 1 to 2 hours.
技术解决方案Technical Solutions
本发明提供了一种恒温扩增检测猴痘病毒的试剂及其检测方法,可以有效解决上述问题。The present invention provides a reagent for detecting monkeypox virus by isothermal amplification and a detection method thereof, which can effectively solve the above-mentioned problems.
本发明是这样实现的:The present invention is achieved in that:
一种恒温扩增检测猴痘病毒的方法,包括以下步骤:A method for isothermal amplification detection of monkeypox virus, comprising the following steps:
S1,提取猴痘病毒核酸,所用的核酸提取试剂包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水;S1, extracting monkeypox virus nucleic acid, the nucleic acid extraction reagents used include hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water;
S2,设计引物和免疫层析探针,应用增强型重组酶依赖扩增技术扩增步骤S1提取的猴痘病毒核酸;所述引物之一的5’端修饰生物素, 所述免疫层析探针的5’端修饰FAM;S2, designing primers and immunochromatographic probes, and applying enhanced recombinase-dependent amplification technology to amplify the monkeypox virus nucleic acid extracted in step S1; the 5' end of one of the primers is modified with biotin, The 5' end of the immunochromatographic probe is modified with FAM;
S3,进行免疫层析结果分析,判断是否有猴痘病毒。S3, analyze the immunochromatography results to determine whether monkeypox virus is present.
作为进一步改进的,所述盐酸、乙酸、氯化钠、吐温20、Tritonx-100的比例为10~30mM、10~20mM、50~100mM、0.05%~1v/v%、0.05%~1v/v%。As a further improvement, the ratios of hydrochloric acid, acetic acid, sodium chloride, Tween 20 and Tritonx-100 are 10-30 mM, 10-20 mM, 50-100 mM, 0.05%-1 v/v%, and 0.05%-1 v/v%.
作为进一步改进的,所述增强型重组酶依赖扩增技术所使用的试剂为冻干试剂,所述冻干试剂包括所述引物、所述免疫层析探针、冻干缓冲液和冻干酶混合物。As a further improvement, the reagent used in the enhanced recombinase-dependent amplification technology is a lyophilized reagent, and the lyophilized reagent includes the primer, the immunochromatographic probe, a lyophilized buffer and a lyophilized enzyme mixture.
作为进一步改进的,所述引物包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示;所述免疫层析探针包括MPV-eRDA-P,其碱基序列分别如SEQ ID NO:4。As a further improvement, the primers include MPV-eRDA-F and MPV-eRDA-R, and their base sequences are respectively shown in SEQ ID NO:2-SEQ ID NO:3; the immunochromatography probe includes MPV-eRDA-P, and its base sequences are respectively shown in SEQ ID NO:4.
作为进一步改进的,所述冻干缓冲液包括Tris-HCl缓冲液、MgAc、聚环氧乙烷、甘露醇、海藻糖、甜菜碱、ATP、dNTPs、四水磷酸肌酸钠。As a further improvement, the lyophilization buffer comprises Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate.
作为进一步改进的,所述冻干酶混合物包括肌酸激酶CKM、重组酶UvsX、重组酶加载因子UvsY、单链结合蛋白GP32、DNA聚合酶、核酸内切酶IV(Nfo)。As a further improvement, the freeze-dried enzyme mixture includes creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo).
一种核酸提取试剂,包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水。 A nucleic acid extraction reagent comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, Triton x-100 and water.
一种核酸提取方法,将样品加入上述的核酸提取试剂中,搅拌后静置即可。A nucleic acid extraction method comprises adding a sample to the nucleic acid extraction reagent, stirring the sample and then allowing the sample to stand.
一种恒温扩增检测猴痘病毒的引物,包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示。A pair of primers for isothermal amplification detection of monkeypox virus, including MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO:2-SEQ ID NO:3, respectively.
一种恒温扩增检测猴痘病毒的冻干试剂,包括增强型重组酶依赖扩增的引物、免疫层析探针、冻干缓冲液和冻干酶混合物。A freeze-dried reagent for isothermal amplification detection of monkeypox virus comprises primers for enhanced recombinase-dependent amplification, immunochromatographic probes, freeze-dried buffer and freeze-dried enzyme mixture.
有益效果Beneficial Effects
本发明的有益效果是:The beneficial effects of the present invention are:
本发明的恒温扩增检测猴痘病毒的方法采用的快速核酸提取试剂为免提裂解液,无繁琐的常规磁珠提取法的操作,如裂解磁吸、洗涤、干燥、洗脱等步骤,对样本的核酸提取只需要一步,即将含待测样本加入核酸提取试剂,搅拌静置就能完成样本核酸的提取,具有操作简便,高效、快速的优点。The rapid nucleic acid extraction reagent used in the method for detecting monkeypox virus by isothermal amplification of the present invention is a hands-free lysis solution, and there is no cumbersome operation of the conventional magnetic bead extraction method, such as lysis magnetic absorption, washing, drying, elution and the like. Only one step is needed for extracting nucleic acid from the sample, that is, adding the sample to be tested into the nucleic acid extraction reagent, stirring and standing to complete the extraction of the sample nucleic acid. The method has the advantages of simple operation, high efficiency and rapidity.
