CN115011671A - Preparation method of respiratory tract nucleic acid composite quality control product - Google Patents
Preparation method of respiratory tract nucleic acid composite quality control product Download PDFInfo
- Publication number
- CN115011671A CN115011671A CN202210883542.2A CN202210883542A CN115011671A CN 115011671 A CN115011671 A CN 115011671A CN 202210883542 A CN202210883542 A CN 202210883542A CN 115011671 A CN115011671 A CN 115011671A
- Authority
- CN
- China
- Prior art keywords
- quality control
- control product
- content
- nucleic acid
- preservation solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000002131 composite material Substances 0.000 title abstract description 14
- 210000002345 respiratory system Anatomy 0.000 title abstract 2
- 238000004321 preservation Methods 0.000 claims abstract description 28
- 239000003761 preservation solution Substances 0.000 claims abstract description 28
- 230000000241 respiratory effect Effects 0.000 claims abstract description 26
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 239000000463 material Substances 0.000 claims abstract description 11
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 7
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- IWBOPFCKHIJFMS-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl) ether Chemical compound NCCOCCOCCN IWBOPFCKHIJFMS-UHFFFAOYSA-N 0.000 abstract 1
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- 208000035473 Communicable disease Diseases 0.000 description 1
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- 229930091371 Fructose Natural products 0.000 description 1
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- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
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- 229920004890 Triton X-100 Polymers 0.000 description 1
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 1
- WTHXTWHYLIZJBH-UHFFFAOYSA-N acetic acid;azane Chemical compound N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O WTHXTWHYLIZJBH-UHFFFAOYSA-N 0.000 description 1
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- 238000011901 isothermal amplification Methods 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
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- YFVGRULMIQXYNE-UHFFFAOYSA-M lithium;dodecyl sulfate Chemical compound [Li+].CCCCCCCCCCCCOS([O-])(=O)=O YFVGRULMIQXYNE-UHFFFAOYSA-M 0.000 description 1
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- ADWNFGORSPBALY-UHFFFAOYSA-M sodium;2-[dodecyl(methyl)amino]acetate Chemical compound [Na+].CCCCCCCCCCCCN(C)CC([O-])=O ADWNFGORSPBALY-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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- 208000011580 syndromic disease Diseases 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
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- 208000020017 viral respiratory tract infection Diseases 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/35—Mycoplasma
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- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及核酸检测质量控制领域，特别涉及呼吸道核酸复合质控品的制备方法。本发明的制备方法是将呼吸道灭活病原体材料高值加入组分为3‑吗啉丙磺酸缓冲液(MOPS)、二甲基亚砜(DMSO)、乙二醇双(2‑氨基乙基醚)四乙酸(EGTA)、脂肪醇聚氧乙烯醚、牛血清白蛋白(BSA)、异硫氰酸胍、鲸蜡醇聚氧乙烯(3)醚、PEG3000和Rnase抑制剂的样品保存液中，分装后放置‑20℃，获得稳定的质控品。本发明质控品均匀性、稳定性良好，不受运输、温度等因素影响，性能稳定。The invention relates to the field of nucleic acid detection quality control, in particular to a preparation method of a respiratory nucleic acid composite quality control product. The preparation method of the present invention comprises the steps of adding high-value respiratory tract inactivating pathogen materials into 3-morpholine propanesulfonic acid buffer (MOPS), dimethyl sulfoxide (DMSO), ethylene glycol bis(2-aminoethyl) ether) tetraacetic acid (EGTA), fatty alcohol polyoxyethylene ether, bovine serum albumin (BSA), guanidine isothiocyanate, cetyl polyoxyethylene (3) ether, PEG3000 and Rnase inhibitor in the sample preservation solution , and place it at -20°C after sub-packaging to obtain a stable quality control product. The quality control product of the invention has good uniformity and stability, is not affected by factors such as transportation and temperature, and has stable performance.
Description
技术领域technical field
本发明涉及核酸检测质量控制领域，特别涉及呼吸道核酸复合质控品的制备方法。The invention relates to the field of nucleic acid detection quality control, in particular to a preparation method of a respiratory nucleic acid composite quality control product.
背景技术Background technique
流感是由流感病毒引起的一种急性病毒性呼吸道感染，传染性强，发病率高，其中甲型流感病毒的流行性比乙型流感病毒更强，症状更严重。在成年人和儿童中，发热伴呼吸道症候群是最普遍的急性传染病，对于患者和公共卫生部门来说，准确快速诊断由哪种病毒引起的呼吸道感染十分重要。新型冠状病毒导致的肺炎与常见的呼吸道病毒以及细菌性病原引起肺炎等有相似之处，单从临床表现、胸部影像学难以鉴别，需依靠病原学检测来区分。因此对于引起的发热等呼吸道症状疾病的病原都需要进行鉴别。临床上常见的呼吸道病原体包括甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等，快速准确对病原体进行鉴定对于临床诊疗以及防止药物滥用具有重要意义。Influenza is an acute viral respiratory tract infection caused by influenza virus, which is highly contagious and has a high morbidity rate. Influenza A virus is more prevalent than influenza B virus and has more severe symptoms. Fever with respiratory syndrome is the most common acute infectious disease in adults and children, and it is important for patients and public health authorities to accurately and quickly diagnose which virus is causing the respiratory infection. Pneumonia caused by the new coronavirus is similar to pneumonia caused by common respiratory viruses and bacterial pathogens. Therefore, it is necessary to identify the pathogens of respiratory symptoms such as fever caused by diseases. Common clinical respiratory pathogens include influenza A virus, influenza B virus, syncytial virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, parainfluenza type I, parainfluenza type II, parainfluenza type III, adenovirus, rhinovirus, metapulmonary Viruses, human Boca virus, Bacillus pertussis, enterovirus, Legionella pneumophila, coronavirus, etc., the rapid and accurate identification of pathogens is of great significance for clinical diagnosis and treatment and prevention of drug abuse.
