WO2023098620A1 - 免洗涤提取核酸的组合物、试剂盒、方法及用途 - Google Patents

免洗涤提取核酸的组合物、试剂盒、方法及用途 Download PDF

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WO2023098620A1
WO2023098620A1 PCT/CN2022/134688 CN2022134688W WO2023098620A1 WO 2023098620 A1 WO2023098620 A1 WO 2023098620A1 CN 2022134688 W CN2022134688 W CN 2022134688W WO 2023098620 A1 WO2023098620 A1 WO 2023098620A1
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nucleic acid
acid extraction
extraction composition
kit
composition according
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French (fr)
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戴立忠
龙凤英
吴康
任小梅
刘佳
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圣湘生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to the field of molecular biology detection; specifically, to the field of nucleic acid extraction; more specifically, the present invention relates to the field of no-wash drop extraction of nucleic acid.
  • Nucleic acid extraction methods mainly include phenol extraction, alkali lysis, CTAB extraction, boiling, spin column extraction, glass bead adsorption and micronano magnetic bead extraction.
  • the mainstream methods used to extract nucleic acid from COVID-19 samples mainly include spin column extraction and magnetic bead extraction.
  • Spin column nucleic acid extraction uses nucleic acid specific adsorption and repeated washing of impurities, and the purity of the nucleic acid obtained at the end has obvious advantages over traditional methods.
  • the magnetic bead nucleic acid extraction technology has become the mainstream method in the industry due to its high degree of automation, large sample throughput, good nucleic acid concentration and purity, and especially high safety.
  • nucleic acid extraction has become more diverse.
  • ethanol washing system The nucleic acid is released and adsorbed to the magnetic beads to remove the waste liquid through the magnetic rack. , and then washed with different concentrations of ethanol salt solution and ethanol solution for 2 to 3 times to remove organic and inorganic impurities in order to elute and obtain a high-purity nucleic acid solution; Salt solution and silicone oil are used to further remove impurities to obtain a high-concentration nucleic acid solution for PCR detection.
  • nucleic acid extractions cannot avoid the need to wash and remove impurities before proceeding to the next step.
  • washing and removing impurities can be avoided by methods such as extraction-free, the nucleic acid obtained by the extraction-free method coexists with impurities.
  • the main purpose of the present invention is to provide a composition for extracting nucleic acid without washing, which does not need to introduce a washing process when extracting nucleic acid, and further simplifies the extraction process.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, dithiothreitol, glycogen, NP-40 and isopropanol, and further includes:
  • Sorbitol polyethylene glycol, and sodium acetate.
  • the entire extraction kit has no washing liquid and does not need to introduce a washing process, which further simplifies the extraction process and greatly shortens the time required for extracting sample nucleic acid, and the manual operation only takes 12 minutes to complete Nucleic acid extraction; at the same time, the reduction of washing liquid further reduces the reagents and consumables in the kit, thereby reducing the cost of the kit.
  • the composition of the present invention can be applied to throat swabs, nasopharyngeal swabs, alveolar lavage fluid, saliva samples, sputum samples, blood samples and other sample types, and is generally applicable to clinical molecular diagnosis of conventional respiratory infectious diseases.
  • the composition of the present invention can extract human DNA/RNA.
  • Non-human DNA/RNA can also be extracted.
  • the composition of the present invention can extract DNA/RNA of human, bacteria, virus, etc.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, dithiothreitol, glycogen, NP-40, isopropanol, guanidine hydrochloride, urea, chlorine Potassium chloride, and Tween 20.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, dithiothreitol, glycogen, NP-40, isopropanol, sorbitol, polyethylene glycol alcohol, and sodium acetate.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, dithiothreitol, glycogen, NP-40, isopropanol, sorbitol, polyethylene glycol alcohol, sodium acetate, and Tris-HCl.
  • the above nucleic acid extraction composition further includes at least one of the following substances: lithium chloride, Triton X-100.
  • the nucleic acid extraction composition further includes at least one of the following substances: lithium chloride, Triton X-100, simethicone, sodium chloride and Tris-HCl.