本发明温扩增检测猴痘病毒的方法设计的引物特异性好,灵敏度高,同时检测试剂为冻干试剂,有着易保存、易运输、稳定性好等特点,该检测试剂对样本的检测时间短,即25min内就能完成样本的检测,对口岸输入性疾病筛查有重要意义,满足当前卫生防疫的需求。The primers designed for the method for detecting monkeypox virus by warm amplification of the present invention have good specificity and high sensitivity. At the same time, the detection reagent is a freeze-dried reagent, which has the characteristics of easy storage, easy transportation, good stability, etc. The detection time of the sample by the detection reagent is short, that is, the sample detection can be completed within 25 minutes, which is of great significance for the screening of imported diseases at ports and meets the current needs of health and epidemic prevention.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施方式的技术方案,下面将对实施方式中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域 普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope of the present invention. For ordinary technicians, other related drawings can be obtained based on these drawings without any creative work.
图1是本发明实施例提供的核酸提取试剂与天根磁珠提取试剂的对比图。FIG. 1 is a comparison diagram of the nucleic acid extraction reagent provided in an embodiment of the present invention and the Tiangen magnetic bead extraction reagent.
图2是本发明实施例提供的恒温扩增检测猴痘病毒核酸检测冻干试剂外观图。FIG. 2 is an appearance diagram of a freeze-dried reagent for isothermal amplification detection of monkeypox virus nucleic acid provided in an embodiment of the present invention.
图3是本发明实施例提供的恒温扩增检测猴痘病毒核酸检测冻干试剂的性能测试图。FIG3 is a performance test diagram of a freeze-dried reagent for isothermal amplification detection of monkeypox virus nucleic acid provided by an embodiment of the present invention.
图4是本发明实施例提供的恒温扩增检测猴痘病毒核酸检测试剂的灵敏度测试图。FIG. 4 is a sensitivity test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided in an embodiment of the present invention.
图5是本发明实施例提供的恒温扩增检测猴痘病毒核酸检测试剂的反应温度测试图。FIG. 5 is a reaction temperature test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided by an embodiment of the present invention.
图6是本发明实施例提供的恒温扩增检测猴痘病毒核酸检测试剂的特异性测试图。FIG6 is a specificity test diagram of a isothermal amplification detection reagent for monkeypox virus nucleic acid provided in an embodiment of the present invention.
本发明的实施方式Embodiments of the present invention
为使本发明实施方式的目的、技术方案和优点更加清楚,下面将结合本发明实施方式中的附图,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式, 都属于本发明保护的范围。因此,以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work, All of them belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts belong to the scope of protection of the present invention.
本发明提供一种恒温扩增检测猴痘病毒的方法,包括以下步骤:The present invention provides a method for isothermal amplification detection of monkeypox virus, comprising the following steps:
S1,提取猴痘病毒核酸,所用的核酸提取试剂包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水;S1, extracting monkeypox virus nucleic acid, the nucleic acid extraction reagents used include hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water;
S2,设计引物和免疫层析探针,应用增强型重组酶依赖扩增技术扩增步骤S1提取的猴痘病毒核酸;所述引物之一的5’端修饰生物素,所述免疫层析探针的5’端修饰FAM;S2, designing primers and immunochromatographic probes, and applying enhanced recombinase-dependent amplification technology to amplify the monkeypox virus nucleic acid extracted in step S1; the 5' end of one of the primers is modified with biotin, and the 5' end of the immunochromatographic probe is modified with FAM;
S3,进行免疫层析结果分析,判断是否有猴痘病毒。所述免疫层析结果分析是将步骤S2扩增的猴痘病毒核酸溶液滴加到免疫层析试纸上,反应一段时间,若免疫层析试纸条上的T线显示红色条带,则判断有猴痘病毒,反之,则无猴痘病毒。S3, performing immunochromatography analysis to determine whether there is monkeypox virus. The immunochromatography analysis is to drop the monkeypox virus nucleic acid solution amplified in step S2 onto the immunochromatography test paper, react for a period of time, and if the T line on the immunochromatography test paper shows a red band, it is determined that there is monkeypox virus, otherwise, there is no monkeypox virus.