目前，检测呼吸道病原体的核酸检测方法主要包扩基因芯片法、核酸等温扩增法和实时逆转录聚合酶链反应(RT-PCR)。基于上述方法已研发出了多种呼吸道病原体基因检测试剂盒，但是目前市面上并没有经济、有效、稳定和多种呼吸道病原体基因检测试剂盒的质控品销售，且现有的核酸质控品类产品多具有不稳定、易降解和不易保存等缺点，而质控品又是衡量和评估一款试剂盒检测准确性和稳定性等性能的重要方法和质量指标。因此，急需提供一种可稳定保存的复合的呼吸道核酸质控品。At present, nucleic acid detection methods for detecting respiratory pathogens mainly include gene chip method, nucleic acid isothermal amplification method and real-time reverse transcription polymerase chain reaction (RT-PCR). Based on the above methods, a variety of respiratory pathogen gene detection kits have been developed, but there are currently no economical, effective, stable and quality control products for various respiratory pathogen gene detection kits on the market, and the existing nucleic acid quality control products Most of the products have shortcomings such as instability, easy degradation and difficult preservation, and quality control materials are important methods and quality indicators to measure and evaluate the performance of a kit such as detection accuracy and stability. Therefore, there is an urgent need to provide a composite respiratory nucleic acid quality control material that can be stably preserved.
临床实验室使用的呼吸道核酸质控品有几大缺陷：①包含项目少：多为针对某项指标的单独质控品，而无复合质控品；②与试剂配套使用：大部分是与其试剂盒配套使用的质控品，不适用所有试剂厂家；③稳定性不好。④材料用质粒，不能监控提取和扩增全过程。因此能够发明一种呼吸道核酸复合质控品，具有很好的临床实用性，也非常有利于临床的推广和发展。The respiratory nucleic acid quality control products used in clinical laboratories have several major defects: ①Includes few items: most of them are single quality control products for a certain indicator, but no composite quality control products; ②Used with reagents: most of them are used with their reagents The quality control products used in the box are not suitable for all reagent manufacturers; ③The stability is not good. ④Materials use plasmids, and the whole process of extraction and amplification cannot be monitored. Therefore, a respiratory nucleic acid composite quality control product can be invented, which has good clinical practicability and is also very beneficial to clinical promotion and development.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明提供了呼吸道核酸复合质控品的制备方法，所述质控品均匀性、稳定性良好，不受运输、温度等因素影响，性能稳定。In view of this, the present invention provides a method for preparing a respiratory nucleic acid composite quality control product. The quality control product has good uniformity and stability, is not affected by factors such as transportation and temperature, and has stable performance.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了样品保存液，包括缓冲液、表面活性剂、酶抑制剂和/或保护剂；The present invention provides a sample preservation solution, including buffer solution, surfactant, enzyme inhibitor and/or protective agent;
所述缓冲液包括MOPS缓冲液；和/或The buffer comprises MOPS buffer; and/or
所述表面活性剂包括脂肪醇聚氧乙烯醚、鲸蜡醇聚氧乙烯(3)醚或PEG3000中的一种或两者以上的组合；和/或The surfactant comprises one or more of fatty alcohol polyoxyethylene ether, cetyl alcohol polyoxyethylene (3) ether or PEG3000; and/or
所述酶抑制剂包括Rnasin和/或异硫氰酸胍；和/或The enzyme inhibitor includes Rnasin and/or guanidine isothiocyanate; and/or
所述保护剂包括蔗糖。The protective agent includes sucrose.
在本发明的一些具体实施方案中，上述缓冲液为咽拭子保存液所替代。In some specific embodiments of the present invention, the above buffer is replaced by a throat swab preservation solution.