  • the above-mentioned nucleic acid extraction composition further includes at least one of the following substances: lithium chloride, Triton X-100, simethicone, sodium chloride, sodium acetate and Tris -HCl.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, dithiothreitol, glycogen, NP-40, isopropanol, guanidine hydrochloride, urea, chlorine Potassium chloride, Tween 20, sodium acetate, lithium chloride, Triton X-100.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, sorbitol, polyethylene glycol, dithiothreitol, sodium acetate, sodium chloride, glycogen , NP-40, Tween 20, Triton X-100, isopropanol, Tris-HCl.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine thiocyanate, sorbitol, polyethylene glycol, dithiothreitol, sodium acetate, glycogen, lithium chloride , sodium chloride, NP-40, isopropanol, simethicone.
  • the present invention provides a nucleic acid extraction composition, which includes: guanidine hydrochloride, guanidine thiocyanate, urea, dithiothreitol, potassium chloride, lithium chloride, glycogen, NP -40, Tween 20, Triton X-100, isopropanol, simethicone.
  • guanidine thiocyanate has a final concentration of 0.5-5M, preferably 1-3M, in the composition.
  • 0.5-5M preferably 1-3M
  • guanidine hydrochloride has a final concentration of 0.5-5M, preferably 3-4M in the composition.
  • 0.5-5M preferably 3-4M in the composition.
  • urea has a final concentration of 0.5-2M, preferably 0.5-1.5M, in the composition.
  • 0.5M, 1M, 1.5M 0.5M, 1M, 1.5M.
  • a final concentration of 1M is more preferred.
  • dithiothreitol has a final concentration of 0.01-1 mM, preferably 0.1-1 mM.
  • potassium chloride has a final concentration of 50-300 mM, preferably 100-200 mM.
  • glycogen has a final concentration of 0.01-0.5 mM, preferably 0.05-0.5 mM.
  • NP-40 has a final concentration of 0.1-10 v/v%, preferably 0.1-5 v/v%.
  • Tween 20 has a final concentration of 0.1-10 v/v%, preferably 0.1-5 v/v%.
  • isopropanol has a final concentration of 15-40 v/v%, preferably 20-30 v/v%. For example 15v/v%, 20v/v%, 22v/v%, 25v/v%, 30v/v%.
  • sorbitol has a final concentration of 1-10 w/v%, preferably 4-6 w/v%. For example, 4w/v%, 5w/v%, 6w/v%. More preferred is a final concentration of 5 w/v%.
  • polyethylene glycol has a final concentration of 1-10 w/v%, preferably 4-6 w/v%. For example, 4w/v%, 5w/v%, 6w/v%. More preferred is a final concentration of 5 w/v%.
  • the molecular weight of polyethylene glycol is 500-100000, preferably 1000-10000, more preferably 2000-5000, most preferably 4000.
  • sodium acetate has a final concentration of 50-1000 mM, preferably 100-500 mM.
  • 50 mM, 100 mM, 200 mM, 500 mM a final concentration of 50-1000 mM, preferably 100-500 mM.
  • sodium chloride has a final concentration of 50-300 mM, preferably 100-200 mM.
  • Tris-HCl has a final concentration of 10-100 mM, preferably 20-80 mM. For example, 10 mM, 20 mM, 50 mM, 100 mM. More preferably 50 mM.
  • lithium chloride has a final concentration of 0.1-2M, preferably 0.2-1M, in the composition.
  • 0.1-2M preferably 0.2-1M
  • 0.2M 0.5M, 1M, 1.5M.
  • Triton X-100 has a final concentration of 0.1-2w/v%, preferably 0.1-1w/v%.
  • simethicone has a final concentration of 10-40 v/v%, preferably 10-30 v/v%. For example 10v/v%, 15v/v%, 20v/v%, 22v/v%, 25v/v%, 30v/v%. More preferred is a final concentration of 25 v/v%.
  • the present invention provides a nucleic acid extraction composition, which includes: 3mol/L guanidine hydrochloride, 2mol/L guanidine thiocyanate, 1mol/L urea, 0.5mmol/L Thiothreitol (DTT), 100mmol/L potassium chloride, 0.01mmol/L glycogen, 0.5% v/v ratio of ethyl phenyl polyethylene glycol (NP-40), v/v ratio 0.5% Tween 20 (tween-20), 22% isopropanol v/v.
  • a nucleic acid extraction composition which includes: 3mol/L guanidine hydrochloride, 2mol/L guanidine thiocyanate, 1mol/L urea, 0.5mmol/L Thiothreitol (DTT), 100mmol/L potassium chloride, 0.01mmol/L glycogen, 0.5% v/v ratio of ethyl phenyl polyethylene glycol (NP-40), v/v ratio
  • the present invention provides a nucleic acid extraction composition, which includes: 3.5mol/L guanidine thiocyanate, 5% sorbitol, 5% PEG4000, 0.1mmol/L dithiothreose Alcohol (DTT), 200mmol/L sodium acetate (NaAc, pH6.0), 0.01mmol/L glycogen, 0.5% volume/volume ratio of ethylphenyl polyethylene glycol (NP-40), volume / 30% isopropanol by volume.