本方法中所用的核酸提取试剂,盐酸和乙酸为蛋白变性剂,吐温20为表面活性剂,Tritonx-100为核酸保护剂及核酸结合剂,该核酸提取试剂舍弃了传统的用胍盐和SDS裂解释放核酸的方法,而是采用盐酸和乙酸裂解细胞变性蛋白的方法来释放核酸,并用Tritonx-100结合核酸和保护核酸,避免核酸被降解。该方法裂解的核酸残留物对 后续核酸扩增试剂的抑制性很小,利于后续核酸的检测,同时,采用该核酸提取试剂无繁琐的常规磁珠提取法的操作,如裂解磁吸、洗涤、干燥、洗脱等步骤,对样本的核酸提取只需要一步,即将含待测样本加入核酸提取试剂,搅拌静置就能完成样本核酸的提取,具有操作简便,高效、快速的优点。The nucleic acid extraction reagent used in this method includes hydrochloric acid and acetic acid as protein denaturants, Tween 20 as a surfactant, and Tritonx-100 as a nucleic acid protective agent and nucleic acid binding agent. This nucleic acid extraction reagent abandons the traditional method of using guanidine salt and SDS to lyse and release nucleic acids, but uses hydrochloric acid and acetic acid to lyse cell denatured proteins to release nucleic acids, and uses Tritonx-100 to bind and protect nucleic acids to prevent nucleic acids from being degraded. The nucleic acid residues lysed by this method have a positive effect on The inhibitory effect of subsequent nucleic acid amplification reagents is very small, which is conducive to the subsequent detection of nucleic acids. At the same time, the use of this nucleic acid extraction reagent does not require the cumbersome operations of conventional magnetic bead extraction methods, such as lysis magnetic absorption, washing, drying, elution and other steps. The nucleic acid extraction of the sample only requires one step, that is, adding the sample to be tested to the nucleic acid extraction reagent, stirring and standing to complete the extraction of the sample nucleic acid. It has the advantages of simple operation, high efficiency and speed.
本发明实施例首次用增强型重组酶依赖扩增技术(enhanced recombinase dependent amplification,eRDA)技术对猴痘病毒的检测。该技术是根据猴痘病毒的目的基因靶基因的核酸序列,设计特异性的eRDA引物,在恒温条件下,引物利用重组酶链置换功能高效并特异性的与模板互补配对的结合并在DNA聚合酶作用下,实现模板的快速扩增。The present invention uses enhanced recombinase dependent amplification (eRDA) technology for the first time to detect monkeypox virus. This technology is based on the nucleic acid sequence of the target gene of the monkeypox virus, and designs specific eRDA primers. Under constant temperature conditions, the primers use the recombinase chain displacement function to efficiently and specifically bind to the template complementary pairing and realize rapid amplification of the template under the action of DNA polymerase.
本发明实施例中,免疫层析探针在重组酶的链置换功能下特异性地结合扩增的模板,并在核酸内切酶的作用下特异性地从探针的THF位点剪切,使得探针3’OH释放,变为特异性的引物,与修饰有生物素的引物反应,产生含有FAM-生物素的复合体,可以与免疫层析试纸条上的T线特异性的结合显示红色条带,未发生特异性扩增则不会形成FAM-生物素的复合体,则不会在T线上显示红色条带,从而完成检测。该检测的原理对仪器要求低,仅需一个简单的恒温加热器即可,比qPCR所需的荧光定量PCR仪便宜,同时结果检测为测流层析试纸显示,因此可以直观的判读结果,结果可视化程度高。In the embodiment of the present invention, the immunochromatographic probe specifically binds to the amplified template under the chain displacement function of the recombinase, and is specifically cut from the THF site of the probe under the action of the nuclease, so that the probe 3'OH is released and becomes a specific primer, which reacts with the primer modified with biotin to produce a complex containing FAM-biotin, which can specifically bind to the T line on the immunochromatographic test strip to display a red band. If no specific amplification occurs, the FAM-biotin complex will not be formed, and the red band will not be displayed on the T line, thereby completing the detection. The principle of this detection has low instrument requirements, and only a simple constant temperature heater is required, which is cheaper than the fluorescent quantitative PCR instrument required for qPCR. At the same time, the result detection is displayed on the flow chromatography test strip, so the result can be intuitively judged, and the result visualization is high.