在本发明的一些具体实施方案中，上述缓冲液还包括磷酸盐缓冲液、Tris缓冲液、Hanks缓冲液、Tricine缓冲液或Bicine缓冲液中的一种或两者以上的组合；In some specific embodiments of the present invention, the above-mentioned buffer also includes one or a combination of two or more of phosphate buffer, Tris buffer, Hanks buffer, Tricine buffer or Bicine buffer;
所述表面活性剂还包括Span-80、Span-20、十二烷基苯磺酸钠、十二烷基硫酸锂、十二烷基硫酸钠、Tween 20、Triton X-100、NP40、SDS、十二烷基肌氨酸钠、PEG6000或PEG8000中的一种或两者以上的组合；The surfactants also include Span-80, Span-20, sodium dodecylbenzenesulfonate, lithium dodecyl sulfate, sodium dodecyl sulfate, Tween 20, Triton X-100, NP40, SDS, One or more combinations of sodium lauryl sarcosinate, PEG6000 or PEG8000;
所述酶抑制剂还包括焦碳酸二乙酯、氧钒核糖核苷复合物、尿素或硅藻土中的一种或两者以上的组合；The enzyme inhibitor also includes one or a combination of two or more of diethyl pyrocarbonate, vanadyl ribonucleoside complex, urea or diatomaceous earth;
所述保护剂还包括海藻糖、果糖或乳糖中的一种或两者以上的组合。The protective agent also includes one or a combination of two or more of trehalose, fructose or lactose.
在本发明的一些具体实施方案中，上述样品保存液中所述缓冲液的pH值包括6.5～10.5；和/或In some specific embodiments of the present invention, the pH value of the buffer solution in the above-mentioned sample preservation solution includes 6.5 to 10.5; and/or
所述缓冲液的浓度包括0～200g/L(w/v)；和/或The concentration of the buffer includes 0 to 200 g/L (w/v); and/or
所述表面活性剂中所述脂肪醇聚氧乙烯醚的含量包括0～2％(w/v)；和/或The content of the fatty alcohol polyoxyethylene ether in the surfactant includes 0-2% (w/v); and/or
所述表面活性剂中所述鲸蜡醇聚氧乙烯(3)醚的含量包括0～2％(w/v)；和/或The content of the cetyl polyoxyethylene (3) ether in the surfactant includes 0-2% (w/v); and/or
所述表面活性剂中所述PEG3000的含量包括0～10％(w/v)；和/或The content of the PEG3000 in the surfactant includes 0-10% (w/v); and/or
所述酶抑制剂中所述Rnasin的含量包括0～1％(w/v)；和/或The content of the Rnasin in the enzyme inhibitor comprises 0-1% (w/v); and/or
所述酶抑制剂中所述异硫氰酸胍的含量包括0～5％(w/v)；和/或The content of the guanidine isothiocyanate in the enzyme inhibitor comprises 0-5% (w/v); and/or
所述保护剂的含量包括0～10％(w/v)；The content of the protective agent includes 0-10% (w/v);
上述样品保存液中各组分含量不同时为0。The content of each component in the above-mentioned sample preservation solution is 0 at the same time.
在本发明的一些具体实施方案中，上述样品保存液中所述缓冲液的pH值为7.4～8.2；和/或In some specific embodiments of the present invention, the pH value of the buffer solution in the above-mentioned sample preservation solution is 7.4-8.2; and/or
所述缓冲液的浓度为50g/L；和/或The concentration of the buffer is 50g/L; and/or
所述表面活性剂中所述脂肪醇聚氧乙烯醚的含量为0.1％(w/v)；和/或The content of the fatty alcohol polyoxyethylene ether in the surfactant is 0.1% (w/v); and/or
所述表面活性剂中所述鲸蜡醇聚氧乙烯(3)醚的含量为0.5％(w/v)；和/或The content of the cetyl polyoxyethylene (3) ether in the surfactant is 0.5% (w/v); and/or
所述表面活性剂中所述PEG3000的含量为5％(w/v)；和/或The content of the PEG3000 in the surfactant is 5% (w/v); and/or
所述酶抑制剂中所述Rnasin的含量为0～1％(w/v)；和/或The content of the Rnasin in the enzyme inhibitor is 0-1% (w/v); and/or
所述酶抑制剂中所述异硫氰酸胍的含量为0.05％(w/v)；和/或The content of the guanidine isothiocyanate in the enzyme inhibitor is 0.05% (w/v); and/or
所述保护剂的含量为5％(w/v)；The content of the protective agent is 5% (w/v);
上述样品保存液中各组分含量不同时为0。The content of each component in the above-mentioned sample preservation solution is 0 at the same time.
在本发明的一些具体实施方案中，上述样品保存液还包括DSMO、EGTA或BSA中的一种或两者以上的组合。In some specific embodiments of the present invention, the above-mentioned sample preservation solution further comprises one or a combination of two or more of DSMO, EGTA or BSA.
本发明还提供了上述样品保存液在保存核酸样品中的应用。The present invention also provides the application of the above-mentioned sample preservation solution in preserving nucleic acid samples.
本发明还提供了上述样品保存液在制备质控品中的应用。The present invention also provides the application of the above-mentioned sample preservation solution in the preparation of quality control products.
本发明还提供了质控品，包括上述样品保存液、核酸和/或可接受的辅料；所述核酸包括：The present invention also provides quality control products, including the above-mentioned sample preservation solution, nucleic acid and/or acceptable auxiliary materials; the nucleic acid includes:
新型冠状病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌或冠状病毒的基因组核酸中的一种或多种。Novel coronavirus, Influenza A virus, Influenza B virus, Syncytial virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, Parainfluenza I, Parainfluenza II, Parainfluenza III, Adenovirus, Rhinovirus, Metapneumovirus, Human One or more of the genomic nucleic acids of Bocavirus, Bacillus pertussis, Enterovirus, Legionella pneumophila, or Coronavirus.