  • DTT dithiothreose Alcohol
  • the present invention provides a nucleic acid extraction composition, which comprises: 4mol/L of guanidine hydrochloride, 2mol/L of guanidine thiocyanate, 1mol/L of urea, 0.5mmol/L of Thiothreitol (DTT), 200mmol/L potassium chloride, 200mmol/L sodium acetate (NaAc, pH6.6), 1mol/L lithium chloride, 0.5mmol/L glycogen, volume/volume ratio 0.5% ethyl phenyl polyethylene glycol (NP-40), volume/volume ratio of 0.5% Tween 20 (tween-20), mass/volume ratio of 0.5% Triton X-100, 22% isopropanol v/v.
  • a nucleic acid extraction composition which comprises: 4mol/L of guanidine hydrochloride, 2mol/L of guanidine thiocyanate, 1mol/L of urea, 0.5mmol/L of Thiothreitol (DTT), 200
  • the present invention provides a nucleic acid extraction composition, which comprises: 3mol/L guanidine hydrochloride, 1.8mol/L guanidine thiocyanate, 1mol/L urea, 1mmol/L di Thiothreitol (DTT), 50mmol/L potassium chloride, 100mmol/L lithium chloride, 0.05mmol/L glycogen, 0.5% volume/volume ratio of ethyl phenyl polyethylene glycol (NP -40), v/v ratio is 0.5% Tween 20 (tween-20), 1% triton X-100, v/v ratio is 30% isopropanol, v/v ratio is 10% Simethicone.
  • the present invention provides a nucleic acid extraction composition, which includes: 3.5mol/L guanidine thiocyanate, 5% sorbitol, 5% PEG4000, 0.1mmol/L dithiothreose Alcohol (DTT), 200mmol/L sodium acetate (NaAc, pH6.0), 0.01mmol/L glycogen, 100mmol/L lithium chloride, 50mmol/L sodium chloride, 0.5% v/v Ethylphenyl polyethylene glycol (NP-40), 30% isopropanol v/v, 20% simethicone v/v.
  • DTT dithiothreose Alcohol
  • the above nucleic acid extraction composition also includes: a sample to be tested.
  • the dosage ratio of the nucleic acid extraction composition and the sample to be tested is 2:2-2:5.
  • the dosage ratio of the nucleic acid extraction composition and the sample to be tested is 2:3-2:5.
  • the dosage ratio of the nucleic acid extraction composition and the sample to be tested is 2:5, under this ratio, the effect of nucleic acid extraction is better, and its Ct value is higher, and its Ct value is usually before 36 , while the 2:3 ratio is usually after 36, and the 2:4 ratio is usually after 36.
  • nucleic acid extraction composition also includes: eluent and magnetic beads.
  • the eluent includes Tris-HCl and EDTA, wherein the concentration of Tris-HCl is 5-20mM; the concentration of EDTA is 0.1-2mM.
  • the present invention provides a nucleic acid extraction kit, which includes the composition as described above.
  • primers and probes for PCR may also include primers and probes for PCR.
  • it may also include a PCR amplification system.
  • it may also include enzymes for PCR, dNTPs, PCR buffers, metal ions and the like.
  • the present invention provides a method for extracting nucleic acid, the method includes the step of mixing the sample with the above composition, wherein the method does not include other washing or impurity removal steps.
  • the method includes the step of eluting the mixture.
  • Fig. 1 is the detection result of the simulated novel coronavirus sample extracted from the throat swab of the composition 1 of the present invention
  • Fig. 2 is the detection result of the throat swab simulated novel coronavirus sample extracted from composition 2 of the present invention
  • Fig. 3 is the detection result of the throat swab simulated novel coronavirus sample extracted from composition 3 of the present invention
  • Fig. 4 is the detection result of the throat swab simulated novel coronavirus sample extracted from composition 4 of the present invention
  • Fig. 5 is the detection result of the throat swab simulated novel coronavirus sample extracted from composition 5 of the present invention
  • Figures 6 to 8 are the detection results of different concentrations of sputum simulated novel coronavirus samples extracted from the composition of the present invention.