本发明实施例用核酸提取试剂进行核酸提取,提取仅需4~6min, 技术手段为增强型重组恒温扩增技术eRDA,该技术仅需8~12min就能完成,同时结果的判读用测流层析试纸条,层析8~12min就能得到结果,即整个检测仅需20~30min就能完成从样本进、结果出的检测,比普通qPCR(3~4h拿到结果)和LAMP技术(90min出结果)快速,特别适合出入境检验检疫检验工作需求。本发明实施例的检测灵敏度高,检测限可至25copies/rxn。The embodiment of the present invention uses a nucleic acid extraction reagent to extract nucleic acid, and the extraction takes only 4 to 6 minutes. The technical means is the enhanced recombinant constant temperature amplification technology eRDA, which can be completed in only 8 to 12 minutes. At the same time, the result is interpreted by the flow chromatography test strip, and the result can be obtained in 8 to 12 minutes. That is, the entire test only takes 20 to 30 minutes to complete the test from sample input to result output, which is faster than ordinary qPCR (3 to 4 hours to get the result) and LAMP technology (90 minutes to get the result), and is particularly suitable for entry-exit inspection and quarantine inspection work requirements. The detection sensitivity of the embodiment of the present invention is high, and the detection limit can be up to 25 copies/rxn.
本发明实施例的方法主要应用于实验室科学研究的目的而进行的猴痘病毒的检测。The method of the embodiment of the present invention is mainly used for the detection of monkeypox virus for the purpose of laboratory scientific research.
作为进一步改进的,所述盐酸、乙酸、氯化钠、吐温20、Tritonx-100的比例为10~30mM、10~20mM、50~100mM、0.05%~1v/v%、0.05%~1v/v%。在此范围内,核酸提取效率最高。As a further improvement, the ratio of hydrochloric acid, acetic acid, sodium chloride, Tween 20, and Tritonx-100 is 10-30 mM, 10-20 mM, 50-100 mM, 0.05%-1 v/v%, and 0.05%-1 v/v%. Within this range, the nucleic acid extraction efficiency is the highest.
作为进一步改进的,所述增强型重组酶依赖扩增技术所使用的试剂为冻干试剂,所述冻干试剂包括所述引物、所述免疫层析探针、冻干缓冲液和冻干酶混合物。冻干试剂具有易保存、易运输、稳定性好的优点,提高了检测效率。As a further improvement, the reagent used in the enhanced recombinase-dependent amplification technology is a lyophilized reagent, which includes the primer, the immunochromatographic probe, a lyophilized buffer and a lyophilized enzyme mixture. The lyophilized reagent has the advantages of easy storage, easy transportation and good stability, thereby improving the detection efficiency.
作为进一步改进的,所述引物包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示;所述免疫层析探针包括MPV-eRDA-P,其碱基序列分别如SEQ ID NO:4。此引物为根据猴痘病毒的标准品的基因片段而设计,适用于猴痘病毒的基因的增强型重组酶依赖扩增(enhanced recombinase dependent amplification,eRDA),引物特异性好,灵敏度高。 As a further improvement, the primers include MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO: 2-SEQ ID NO: 3 respectively; the immunochromatographic probe includes MPV-eRDA-P, whose base sequences are shown in SEQ ID NO: 4 respectively. This primer is designed based on the gene fragment of the standard of monkeypox virus, suitable for enhanced recombinase dependent amplification (enhanced recombinase dependent amplification, eRDA) of the gene of monkeypox virus, and has good primer specificity and high sensitivity.
作为进一步改进的,所述冻干缓冲液包括Tris-HCl缓冲液、MgAc、聚环氧乙烷、甘露醇、海藻糖、甜菜碱、ATP、dNTPs、四水磷酸肌酸钠。As a further improvement, the lyophilization buffer comprises Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate.
作为进一步改进的,所述冻干缓冲液中Tris-HCl缓冲液、MgAc、聚环氧乙烷、甘露醇、海藻糖、甜菜碱、ATP、dNTPs、四水磷酸肌酸钠的比例为100~150mM、10~14mM、2%~5%(w/v)、12%~16%(w/v)、1%~10%(w/v)、30~50μM、10mM、300~420mM、30~40mM。As a further improvement, the ratios of Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate in the lyophilization buffer are 100-150 mM, 10-14 mM, 2%-5% (w/v), 12%-16% (w/v), 1%-10% (w/v), 30-50 μM, 10 mM, 300-420 mM, and 30-40 mM.
作为进一步改进的,所述冻干酶混合物包括肌酸激酶CKM、重组酶UvsX、重组酶加载因子UvsY、单链结合蛋白GP32、DNA聚合酶、核酸内切酶IV(Nfo)。As a further improvement, the freeze-dried enzyme mixture includes creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo).
作为进一步改进的,所述冻干酶混合物中肌酸激酶CKM、重组酶UvsX、重组酶加载因子UvsY、单链结合蛋白GP32、DNA聚合酶、核酸内切酶IV(Nfo)的比例为130~180ng/μL、200~250ng/μL、25~60ng/μL、500~750ng/μL、100~200ng/μL、20~50ng/μL。As a further improvement, the ratios of creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and nuclease IV (Nfo) in the freeze-dried enzyme mixture are 130-180 ng/μL, 200-250 ng/μL, 25-60 ng/μL, 500-750 ng/μL, 100-200 ng/μL, and 20-50 ng/μL.