在本发明的一些具体实施方案中，上述核酸来源于质粒、噬菌体假病毒、腺病毒和/或慢病毒。In some specific embodiments of the present invention, the aforementioned nucleic acids are derived from plasmids, bacteriophage pseudoviruses, adenoviruses and/or lentiviruses.
本发明还提供了上述质控品的制备方法，包括如下步骤：The present invention also provides a preparation method of the above-mentioned quality control product, comprising the following steps:
步骤(a)、取所述缓冲液与所述表面活性剂、所述酶抑制剂和/或所述保护剂混合，获得所述样品保存液；Step (a), mixing the buffer with the surfactant, the enzyme inhibitor and/or the protective agent to obtain the sample preservation solution;
步骤(b)、取步骤(a)所述样品保存液与所述核酸混合，获得所述质控品。In step (b), the sample preservation solution in step (a) is mixed with the nucleic acid to obtain the quality control product.
本发明还提供了上述样品保存液、上述质控品或上述制备方法制得的质控品在制备检测呼吸道病原体的试剂盒中的应用。The present invention also provides the application of the above-mentioned sample preservation solution, the above-mentioned quality control product or the quality control product prepared by the above-mentioned preparation method in the preparation of a kit for detecting respiratory pathogens.
本发明还提供了试剂盒，其特征在于，上述样品保存液、上述质控品或上述制备方法制得的质控品，以及可接受的辅料和/或助剂。The present invention also provides a kit, which is characterized in that the above-mentioned sample preservation solution, the above-mentioned quality control product or the quality control product prepared by the above-mentioned preparation method, and acceptable auxiliary materials and/or auxiliaries.
本发明还提供了装置，其特征在于，包被有上述的样品保存液、上述的质控品或上述制备方法制得的质控品，以及可接受的部件。The present invention also provides a device, characterized in that it is coated with the above-mentioned sample preservation solution, the above-mentioned quality control product or the quality control product prepared by the above-mentioned preparation method, and acceptable components.
在本发明的一些具体实施方案中，上述辅料和/或助剂包括但不限于黏合剂、填充剂、崩解剂、润滑剂、等渗调节剂、增溶剂、助溶剂、防腐剂、着色剂、助悬剂、润湿剂、乳化剂和/或表面活性剂。In some specific embodiments of the present invention, the above-mentioned adjuvants and/or adjuvants include, but are not limited to, binders, fillers, disintegrants, lubricants, isotonicity regulators, solubilizers, cosolvents, preservatives, colorants , suspending agents, wetting agents, emulsifying agents and/or surfactants.
在本发明的一些具体实施方案中，对上述质控品进行了冻干。In some specific embodiments of the present invention, the above-mentioned quality control substance is lyophilized.
在本发明的一些具体实施方案中，上述冻干的方法如下：In some specific embodiments of the present invention, the method for above-mentioned lyophilization is as follows:
降温过程5min温度降至-5℃，保持45min；During the cooling process, the temperature dropped to -5°C for 5 minutes and kept for 45 minutes;
降温过程5min温度降至-45℃，保持120min；During the cooling process, the temperature dropped to -45°C for 5 minutes and kept for 120 minutes;
升温过程50min温度升至-5℃，保持750min，真空度10～14Pa；During the heating process, the temperature rises to -5°C for 50 minutes, and the temperature is maintained for 750 minutes. The vacuum degree is 10-14Pa;
升温过程50min温度升至10℃，保持60min；During the heating process, the temperature was raised to 10°C for 50min and kept for 60min;
升温过程50min温度升至25℃，保持180min，真空度13～17Pa。During the heating process, the temperature rose to 25°C for 50 minutes, and was maintained for 180 minutes with a vacuum degree of 13-17Pa.
本发明的质控品有如下效果：The quality control product of the present invention has the following effects:
1、本发明呼吸道核酸复合质控品包含多种病原体项目，1支质控品可同时满足临床多种需求；1. The respiratory nucleic acid composite quality control product of the present invention contains a variety of pathogen items, and one quality control product can meet various clinical needs at the same time;
2、本发明选用灭活病原体和慢病毒，厂家适用性强，可同时监控提取和扩增全过程；2. The present invention selects inactivated pathogens and lentiviruses, has strong applicability to manufacturers, and can monitor the entire process of extraction and amplification at the same time;
3、本发明质控品为高、中、低三水平，所涵盖的检测范围广泛；3. The quality control product of the present invention has three levels of high, medium and low, covering a wide range of detection;
4、实验表明本发明优选的质控品37℃14天后的病原体核酸拷贝数降幅不高于3％，本发明质控品均匀性、稳定性良好，不受运输、温度等因素影响；4. Experiments show that the copy number of pathogen nucleic acid of the preferred quality control product of the present invention after 14 days at 37°C decreases by no more than 3%. The quality control product of the present invention has good uniformity and stability, and is not affected by factors such as transportation and temperature;
5、本发明质控品可用于呼吸道核酸室内质控使用，同时也可用于室间质量评价组织者开展的呼吸道核酸室间质评活动。5. The quality control product of the present invention can be used for the indoor quality control of respiratory nucleic acid, and can also be used for the inter-laboratory quality assessment activity of respiratory nucleic acid carried out by the organizer of the inter-laboratory quality assessment.