  • Figures 9 to 11 are the detection results of different concentrations of saliva simulated novel coronavirus samples extracted from the composition of the present invention.
  • Fig. 12 is the detection result of the negative swab extracted from the composition of the present invention and the sample of new crown A and B flow diluted by the negative swab.
  • Embodiment 1 nucleic acid extraction method
  • the washing-free nucleic acid extraction composition of the present invention is used to extract nucleic acid from unknown samples in oropharyngeal swabs, sputum, alveolar lavage fluid, and saliva.
  • the process is as follows:
  • the eluted nucleic acid is further used for detection by PCR.
  • Example 2 the composition of the present invention extracts a throat swab to simulate a new coronavirus sample
  • composition 1-3 shown in the following table 1 to extract the negative throat swab sample mixture to dilute the calibrated new coronavirus culture (10 copies/mL), and the nucleic acid detection results are shown in Figure 1 ⁇ 5 shown. It can be seen from the figure that all five compositions can extract as low as 10 copies/mL of novel coronavirus in throat swab samples, and can be accurately detected.
  • composition 1-5 extracted negative swab sample mixture diluted to 10 copies/mL of the new coronavirus culture, the results showed that the internal standard gene amplification, N gene amplification and ORF1ab amplification detection results were good, and the detection sensitivity could reach 10 copies/mL. Furthermore, the Ct value of composition 3 is higher than that of compositions 1 and 2, indicating that the extraction effect of composition 3 is better than that of compositions 1 and 2.
  • Example 3 the composition of the present invention extracts sputum to simulate a new coronavirus sample
  • the new coronavirus culture (1000 copies/mL, 100 copies/mL, 10 copies/mL) of the negative sputum sample was extracted using the composition 3 shown in Table 1 below, and the nucleic acid
  • the test results are shown in Figures 6-8.
  • the composition of the present invention can extract novel coronaviruses as low as 10 copies/mL in sputum samples, and can accurately detect them.
  • Example 4 the composition of the present invention extracts saliva to simulate a new coronavirus sample