作为进一步改进的,所述增强型重组酶依赖扩增技术扩增反应的温度为40~44℃。As a further improvement, the temperature of the amplification reaction of the enhanced recombinase-dependent amplification technology is 40-44°C.
一种核酸提取试剂,包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水。A nucleic acid extraction reagent comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water.
一种核酸提取方法,将样品加入上述的核酸提取试剂中,搅拌后静置即可。优选的,静置的时间为4~6min。 A nucleic acid extraction method comprises adding a sample to the nucleic acid extraction reagent, stirring and then standing. Preferably, the standing time is 4 to 6 minutes.
一种恒温扩增检测猴痘病毒的引物,包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示。A pair of primers for isothermal amplification detection of monkeypox virus, including MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO:2-SEQ ID NO:3, respectively.
一种恒温扩增检测猴痘病毒的冻干试剂,包括增强型重组酶依赖扩增的引物、免疫层析探针、冻干缓冲液和冻干酶混合物。A freeze-dried reagent for isothermal amplification detection of monkeypox virus comprises primers for enhanced recombinase-dependent amplification, immunochromatographic probes, freeze-dried buffer and freeze-dried enzyme mixture.
实施例1Example 1
快速核酸提取试剂制备Preparation of rapid nucleic acid extraction reagents
将以下原料:20mM的HCL,15mM的乙酸,80mM的NaCl,体积比为0.85%的Tween20,体积比为0.65%的Tritonx-100和纯化水进行混合均匀即可得到快速核酸提取试剂,按每人份500μL分装于提取管中,并封口。The following raw materials: 20mM HCL, 15mM acetic acid, 80mM NaCl, 0.85% by volume of Tween20, 0.65% by volume of Tritonx-100 and purified water are mixed evenly to obtain a rapid nucleic acid extraction reagent, which is dispensed into extraction tubes at a volume of 500 μL per person and sealed.
对比例1Comparative Example 1
核酸提取试剂与天根磁珠提取试剂的对比Comparison between nucleic acid extraction reagent and Tiangen magnetic bead extraction reagent
人工合成猴痘病毒的标准品的基因片段,所述基因片段的碱基序列为:

The gene fragment of the standard product of artificially synthesized monkeypox virus, the base sequence of the gene fragment is:

将该基因片段克隆入PGEM-T easy载体得到的重组质粒载体(目的基因载体)。将重组质粒同时用本发明实施例1制备的核酸提取试剂及天根磁珠提取试剂提取,提取的DNA进行稀释后用圣湘生物公司的猴痘病毒核酸检测试剂盒(PCR-荧光法)进行检测,检测结果如图1所示。结果表明核酸快速提取试剂的提取效率比天根磁珠提取试剂的效率略靠后,但是在1Ct值内,即2种提取方式效率差别不大。The gene fragment was cloned into the PGEM-T easy vector to obtain a recombinant plasmid vector (target gene vector). The recombinant plasmid was extracted with the nucleic acid extraction reagent prepared in Example 1 of the present invention and the Tiangen magnetic bead extraction reagent at the same time. The extracted DNA was diluted and then detected with the monkeypox virus nucleic acid detection kit (PCR-fluorescence method) of Shengxiang Biotechnology Co., Ltd. The detection results are shown in Figure 1. The results show that the extraction efficiency of the nucleic acid rapid extraction reagent is slightly lower than that of the Tiangen magnetic bead extraction reagent, but within the 1Ct value, that is, the efficiency of the two extraction methods is not much different.
实施例2Example 2
恒温扩增检测猴痘病毒的冻干试剂(MPV冻干试剂)的制备Preparation of freeze-dried reagent for isothermal amplification detection of monkeypox virus (MPV freeze-dried reagent)
MPV冻干试剂的配方如下表1所示。The formula of MPV lyophilized reagent is shown in Table 1 below.
表1

Table 1

将上述配好的试剂按40μL每人份分装于八联管中,开盖并放入冻干机中进行冻干。将所得的冻干试剂于湿度30%以下,温度25℃以下的环境下进行封盖,并对试剂卡进行封口,并将密封好的试剂于2-8℃中保存。The prepared reagents were dispensed into eight tubes at 40 μL per person, opened and placed in a freeze dryer for freeze drying. The freeze-dried reagents were capped and sealed in an environment with a humidity below 30% and a temperature below 25°C, and the reagent cards were sealed, and the sealed reagents were stored at 2-8°C.