具体实施方式Detailed ways
本发明公开了呼吸道核酸复合质控品的制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a preparation method of a respiratory nucleic acid composite quality control product, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
该技术应用产品：新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等其他呼吸道病原体。Products of this technology: new coronavirus, influenza A virus, influenza B virus, syncytial virus, chlamydia pneumoniae, mycoplasma pneumoniae, parainfluenza I, parainfluenza II, parainfluenza III, adenovirus, rhinovirus, parainfluenza Pneumovirus, Human Bocavirus, Bacillus pertussis, Enterovirus, Legionella pneumophila, Coronavirus and other respiratory pathogens.
术语解释：Terminology Explanation:
FluA： 甲型流感病毒FluA: Influenza A virus
FluB： 乙型流感病毒FluB: Influenza B virus
RSV： 合胞病毒RSV: Syncytial Virus
CP： 肺炎衣原体CP: Chlamydia pneumoniae
MP： 肺炎支原体MP: Mycoplasma pneumoniae
HPIV： 副流感IHPIV: Parainfluenza I
HPIV： 副流感IIHPIV: Parainfluenza II
HPIV： 副流感III型HPIV: Parainfluenza Type III
HAdV： 腺病毒HAdV: Adenovirus
RhV： 鼻病毒RhV: Rhinovirus
hMPV： 偏肺病毒hMPV: Metapneumovirus
HBoV： 人博卡病毒HBoV: Human Bocavirus
BPA： 百日咳杆菌BPA: Bacillus pertussis
EV： 肠道病毒EV: Enterovirus
LP： 嗜肺军团菌LP: Legionella pneumophila
OC43： 冠状病毒OC43: Coronavirus
FluA： 甲型流感病毒FluA: Influenza A virus
FluB： 乙型流感病毒FluB: Influenza B virus
RSV： 合胞病毒RSV: Syncytial Virus
CP： 肺炎衣原体CP: Chlamydia pneumoniae
MP： 肺炎支原体MP: Mycoplasma pneumoniae
HPIV： 副流感IHPIV: Parainfluenza I
本发明涉及一种呼吸道核酸复合质控品的制备方法。The invention relates to a preparation method of a respiratory nucleic acid composite quality control product.
本发明的制备方法是将新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等呼吸道灭活病原体材料高值加入样品保存液中(组分为：3-吗啉丙磺酸缓冲液(MOPS)、二甲基亚砜(DMSO)、乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、脂肪醇聚氧乙烯醚、牛血清白蛋白(BSA)、异硫氰酸胍、鲸蜡醇聚氧乙烯(3)醚、PEG3000、Rnase抑制剂)，分装后放置-20℃，获得稳定的质控品。The preparation method of the present invention is to combine new coronavirus, influenza A virus, influenza B virus, syncytial virus, chlamydia pneumoniae, mycoplasma pneumoniae, parainfluenza type I, parainfluenza type II, parainfluenza type III, adenovirus, rhinovirus , metapneumovirus, human boca virus, bacillus pertussis, enterovirus, legionella pneumophila, coronavirus and other respiratory inactivated pathogen materials are added to the sample preservation solution (component: 3-morpholine propanesulfonic acid buffer) Liquid (MOPS), dimethyl sulfoxide (DMSO), ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), fatty alcohol polyoxyethylene ether, bovine serum albumin (BSA), isothiocyanate Acid guanidine, cetyl alcohol polyoxyethylene (3) ether, PEG3000, Rnase inhibitor), placed in -20 ℃ after packaging, to obtain stable quality control products.