  • the new coronavirus culture (1000 copies/mL, 100 copies/mL, 10 copies/mL) of the negative saliva sample was extracted using the composition 3 shown in Table 1 above, and the nucleic acid detection The results are shown in Figures 9-11.
  • the composition of the present invention can extract novel coronaviruses as low as 10 copies/mL in saliva samples, and can accurately detect them.
  • Example 5 the composition of the present invention extracts negative swabs and samples of new crown A and B flow diluted by negative swabs
  • negative swab samples were diluted and calibrated for new crown A and B flow sensitivity reference products (including new coronavirus, influenza A virus and influenza B virus detection) and negative swab samples, and the nucleic acid detection results are shown in Figure 12 shown.
  • the composition of the present invention can extract novel coronavirus, influenza A virus and influenza B virus with a concentration as low as 100 copies/mL in negative swab samples, and can accurately detect them.

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Abstract

提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40和异丙醇，并进一步包括：盐酸胍、尿素、氯化钾，和吐温20，或者山梨醇、聚乙二醇，和醋酸钠。并进一步提供了包括该核酸提取组合物的试剂盒，及其使用方法。使用该核酸提取组合物，整个提取试剂盒无洗涤液，不需要引入洗涤过程，进一步简化了提取流程，并缩短了提取样品核酸所需要的时间，手工操作只需要12分钟即可完成核酸的提取；同时，洗涤液的减少，进一步减少了试剂盒内试剂与耗材，从而降低了试剂盒的成本。

Description

免洗涤提取核酸的组合物、试剂盒、方法及用途
相关申请
本申请要求2021年12月02日申请的，申请号为202111460821.X，名称为“免洗涤提取核酸的组合物、试剂盒、方法及用途”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本发明涉及分子生物学检测领域；具体地，涉及核酸提取的领域；更具体地，本发明涉及免洗滴提取核酸的领域。
背景技术
核酸提取的方法主要包括酚抽提法、碱裂解法、CTAB抽提法、煮沸法、离心柱提取法、玻璃珠吸附法及微纳米磁珠提取法。目前应用与新冠病毒样本核酸提取的主流方法主要包括离心柱提取法和磁珠提取法，离心柱核酸提取法由于采用核酸特异吸附和反复洗涤杂质，最后得到的核酸纯度较传统方法具有明显优势，而磁珠法核酸提取技术由于自动化程度高、样本通量大、核酸浓度和纯度好，尤其是安全性高，已成为业内主流方法。国内核酸提取主流厂家拥有全自动核酸提取系统采用磁珠法原理，无需离心且操作自动化，可减少人员接触，并使用深孔板耗材，可避免交叉污染。当面对突发疫情时，更是需要有高通量、自动化、安全的设备与方法才能更有效准确的监测疫情、控制疫情。
随着研究的不断深入，核酸提取也变得多种多样，目前已有的磁珠法提取技术主要有两种，一是乙醇洗涤体系，核酸释放并吸附到磁珠上通过磁架去掉废液，然后用不同浓度的乙醇盐溶液和乙醇溶液洗涤2～3次依次去除有机和无机的杂质，从而洗脱获得高纯度的核酸溶液；二是无乙醇洗涤体系，磁珠上吸附得到的核酸通过盐溶液和硅油等进一步去除杂质获得高浓度核酸溶液供PCR检测。
然而，所有这些核酸提取都避免不了需要通过洗涤除杂才能进行下一步操作，虽然可以通过免提取等方法规避洗涤除杂，但是免提取法得到的核酸与杂质共存。
因此，随着临床分子诊断的发展，在某些传染病筛查领域，对于操作时间和检测通量提出了新的要求，亟需一种简单、快速的样本处理技术。
发明内容
有鉴于此，本发明的主要目的在于提供一种免洗涤提取核酸的组合物，其在提取核酸时，不需要引入洗涤过程，进一步简化了提取流程。
第一方面，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40和异丙醇，并进一步包括：