其中,引物F/R/探针的设计如下:Among them, the design of primer F/R/probe is as follows:
根据人工合成猴痘病毒的标准品的基因片段(SEQ ID NO:1),用Oligo 7设计一套特异性引物,其基因碱基序列:Based on the gene fragment of the standard product of artificially synthesized monkeypox virus (SEQ ID NO: 1), a set of specific primers was designed using Oligo 7, and its gene base sequence is:
MPV-eRDA-F:Biotin-5-AAGACTTATTATCCTCTCTCATTGATTTTTCGC(SEQ ID NO:2),其中5’端修饰基团为生物素(Biotin);MPV-eRDA-F: Biotin-5-AAGACTTATTATCCTCTCTCATTGATTTTTCGC (SEQ ID NO: 2), in which the 5'-end modification group is biotin;
MPV-eRDA-R:AACGTAGTACTATGGTGTACAGTTCCGAC(SEQ ID NO:3);MPV-eRDA-R:AACGTAGTACTATGGTGTACAGTTCCGAC (SEQ ID NO:3);
免疫层析探针的序列如下: The sequence of the immunochromatographic probe is as follows:
MPV-eRDA-P:FAM-5-CAGCATCAGAATCTGTAGGCCGTGTATCAG(THF)CATCCATTGTCGTAG-3-C3(SEQ ID NO:4),其中5’端修饰基团为FAM,3'-末端为阻断剂,用于防止聚合酶从3'-末端延伸;THF为四氢呋喃。MPV-eRDA-P:FAM-5-CAGCATCAGAATCTGTAGGCCGTGTATCAG(THF)CATCCATTGTCGTAG-3-C3 (SEQ ID NO:4), wherein the 5'-end modification group is FAM and the 3'-end is a blocker to prevent the polymerase from extending from the 3'-end; THF is tetrahydrofuran.
冻干试剂外观及检测结果考察:Inspection of freeze-dried reagent appearance and test results:
观察冻干试剂的外形,冻型外观表面平整,与管壁贴合且不易脱落,结果如图2所示,即冻干试剂冻型符合要求。将冻干好的试剂分别用40μL的100cpsies及50cpsies的DNA模板(实施例1制备的目的基因载体)复溶,上下颠倒试剂10次,并于掌上离心机上瞬离,离心好的试剂于42℃扩增10min,扩增结束后进行层析测试,从图3结果可见,本发明的冻干试剂性能良好。The appearance of the lyophilized reagent was observed. The lyophilized reagent had a smooth surface, adhered to the tube wall and was not easy to fall off. The result is shown in Figure 2, that is, the lyophilized reagent met the requirements. The lyophilized reagent was reconstituted with 40 μL of 100 cpsies and 50 cpsies DNA templates (target gene vectors prepared in Example 1), respectively, and the reagent was turned upside down 10 times and centrifuged on a handheld centrifuge. The centrifuged reagent was amplified at 42°C for 10 minutes. After the amplification, a chromatography test was performed. From the results in Figure 3, it can be seen that the lyophilized reagent of the present invention has good performance.
实施例3Example 3
恒温扩增检测猴痘病毒的方法如下:The method for isothermal amplification detection of monkeypox virus is as follows:
步骤一:样本提取:Step 1: Sample extraction:
打开提取管喷嘴,将采集的含待测样本的拭子(样本包括病变皮疹、痘疱表面或渗出物等)放入提取管中搅拌15圈,舍弃拭子;或加入全血或血清等液体样本5μL,加完样本后将提取管的喷嘴盖盖至提取管上,混匀,室温静置5min。提取管中装有实施例1制备的核酸提取试剂(提取试剂500μL/管/人份)。 Open the nozzle of the extraction tube, put the collected swab containing the sample to be tested (the sample includes the lesion rash, acne surface or exudate, etc.) into the extraction tube and stir for 15 circles, and discard the swab; or add 5 μL of liquid sample such as whole blood or serum, cover the nozzle cover of the extraction tube on the extraction tube after adding the sample, mix well, and stand at room temperature for 5 minutes. The extraction tube is filled with the nucleic acid extraction reagent prepared in Example 1 (extraction reagent 500 μL/tube/person).
步骤二:样品制备:Step 2: Sample preparation:
上样:将上速提取完的样本滴2滴(40μL)至实施例2制备的MPV冻干试剂中(1test为1反应孔),上下摇10下。Sample loading: Add 2 drops (40 μL) of the sample extracted from the upper speed to the MPV freeze-dried reagent prepared in Example 2 (1 test is 1 reaction well), and shake up and down 10 times.
步骤三:反应:Step 3: Reaction:
将混匀试剂放入恒温仪中,42℃反应10min。Place the mixed reagent in a thermostat and react at 42°C for 10 min.