本发明提所用原料及试剂均可由市场购得。The raw materials and reagents used in the present invention can be purchased from the market.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, the present invention is further elaborated:
实施例1Example 1
1)制备1.20％的Tris-HCl缓冲液，pH调至7.4；1) Prepare 1.20% Tris-HCl buffer and adjust the pH to 7.4;
2)在Tris-HCl缓冲液中加入体积分数为5％的二甲基亚砜(DMSO)、浓度为0.5M的乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、质量百分比为0.11％的十二烷基肌氨酸钠(SDS)、质量百分比为1％的牛血清白蛋白(BSA)、质量百分比为1％的异硫氰酸胍、浓度为5mol/L的乙二胺四乙酸(EDTA)和体积分数为5％的丙三醇，搅拌均匀使其完全溶解；2) Add 5% dimethyl sulfoxide (DMSO) and 0.5M ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA) with a volume fraction of 5% in Tris-HCl buffer. 0.11% sodium dodecyl sarcosinate (SDS), 1% bovine serum albumin (BSA), 1% guanidine isothiocyanate, 5mol/L ethylene glycol Aminetetraacetic acid (EDTA) and glycerol with a volume fraction of 5%, stir evenly to make it completely dissolved;
3)加入新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等灭活病原体高值，可添加不同的浓度；3) Add New Coronavirus, Influenza A, Influenza B, Syncytial Virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, Parainfluenza I, Parainfluenza II, Parainfluenza III, Adenovirus, Rhinovirus, Metapneumovirus , Human Boca virus, Bacillus pertussis, enterovirus, Legionella pneumophila, coronavirus and other high values of inactivated pathogens, different concentrations can be added;
4)将添加高值后的呼吸道核酸复合质控品分装至棕色玻璃瓶，进行真空冷冻干燥，具体过程：4) Dispense the respiratory nucleic acid composite quality control product with high value into a brown glass bottle for vacuum freeze-drying. The specific process is as follows:
降温过程5min温度降至-5℃，保持45min；During the cooling process, the temperature dropped to -5°C for 5 minutes and kept for 45 minutes;
降温过程5min温度降至-45℃，保持120min；During the cooling process, the temperature dropped to -45°C for 5 minutes and kept for 120 minutes;
升温过程50min温度升至-5℃，保持750min，真空度10～14Pa；During the heating process, the temperature rises to -5°C for 50 minutes, and the temperature is maintained for 750 minutes. The vacuum degree is 10-14Pa;
升温过程50min温度升至10℃，保持60min；During the heating process, the temperature was raised to 10°C for 50min and kept for 60min;
升温过程50min温度升至25℃，保持180min，真空度13～17Pa。During the heating process, the temperature rose to 25°C for 50 minutes, and was maintained for 180 minutes with a vacuum degree of 13-17Pa.
5)获得的质控品放置于2～8℃保存。5) The obtained quality control product is stored at 2-8°C.
6)取一部分所述质控品，复溶后提取核酸，对所包含的病原体分别进行数字PCR定值检测。6) Take a part of the quality control material, extract nucleic acid after reconstitution, and perform digital PCR quantitative detection on the contained pathogens respectively.
对余下质控品进行如下处理：The remaining controls were processed as follows:
a、不复溶，置于37℃放置14天；或a. Not reconstituted, placed at 37°C for 14 days; or
b、复溶后，置于4℃放置14天。b. After reconstitution, place it at 4°C for 14 days.
随后对经a或b处理的质控品分别进行稳定性验证，数据如表1所示。Subsequently, the quality control products treated with a or b were subjected to stability verification respectively, and the data are shown in Table 1.
实施例2Example 2
1)制备50g/L 3-吗啉丙磺酸缓冲液(MOPS)，pH调至8.2；1) prepare 50g/L 3-morpholine propanesulfonic acid buffer solution (MOPS), adjust pH to 8.2;
2)在3-吗啉丙磺酸缓冲液(MOPS)中，加入2％二甲基亚砜(DMSO)、0.2％乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、0.1％脂肪醇聚氧乙烯醚、2％牛血清白蛋白(BSA)、1％异硫氰酸胍、0.5％鲸蜡醇聚氧乙烯(3)醚、5％PEG3000、5％蔗糖、0.05％Rnasin，搅拌均匀使其完全溶解；2) In 3-morpholine propanesulfonic acid buffer (MOPS), add 2% dimethyl sulfoxide (DMSO), 0.2% ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 0.1 % fatty alcohol polyoxyethylene ether, 2% bovine serum albumin (BSA), 1% guanidine isothiocyanate, 0.5% cetyl polyoxyethylene (3) ether, 5% PEG3000, 5% sucrose, 0.05% Rnasin , stir evenly to dissolve it completely;
3)加入新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等灭活病原体高值，可添加不同的浓度；3) Add New Coronavirus, Influenza A, Influenza B, Syncytial Virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, Parainfluenza I, Parainfluenza II, Parainfluenza III, Adenovirus, Rhinovirus, Metapneumovirus , Human Boca virus, Bacillus pertussis, enterovirus, Legionella pneumophila, coronavirus and other high values of inactivated pathogens, different concentrations can be added;
4)将添加高值后的呼吸道核酸复合质控品分装至棕色玻璃瓶，进行真空冷冻干燥，具体过程：4) Dispense the respiratory nucleic acid composite quality control product with high value into a brown glass bottle for vacuum freeze-drying. The specific process is as follows:
降温过程5min温度降至-5℃，保持45min；During the cooling process, the temperature dropped to -5°C for 5 minutes and kept for 45 minutes;
降温过程5min温度降至-45℃，保持120min；During the cooling process, the temperature dropped to -45°C for 5 minutes and kept for 120 minutes;
升温过程50min温度升至-5℃，保持750min，真空度10～14Pa；During the heating process, the temperature rises to -5°C for 50 minutes, and the temperature is maintained for 750 minutes. The vacuum degree is 10-14Pa;
升温过程50min温度升至10℃，保持60min；During the heating process, the temperature was raised to 10°C for 50min and kept for 60min;
升温过程50min温度升至25℃，保持180min，真空度13～17Pa。During the heating process, the temperature rose to 25°C for 50 minutes, and was maintained for 180 minutes with a vacuum degree of 13-17Pa.