盐酸胍、尿素、氯化钾，和吐温20，或者
山梨醇、聚乙二醇，和醋酸钠。
使用本发明的核酸提取组合物，整个提取试剂盒无洗涤液，不需要引入洗涤过程，进一步简化了提取流程，并大大缩短了提取样品核酸所需要的时间，手工操作只需要12分钟即可完成核酸的提取；同时，洗涤液的减少，进一步减少了试剂盒内试剂与耗材，从而降低了试剂盒的成本。
本发明组合物可适用于咽拭子、鼻咽拭子、肺泡灌洗液、唾液样本、痰液样本、血液样本等样本类型，普遍适用于常规呼吸道传染病临床分子诊断。本发明组合物既可以提取人的DNA/RNA。也可以提取非人的DNA/RNA。举例来说，本发明组合物可以提取人、细菌、病毒等的DNA/RNA。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40、异丙醇、盐酸胍、尿素、氯化钾，和吐温20。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40、异丙醇、山梨醇、聚乙二醇，和醋酸钠。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40、异丙醇、山梨醇、聚乙二醇、醋酸钠，和Tris-HCl。
进一步地，在一些具体的实施方案中，上述核酸提取组合物进一步包括下列物质中的至少一种：氯化锂、曲拉通X-100。
进一步地，在一些具体的实施方案中，上述核酸提取组合物进一步包括下列物质中的至少一种：氯化锂、曲拉通X-100、二甲基硅油、氯化钠和Tris-HCl。
进一步地，在一些具体的实施方案中，上述核酸提取组合物进一步包括下列物质中的至少一种：氯化锂、曲拉通X-100、二甲基硅油、氯化钠、醋酸钠和Tris-HCl。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40、异丙醇、盐酸胍、尿素、氯化钾、吐温20、醋酸钠、氯化锂、曲拉通X-100。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、 山梨醇、聚乙二醇、二硫苏糖醇、醋酸钠、氯化钠、糖原、NP-40、吐温20、曲拉通X-100、异丙醇、Tris-HCl。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：硫氰酸胍、山梨醇、聚乙二醇、二硫苏糖醇、醋酸钠、糖原、氯化锂、氯化钠、NP-40、异丙醇、二甲基硅油。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：盐酸胍、硫氰酸胍、尿素、二硫苏糖醇、氯化钾、氯化锂、糖原、NP-40、吐温20、曲拉通X-100、异丙醇、二甲基硅油。
在本发明中，硫氰酸胍在组合物中具有0.5-5M，优选1-3M的终浓度。例如，1M、2M、2.5M、3M。
在本发明中，盐酸胍在组合物中具有0.5-5M，优选3-4M的终浓度。例如，1M、2M、3M、3.5M、4mM。
在本发明中，尿素在组合物中具有0.5-2M，优选0.5-1.5M的终浓度。例如，0.5M、1M、1.5M。更优选为1M的终浓度。
在本发明中，二硫苏糖醇具有0.01-1mM，优选0.1-1mM的终浓度。例如，0.1mM、0.2mM、0.3mM、0.5mM、1mM。
在本发明中，氯化钾具有50-300mM，优选100-200mM的终浓度。例如，50mM、100mM、200mM、300mM。
在本发明中，糖原具有0.01-0.5mM，优选0.05-0.5mM的终浓度。例如，0.1mM、0.2mM、0.3mM、0.5mM。
在本发明中，NP-40具有0.1-10v/v％，优选0.1-5v/v％的终浓度。例如0.1v/v％、0.5v/v％、1v/v％、2v/v％、5v/v％。更优选为0.5v/v％的终浓度。
在本发明中，吐温20具有0.1-10v/v％，优选0.1-5v/v％的终浓度。例如0.1v/v％、0.5v/v％、1v/v％、2v/v％、5v/v％。更优选为0.5v/v％的终浓度。
在本发明中，异丙醇具有15-40v/v％，优选20-30v/v％的终浓度。例如15v/v％、20v/v％、22v/v％、25v/v％、30v/v％。
在本发明中，山梨醇具有1-10w/v％，优选4-6w/v％的终浓度。例如，4w/v％、5w/v％、6w/v％。更优选为5w/v％的终浓度。
在本发明中，聚乙二醇具有1-10w/v％，优选4-6w/v％的终浓度。例如，4w/v％、5w/v％、6w/v％。更优选为5w/v％的终浓度。聚乙二醇的分子量为500-100000，优选1000-10000，更优选2000-5000，最优选4000。
在本发明中，醋酸钠具有50-1000mM，优选100-500mM的终浓度。例如，50mM、100mM、200mM、500mM。
在本发明中，氯化钠具有50-300mM，优选100-200mM的终浓度。例如，50mM、100mM、200mM、300mM。更优选为200mM。
在本发明中，Tris-HCl具有10-100mM，优选20-80mM的终浓度。例如，10mM、20mM、50mM、100mM。更优选为50mM。
在本发明中，氯化锂在组合物中具有0.1-2M，优选0.2-1M的终浓度。例如，0.2M、0.5M、1M、1.5M。
在本发明中，曲拉通X-100具有0.1-2w/v％，优选0.1-1w/v％的终浓度。例如，0.1w/v％、0.5w/v％、1w/v％。更优选为0.5w/v％的终浓度。