步骤四:结果检测:Step 4: Result detection:
反应结束后,进行层析试纸条结果分析,层析结果中,C线均有条带产生,如果T线无条带,则没有猴痘病毒;若T线有条带则有猴痘病毒。After the reaction is completed, the chromatography test strip results are analyzed. In the chromatography results, if the C line has a band, if the T line has no band, there is no monkeypox virus; if the T line has a band, there is monkeypox virus.
实施例4Example 4
灵敏度考察:Sensitivity test:
将克隆含有目的基因片段的PGEM-T easy质粒(实施例1制备的目的基因载体)作为阳性样本标准品DNA模板,做灵敏度考察:取已知浓度的阳性模板,用TE梯度稀释至10cpsies/rxn、25cpsies/rxn、50cpsies/rxn、100cpsies/rxn。各浓度梯度加40μL至体系中,每个浓度各2重复,按照实施例3的方法进行测试,测试结果如图4所示。结果可见,本发明实施例的方法可稳定检出25cpsies/rxn的质粒DNA。The PGEM-T easy plasmid (the target gene vector prepared in Example 1) containing the target gene fragment was cloned as a positive sample standard DNA template for sensitivity test: a positive template of known concentration was taken and diluted with TE gradient to 10cpsies/rxn, 25cpsies/rxn, 50cpsies/rxn, and 100cpsies/rxn. 40μL of each concentration gradient was added to the system, and each concentration was repeated 2 times. The test was performed according to the method of Example 3, and the test results are shown in Figure 4. As can be seen from the results, the method of the embodiment of the present invention can stably detect plasmid DNA of 25cpsies/rxn.
实施例5 Example 5
猴痘病毒核酸检测试剂的反应温度测试:Reaction temperature test of monkeypox virus nucleic acid detection reagent:
比较不同温度下的扩增效率。将克隆含有目的基因片段的PGEM-T easy质粒(实施例1制备的目的基因载体)作为阳性样本标准品DNA模板。将40μL的100cpsies/rxn的DNA模板按照实施例3的方法进行测试,测试温度设置为37℃、42℃、45℃,反应时间10min,反应后进行免疫层析测试。每个温度2个重复。测试结果如图5所示。结果可知,反应温度37~45℃下效率差别不大,但整体42℃下色度最稳定。Compare the amplification efficiency at different temperatures. The PGEM-T easy plasmid (the target gene vector prepared in Example 1) containing the cloned target gene fragment was used as the positive sample standard DNA template. 40 μL of 100 cpsies/rxn DNA template was tested according to the method of Example 3. The test temperature was set to 37°C, 42°C, and 45°C, the reaction time was 10 min, and an immunochromatography test was performed after the reaction. Two replicates were performed at each temperature. The test results are shown in Figure 5. The results show that the efficiency is not much different at the reaction temperature of 37-45°C, but the chromaticity is most stable at 42°C overall.
实施例6Example 6
猴痘病毒核酸检测试剂的特异性测试:Specificity test of monkeypox virus nucleic acid detection reagent:
将克隆含有目的基因片段的PGEM-T easy质粒(实施例1制备的目的基因载体)作为阳性样本标准品与水痘-带状疱疹病毒、风疹病毒、单纯疱疹病毒、痘苗病毒、牛痘病毒、化脓性链球菌等按照本发明实施例3的方法进行测试分析,结果如图6所示。可知,仅阳性样本标准品的T线有显色,其余病毒T线未显色。结果表明本发明实施例的方法具有很好的检测特异性。The PGEM-T easy plasmid (target gene vector prepared in Example 1) containing the target gene fragment cloned therein was used as a positive sample standard and tested and analyzed with varicella-zoster virus, rubella virus, herpes simplex virus, vaccinia virus, vaccinia virus, Streptococcus pyogenes, etc. according to the method of Example 3 of the present invention, and the results are shown in Figure 6. It can be seen that only the T line of the positive sample standard is colored, and the T lines of the other viruses are not colored. The results show that the method of the embodiment of the present invention has good detection specificity.
以上所述仅为本发明的优选实施方式而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。 The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

  1. 一种恒温扩增检测猴痘病毒的方法,其特征在于,包括以下步骤:A method for isothermal amplification detection of monkeypox virus, characterized in that it comprises the following steps:
    S1,提取猴痘病毒核酸,所用的核酸提取试剂包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水;S1, extracting monkeypox virus nucleic acid, the nucleic acid extraction reagents used include hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water;
    S2,设计引物和免疫层析探针,应用增强型重组酶依赖扩增技术扩增步骤S1提取的猴痘病毒核酸;所述引物之一的5’端修饰生物素,所述免疫层析探针的5’端修饰FAM;S2, designing primers and immunochromatographic probes, and applying enhanced recombinase-dependent amplification technology to amplify the monkeypox virus nucleic acid extracted in step S1; the 5' end of one of the primers is modified with biotin, and the 5' end of the immunochromatographic probe is modified with FAM;
    S3,进行免疫层析结果分析,判断是否有猴痘病毒。S3, analyze the immunochromatography results to determine whether monkeypox virus is present.