5)获得的质控品放置于2～8℃保存。5) The obtained quality control product is stored at 2-8°C.
6)取一部分所述质控品，复溶后提取核酸，对所包含的病原体分别进行数字PCR定值检测。6) Take a part of the quality control material, extract nucleic acid after reconstitution, and perform digital PCR quantitative detection on the contained pathogens respectively.
对余下质控品进行如下处理：The remaining controls were processed as follows:
a、不复溶，37℃放置14天；或a. Not reconstituted, placed at 37°C for 14 days; or
b、复溶后，4℃放置14天。b. After reconstitution, place at 4°C for 14 days.
随后对经a或b处理的质控品分别进行稳定性验证，数据如表1所示：Subsequently, the stability verification of the quality control products treated by a or b was carried out respectively, and the data are shown in Table 1:
表1Table 1
从数据显示，实施例2制备的质控品与实施例1配方对比，两者差别为更换了缓冲液，其缓冲液pH设置在8.2；表面活性剂更换了脂肪醇聚氧乙烯醚、鲸蜡醇聚氧乙烯(3)醚、PEG3000，另外增加了Rnase抑制剂和蔗糖，其稳定性明显好于实施例1配方的稳定性。The data shows that the quality control product prepared in Example 2 is compared with the formula of Example 1. The difference between the two is that the buffer solution is replaced, and the pH of the buffer solution is set at 8.2; the surfactant is replaced by fatty alcohol polyoxyethylene ether, spermaceti Alcohol polyoxyethylene (3) ether, PEG3000, additionally added Rnase inhibitor and sucrose, the stability is obviously better than that of the formula in Example 1.
实施例3Example 3
1)制备50g/L 3-吗啉丙磺酸缓冲液(MOPS)，pH调至7.8；1) prepare 50g/L 3-morpholine propanesulfonic acid buffer solution (MOPS), adjust pH to 7.8;
2)在3-吗啉丙磺酸缓冲液(MOPS)中，加入2％二甲基亚砜(DMSO)、0.2％乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、0.1％脂肪醇聚氧乙烯醚、2％牛血清白蛋白(BSA)、1％异硫氰酸胍、0.5％鲸蜡醇聚氧乙烯(3)醚、5％PEG3000、5％蔗糖、0.05％Rnasin，搅拌均匀使其完全溶解；2) In 3-morpholine propanesulfonic acid buffer (MOPS), add 2% dimethyl sulfoxide (DMSO), 0.2% ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 0.1 % fatty alcohol polyoxyethylene ether, 2% bovine serum albumin (BSA), 1% guanidine isothiocyanate, 0.5% cetyl polyoxyethylene (3) ether, 5% PEG3000, 5% sucrose, 0.05% Rnasin , stir evenly to dissolve it completely;
3)加入新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等灭活病原体高值，可添加不同的浓度；3) Add New Coronavirus, Influenza A, Influenza B, Syncytial Virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, Parainfluenza I, Parainfluenza II, Parainfluenza III, Adenovirus, Rhinovirus, Metapneumovirus , Human Boca virus, Bacillus pertussis, enterovirus, Legionella pneumophila, coronavirus and other high values of inactivated pathogens, different concentrations can be added;
4)将添加高值后的呼吸道核酸复合质控品分装至棕色玻璃瓶，进行真空冷冻干燥，具体过程：4) Dispense the respiratory nucleic acid composite quality control product with high value into a brown glass bottle for vacuum freeze-drying. The specific process is as follows:
降温过程5min温度降至-5℃，保持45min；During the cooling process, the temperature dropped to -5°C for 5 minutes and kept for 45 minutes;
降温过程5min温度降至-45℃，保持120min；During the cooling process, the temperature dropped to -45°C for 5 minutes and kept for 120 minutes;
升温过程50min温度升至-5℃，保持750min，真空度10～14Pa；During the heating process, the temperature rises to -5°C for 50 minutes, and the temperature is maintained for 750 minutes. The vacuum degree is 10-14Pa;
升温过程50min温度升至10℃，保持60min；During the heating process, the temperature was raised to 10°C for 50min and kept for 60min;
升温过程50min温度升至25℃，保持180min，真空度13～17Pa。During the heating process, the temperature rose to 25°C for 50 minutes, and was maintained for 180 minutes with a vacuum degree of 13-17Pa.
5)获得的质控品放置于2～8℃保存。5) The obtained quality control product is stored at 2-8°C.
6)取一部分所述质控品，复溶后提取核酸，对所包含的病原体分别进行数字PCR定值检测。6) Take a part of the quality control material, extract nucleic acid after reconstitution, and perform digital PCR quantitative detection on the contained pathogens respectively.
对余下质控品进行如下处理：The remaining controls were processed as follows:
a、不复溶，37℃放置14天；或a. Not reconstituted, placed at 37°C for 14 days; or
b、复溶后，4℃放置14天。b. After reconstitution, place at 4°C for 14 days.