在本发明中，二甲基硅油具有10-40v/v％，优选10-30v/v％的终浓度。例如10v/v％、15v/v％、20v/v％、22v/v％、25v/v％、30v/v％。更优选为25v/v％的终浓度。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：3mol/L的盐酸胍、2mol/L的硫氰酸胍、1mol/L的尿素、0.5mmol/L的二硫苏糖醇(DTT)、100mmol/L的氯化钾、0.01mmol/L的糖原、体积/体积比为0.5％的乙基苯基聚乙二醇(NP-40)、体积/体积比为0.5％的吐温20(tween-20)、体积/体积比为22％的异丙醇。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：3.5mol/L的硫氰酸胍、5％山梨醇、5％PEG4000、0.1mmol/L的二硫苏糖醇(DTT)、200mmol/L的醋酸钠(NaAc，pH6.0)、0.01mmol/L的糖原、体积/体积比为0.5％的乙基苯基聚乙二醇(NP-40)、体积/体积比为30％的异丙醇。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：4mol/L的盐酸胍、2mol/L的硫氰酸胍、1mol/L的尿素、0.5mmol/L的二硫苏糖醇(DTT)、200mmol/L的氯化钾、200mmol/L的醋酸钠(NaAc，pH6.6)、1mol/L的氯化锂、0.5mmol/L的糖原、体积/体积比为0.5％的乙基苯基聚乙二醇(NP-40)、体积/体积比为0.5％的吐温20(tween-20)、质量/体积比为0.5％的曲拉通X-100、体积/体积比为22％的异丙醇。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：3mol/L的盐酸胍、1.8mol/L的硫氰酸胍、1mol/L的尿素、1mmol/L的二硫苏糖醇(DTT)、50mmol/L的氯化钾、100mmol/L的氯化锂、0.05mmol/L的糖原、体积/体积比为0.5％的乙基苯基聚乙二醇(NP-40)、体积/体积比为0.5％的吐温20(tween-20)、1％的曲拉通X-100、体积/体积比为30％的异丙醇、体积/体积比为10％二甲基硅油。
在一个具体的实施方案中，本发明提供了一种核酸提取组合物，其包括：3.5mol/L的 硫氰酸胍、5％山梨醇、5％PEG4000、0.1mmol/L的二硫苏糖醇(DTT)、200mmol/L的醋酸钠(NaAc，pH6.0)、0.01mmol/L的糖原、100mmol/L的氯化锂、50mmol/L的氯化钠、体积/体积比为0.5％的乙基苯基聚乙二醇(NP-40)、体积/体积比为30％的异丙醇、体积/体积比为20％二甲基硅油。
进一步地，上述核酸提取组合物还包括：待测样本。
进一步地，核酸提取组合物和待测样本的用量比例为2:2-2:5。
进一步地，核酸提取组合物和待测样本的用量比例为2:3-2:5。
在一个优选的实施方案中，核酸提取组合物和待测样本的用量比例为2:5，在该比例下，核酸提取的效果更好，其Ct值较为靠前，其通常Ct值在36之前，而2:3的比例通常在36，2:4的比例通常在36之后。
进一步地，上述核酸提取组合物还包括：洗脱液和磁珠。
进一步地，洗脱液包括Tris-HCl、EDTA，其中，Tris-HCl的浓度为5-20mM；EDTA的浓度为0.1-2mM。
第二方面，本发明提供一种核酸提取试剂盒，其包括如上所述的组合物。
进一步地，其还可以包括用于PCR的引物和探针。
进一步地，其还可以包括PCR扩增体系。
进一步地，其还可以包括用于PCR的酶、dNTP、PCR缓冲液、金属离子等。
第三方面，本发明提供一种提取核酸的方法，所述方法包括将样本与上述组合物混合的步骤，其中，所述方法不包括其它洗涤或者除杂步骤。
进一步地，所述方法包括混合物洗脱的步骤。
附图说明
图1为本发明组合物1提取咽拭子模拟新型冠状病毒样品的检测结果；
图2为本发明组合物2提取咽拭子模拟新型冠状病毒样品的检测结果；
图3为本发明组合物3提取咽拭子模拟新型冠状病毒样品的检测结果；
图4为本发明组合物4提取咽拭子模拟新型冠状病毒样品的检测结果；
图5为本发明组合物5提取咽拭子模拟新型冠状病毒样品的检测结果；
图6～8为本发明组合物提取不同浓度的痰液模拟新型冠状病毒样品的检测结果；
图9～11为本发明组合物提取不同浓度的唾液模拟新型冠状病毒样品的检测结果；
图12为本发明组合物提取阴性拭子及阴性拭子稀释的新冠甲乙流样品的检测结果。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1、核酸提取方法
将本发明免洗涤的核酸提取组合物用于提取口咽拭子、痰液、肺泡灌洗液、唾液中未知样本中核酸。其过程如下所述：
1.1、根据待测样本数量取1.5mL离心管若干，每管加入400μL样品；
1.2、加入400μL本发明核酸提取组合物和30μL磁珠溶液；盖上管盖，震荡混匀30s，60℃加热4min。
1.3、低速瞬时离心，将离心管置于磁性分离器上，30s后从管底缓慢吸弃废液(*注意不要碰到吸附于管壁内侧的磁珠)；
1.4、低速瞬时离心，将离心管置于磁性分离器上，30s后将废液完全吸出丢弃(*注意不要碰到吸附于管壁内侧的磁珠)；；