  2. 根据权利要求1所述恒温扩增检测猴痘病毒的方法,其特征在于,所述盐酸、乙酸、氯化钠、吐温20、Tritonx-100的比例为10~30mM、10~20mM、50~100mM、0.05%~1v/v%、0.05%~1v/v%。The method for isothermal amplification detection of monkeypox virus according to claim 1, characterized in that the ratios of hydrochloric acid, acetic acid, sodium chloride, Tween 20, and Tritonx-100 are 10-30 mM, 10-20 mM, 50-100 mM, 0.05%-1 v/v%, and 0.05%-1 v/v%.
  3. 根据权利要求1所述恒温扩增检测猴痘病毒的方法,其特征在于,所述增强型重组酶依赖扩增技术所使用的试剂为冻干试剂,所述冻干试剂包括所述引物、所述免疫层析探针、冻干缓冲液和冻干酶混合物。The method for detecting monkeypox virus by isothermal amplification according to claim 1 is characterized in that the reagent used in the enhanced recombinase-dependent amplification technology is a lyophilized reagent, and the lyophilized reagent includes the primer, the immunochromatographic probe, a lyophilized buffer and a lyophilized enzyme mixture.
  4. 根据权利要求3所述恒温扩增检测猴痘病毒的方法,其特征在于,所述引物包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示;所述免疫层析探针为MPV-eRDA-P, 其碱基序列分别如SEQ ID NO:4所示。The method for detecting monkeypox virus by isothermal amplification according to claim 3, characterized in that the primers include MPV-eRDA-F and MPV-eRDA-R, whose base sequences are shown in SEQ ID NO:2-SEQ ID NO:3 respectively; the immunochromatographic probe is MPV-eRDA-P, The base sequences thereof are shown in SEQ ID NO: 4.
  5. 根据权利要求3所述恒温扩增检测猴痘病毒的方法,其特征在于,所述冻干缓冲液包括Tris-HCl缓冲液、MgAc、聚环氧乙烷、甘露醇、海藻糖、甜菜碱、ATP、dNTPs、四水磷酸肌酸钠。The method for isothermal amplification detection of monkeypox virus according to claim 3, characterized in that the lyophilization buffer comprises Tris-HCl buffer, MgAc, polyethylene oxide, mannitol, trehalose, betaine, ATP, dNTPs, and sodium creatine phosphate tetrahydrate.
  6. 根据权利要求3所述恒温扩增检测猴痘病毒的方法,其特征在于,所述冻干酶混合物包括肌酸激酶CKM、重组酶UvsX、重组酶加载因子UvsY、单链结合蛋白GP32、DNA聚合酶、核酸内切酶IV(Nfo)。The method for isothermal amplification detection of monkeypox virus according to claim 3, characterized in that the lyophilized enzyme mixture includes creatine kinase CKM, recombinase UvsX, recombinase loading factor UvsY, single-stranded binding protein GP32, DNA polymerase, and endonuclease IV (Nfo).
  7. 一种核酸提取试剂,其特征在于,包括盐酸、乙酸、氯化钠、吐温20、Tritonx-100及水。A nucleic acid extraction reagent, characterized in that it comprises hydrochloric acid, acetic acid, sodium chloride, Tween 20, Tritonx-100 and water.
  8. 一种核酸提取方法,其特征在于,将样品加入权利要求7所述的核酸提取试剂中,搅拌后静置即可。A method for extracting nucleic acid, characterized in that a sample is added to the nucleic acid extraction reagent according to claim 7, stirred and then allowed to stand.
  9. 一种恒温扩增检测猴痘病毒的引物,其特征在于,包括MPV-eRDA-F、MPV-eRDA-R,其碱基序列分别如SEQ ID NO:2-SEQ ID NO:3所示。A primer for isothermal amplification detection of monkeypox virus, characterized in that it includes MPV-eRDA-F and MPV-eRDA-R, and their base sequences are shown in SEQ ID NO:2-SEQ ID NO:3 respectively.
  10. 一种恒温扩增检测猴痘病毒的冻干试剂,其特征在于,包括增强型重组酶依赖扩增的引物、免疫层析探针、冻干缓冲液和冻干酶混合物。 A freeze-dried reagent for isothermal amplification detection of monkeypox virus, characterized in that it comprises primers for enhanced recombinase-dependent amplification, immunochromatographic probes, a freeze-dried buffer and a freeze-dried enzyme mixture.
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