随后对经a或b处理的质控品分别进行稳定性验证，数据如表2所示：Subsequently, the stability verification of the quality control products treated by a or b was carried out respectively, and the data are shown in Table 2:
表2Table 2
实施例2制备的质控品与实施例3配方对比，两者差别在pH不同，实施例2为8.2，实施例3为7.8，实施例2配方稳定性明显好于实施例3配方的稳定性。The quality control product prepared in Example 2 is compared with the formulation of Example 3. The difference between the two is in pH. Example 2 is 8.2, and Example 3 is 7.8. The stability of the formulation of Example 2 is significantly better than that of the formulation of Example 3. .
实施例4Example 4
1)制备50g/L 3-吗啉丙磺酸缓冲液(MOPS)，pH调至8.2；1) prepare 50g/L 3-morpholine propanesulfonic acid buffer solution (MOPS), adjust pH to 8.2;
2)在3-吗啉丙磺酸缓冲液(MOPS)中，加入2％二甲基亚砜(DMSO)、0.2％乙二醇双(2-氨基乙基醚)四乙酸(EGTA)、0.1％脂肪醇聚氧乙烯醚、2％牛血清白蛋白(BSA)、1％异硫氰酸胍、5％PEG3000、5％蔗糖、0.05％Rnasin，搅拌均匀使其完全溶解；2) In 3-morpholine propanesulfonic acid buffer (MOPS), add 2% dimethyl sulfoxide (DMSO), 0.2% ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 0.1 % fatty alcohol polyoxyethylene ether, 2% bovine serum albumin (BSA), 1% guanidine isothiocyanate, 5% PEG3000, 5% sucrose, 0.05% Rnasin, stir well to dissolve it completely;
3)加入新冠病毒、甲型流感病毒、乙型流感病毒、合胞病毒、肺炎衣原体、肺炎支原体、副流感I型、副流感II型、副流感III型、腺病毒、鼻病毒、偏肺病毒、人博卡病毒、百日咳杆菌、肠道病毒、嗜肺军团菌、冠状病毒等灭活病原体高值，可添加不同的浓度；3) Add New Coronavirus, Influenza A, Influenza B, Syncytial Virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, Parainfluenza I, Parainfluenza II, Parainfluenza III, Adenovirus, Rhinovirus, Metapneumovirus , Human Boca virus, Bacillus pertussis, enterovirus, Legionella pneumophila, coronavirus and other high values of inactivated pathogens, different concentrations can be added;
4)将添加高值后的呼吸道核酸复合质控品分装至棕色玻璃瓶，进行真空冷冻干燥，具体过程：4) Dispense the respiratory nucleic acid composite quality control product with high value into a brown glass bottle for vacuum freeze-drying. The specific process is as follows:
降温过程5min温度降至-5℃，保持45min；During the cooling process, the temperature dropped to -5°C for 5 minutes and kept for 45 minutes;
降温过程5min温度降至-45℃，保持120min；During the cooling process, the temperature dropped to -45°C for 5 minutes and kept for 120 minutes;
升温过程50min温度升至-5℃，保持750min，真空度10～14Pa；During the heating process, the temperature rises to -5°C for 50 minutes, and the temperature is maintained for 750 minutes. The vacuum degree is 10-14Pa;
升温过程50min温度升至10℃，保持60min；During the heating process, the temperature was raised to 10°C for 50min and kept for 60min;
升温过程50min温度升至25℃，保持180min，真空度13～17Pa。During the heating process, the temperature rose to 25°C for 50 minutes, and was maintained for 180 minutes with a vacuum degree of 13-17Pa.
5)获得的质控品放置于2～8℃保存。5) The obtained quality control product is stored at 2-8°C.
6)取一部分所述质控品，复溶后提取核酸，对所包含的病原体分别进行数字PCR定值检测。6) Take a part of the quality control material, extract nucleic acid after reconstitution, and perform digital PCR quantitative detection on the contained pathogens respectively.
对余下质控品进行如下处理：The remaining controls were processed as follows:
a、不复溶，37℃放置14天；或a. Not reconstituted, placed at 37°C for 14 days; or
b、复溶后，4℃放置14天。b. After reconstitution, place at 4°C for 14 days.
随后对经a或b处理的质控品分别进行稳定性验证，数据如表3所示：Subsequently, the stability verification of the quality control products treated by a or b was carried out respectively, and the data are shown in Table 3:
表3table 3
实施例2制备的质控品与实施例4配方对比，两者差别在实施例2添加鲸蜡醇聚氧乙烯(3)醚，实施例2配方稳定性明显好于实施例3配方的稳定性。The quality control product prepared in Example 2 is compared with the formulation of Example 4. The difference between the two is that cetyl alcohol polyoxyethylene (3) ether is added in Example 2. The stability of the formulation of Example 2 is obviously better than that of the formulation of Example 3. .
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
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|---|---|---|---|---|
| CN115541881A (en) * | 2022-10-28 | 2022-12-30 | 山东康华生物医疗科技股份有限公司 | Detection kit capable of inactivating neocoronal antigen reagent |
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