1.5、加入50μL洗脱液S，震荡混匀10s，将离心管壁上磁珠洗脱到管底；低速瞬时离心，将离心管再次置于磁性分离器上磁吸30s，然后将洗脱下来的核酸转移到干净的1.5mL离心管中。
1.6、洗脱的核酸进一步用于PCR进行检测。
实施例2、本发明组合物提取咽拭子模拟新型冠状病毒样品
按照实施例1所述的方法，使用如下表1所示的组合物1-3提取阴性咽拭子样本混合液稀释标定的新型冠状病毒培养物(10拷贝/mL)，核酸检测结果如图1～5所示。从图中可以看出，5种组合物均能够提取咽拭子样本中低至10拷贝/mL的新型冠状病毒，并且可以准确检出。
组合物1-5提取阴性拭子样本混合液稀释至10拷贝/mL的新型冠状病毒培养物，结果显示内标基因扩增、N基因扩增和ORF1ab扩增检测结果良好，检测灵敏度可以达到10拷贝/mL。进一步地，组合物3的Ct值较组合物1和2更为靠前，表明组合物3的提取效果相较于组合物1和2更好。
表1
Figure PCTCN2022134688-appb-000001
Figure PCTCN2022134688-appb-000002
Figure PCTCN2022134688-appb-000003
实施例3、本发明组合物提取痰液模拟新型冠状病毒样品
按照实施例1所述的方法，使用如下表1所示的组合物3提取阴性痰液样本稀释标定的新型冠状病毒培养物(1000拷贝/mL、100拷贝/mL、10拷贝/mL)，核酸检测结果如图6～8所示。本发明组合物能够提取痰液样本中低至10拷贝/mL的新型冠状病毒，并且可以准确检出。
实施例4、本发明组合物提取唾液模拟新型冠状病毒样品
按照实施例1所述的方法，使用如上表1所示的组合物3提取阴性唾液样本稀释标定的新型冠状病毒培养物(1000拷贝/mL、100拷贝/mL、10拷贝/mL)，核酸检测结果如图9～11所示。本发明组合物能够提取唾液样本中低至10拷贝/mL的新型冠状病毒，并且可以准确检出。
实施例5、本发明组合物提取阴性拭子及阴性拭子稀释的新冠甲乙流样品
按照本发明提供的实验方案提取阴性拭子样本稀释标定的新冠甲乙流灵敏度参考品(包含新型冠状病毒、甲型流感病毒和乙型流感病毒检测)和阴性拭子样本，核酸检测结果如图12所示。本发明组合物能够提取阴性拭子样本中浓度低至100拷贝/mL的新型冠状病毒、甲型流感病毒和乙型流感病毒，并且可以准确检出。

Claims (15)

  1. 一种核酸提取组合物，其包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40和异丙醇，并进一步包括：
    盐酸胍、尿素、氯化钾，和吐温20，或者
    山梨醇、聚乙二醇，和醋酸钠。
  2. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物进一步包括下列物质中的至少一种：氯化锂、曲拉通X-100、二甲基硅油、氯化钠、Tris-HCl。
  3. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物进一步包括下列物质：氯化锂、曲拉通X-100。
  4. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物包括：硫氰酸胍、二硫苏糖醇、糖原、NP-40、异丙醇、盐酸胍、尿素、氯化钾、吐温20、醋酸钠、氯化锂、曲拉通X-100。
  5. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物还包括：待测样本，进一步地，所述核酸提取组合物和所述待测样本的用量比例为2:3～2:5。
  6. 根据权利要求1～5中任一项所述的核酸提取组合物，其中，所述核酸提取组合物还包括：洗脱液和磁珠。
  7. 一种核酸提取试剂盒，其包括如权利要求1～6中任一项所述的核酸提取组合物。
  8. 根据权利要求7所述的试剂盒，其中，所述试剂盒进一步包括PCR扩增体系。
  9. 一种核酸提取的方法，所述方法包括将待测样本与如权利要求1～7中任一项所述的核酸提取组合物混合的步骤，其中，所述方法不包括其它洗涤或者除杂步骤。
  10. 根据权利要求9所述的方法，其中，所述方法包括混合物洗脱的步骤。
  11. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物包括：硫氰酸胍、山梨醇、聚乙二醇、二硫苏糖醇、醋酸钠、氯化钠、糖原、NP-40、吐温20、曲拉通X-100、异丙醇、和Tris-HCl。
  12. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物包括：硫氰酸胍、山梨醇、聚乙二醇、二硫苏糖醇、醋酸钠、糖原、氯化锂、氯化钠、NP-40、异丙醇、和二甲基硅油。
  13. 根据权利要求1所述的核酸提取组合物，其中，所述核酸提取组合物包括：盐酸胍、硫氰酸胍、尿素、二硫苏糖醇、氯化钾、氯化锂、糖原、NP-40、吐温20、曲拉通X-100、异丙醇、和二甲基硅油。
  14. 根据权利要求7所述的试剂盒，其中，所述试剂盒进一步包括用于PCR的酶、dNTP、PCR缓冲液、和金属离子等。
  15. 根据权利要求1-6中任一项或11-13中任一项所述的核酸提取组合物，其中，所述组合物适用于咽拭子、鼻咽拭子、肺泡灌洗液、唾液样本、痰液样本、血液样本中的任一样本。
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