用于MERS-CoV核酸检测的假病毒阳性对照品制备及鉴定 : = Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-CoV
目的制备安全、稳定的含有中东呼吸综合征冠状病毒(MERS-CoV)靶基因片段的假病毒颗粒阳性对照品。方法人工合成含up E、ORF1b以及N基因片段的MERS-CoV核苷酸序列,连入慢病毒表达载体,将重组慢病毒表达载体与包装质粒同时转染HEK293T-17细胞,利用透射电镜观察重组假病毒颗粒形态,用荧光定量RT-PCR方法检测假病毒包装情况,并进行稳定性、均一性以及线性试验。结果测序分析显示,3个基因片段正确克隆至载体pCMVMCS-GFP-PURO,经大肠杆菌表达可形成直径约为100 nm的均一、球形重组假病毒颗粒。不同浓度样品在不同温度条件下存放数日仍保持稳定,不同浓度样品、不同组别的Ct值的变异系数均小于0.1%。结论成功构建了含有upE、ORF1b以及N基因片段的MERS-CoV假病毒颗粒,可作为阳性对照品实现对核酸提取与检测过程全程监控。.
Objective To prepare quality control samples for Middle East Respiratory Syndrome Coronavirus(MERSCoV) molecular detection by constructing pseudovirus containing upE,ORF1 b and N gene sequences of MERS-CoV.Methods The nucleic acid sequence containing amplification target of MERS-CoV was synthesized and inserted into a lentiviral vector,the recombinant lentiviral vector and the lentivirus-packaging vector were co-transfected into HEK293 T-17 cells. The pseudoviral particles were characterized by transmission electron microscopy,and Real-time PCR assay was used for the detection of the packaging and production. The determination of stability,homogeneity and linearity was conducted. Result Sequencing analysis confirmed that these upE,ORF1 b and N gene sequence of MERSCoV was cloned into vector pCMV-MCS-GFP-PURO.Transmission electron microscopy showed that homogenous spherical particles with a diameter of about 100 nm. The MERS-CoV genomic RNA within the pseudovirus particles remained stable during different temperature, and the result of homogeneity and linearity showed good. Conclusion The pseudoviral particles containing upE, ORF1 b and N gene produced by using lentiviral packaging technology in this study are constructed successfully,which can be used as positive quality control samples for RNA extraction and molecular detection..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2019-08-20 2019 |
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| Erschienen: |
2019-08-20 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2019 |
|---|---|
| Enthalten in: |
Zhong guo guo jing wei sheng jian yi za zhi - (2019), 04 vom: 20. Aug., Seite 233-238 Original Letters: Enthalten in 中国国境卫生检疫杂志 (DE-600)2993033-9 (DE-600)2993033-9 北京市 |
| Reihe: |
China Academic Journals (CAJ), E, 医药卫生科技 = Medicine & Public Health |
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| Sprache: |
Chinesisch |
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| Weiterer Titel: |
Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-CoV |
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| Beteiligte Personen: |
周冬根 [VerfasserIn] |
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| Links: |
oversea.cnki.net [lizenzpflichtig] |
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| Themen: |
中东呼吸综合征冠状病毒 |
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| Anmerkungen: |
Author info:ZHOU Dong-gen;LI Yun-peng;LUO Jie;LI Jun-hua;YU Jun-ping;WEI Hong-ping;Ningbo International Travel Healthcare Center |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
CAJ637465407 |
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| 245 | 1 | 0 | |a 用于MERS-CoV核酸检测的假病毒阳性对照品制备及鉴定 |b = Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-CoV |
| 246 | 3 | 1 | |a Preparation and characterization of pseudoviral positive control for nucleic acid detection of MERS-CoV |
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| 500 | |a Author info:ZHOU Dong-gen;LI Yun-peng;LUO Jie;LI Jun-hua;YU Jun-ping;WEI Hong-ping;Ningbo International Travel Healthcare Center | ||
| 520 | |a 目的制备安全、稳定的含有中东呼吸综合征冠状病毒(MERS-CoV)靶基因片段的假病毒颗粒阳性对照品。方法人工合成含up E、ORF1b以及N基因片段的MERS-CoV核苷酸序列,连入慢病毒表达载体,将重组慢病毒表达载体与包装质粒同时转染HEK293T-17细胞,利用透射电镜观察重组假病毒颗粒形态,用荧光定量RT-PCR方法检测假病毒包装情况,并进行稳定性、均一性以及线性试验。结果测序分析显示,3个基因片段正确克隆至载体pCMVMCS-GFP-PURO,经大肠杆菌表达可形成直径约为100 nm的均一、球形重组假病毒颗粒。不同浓度样品在不同温度条件下存放数日仍保持稳定,不同浓度样品、不同组别的Ct值的变异系数均小于0.1%。结论成功构建了含有upE、ORF1b以及N基因片段的MERS-CoV假病毒颗粒,可作为阳性对照品实现对核酸提取与检测过程全程监控。 | ||
| 520 | |a Objective To prepare quality control samples for Middle East Respiratory Syndrome Coronavirus(MERSCoV) molecular detection by constructing pseudovirus containing upE,ORF1 b and N gene sequences of MERS-CoV.Methods The nucleic acid sequence containing amplification target of MERS-CoV was synthesized and inserted into a lentiviral vector,the recombinant lentiviral vector and the lentivirus-packaging vector were co-transfected into HEK293 T-17 cells. The pseudoviral particles were characterized by transmission electron microscopy,and Real-time PCR assay was used for the detection of the packaging and production. The determination of stability,homogeneity and linearity was conducted. Result Sequencing analysis confirmed that these upE,ORF1 b and N gene sequence of MERSCoV was cloned into vector pCMV-MCS-GFP-PURO.Transmission electron microscopy showed that homogenous spherical particles with a diameter of about 100 nm. The MERS-CoV genomic RNA within the pseudovirus particles remained stable during different temperature, and the result of homogeneity and linearity showed good. Conclusion The pseudoviral particles containing upE, ORF1 b and N gene produced by using lentiviral packaging technology in this study are constructed successfully,which can be used as positive quality control samples for RNA extraction and molecular detection. | ||
| 610 | 2 | 4 | |a 宁波国际旅行卫生保健中心 |
| 610 | 2 | 4 | |a 中科院武汉病毒研究所 |
| 650 | 4 | |a 传染病 | |
| 650 | 4 | |a 内科学 | |
| 650 | 4 | |a 医药、卫生 | |
| 650 | 4 | |a 诊断学 | |
| 650 | 4 | |a 临床医学 | |
| 650 | 4 | |a Clinical Medicine | |
| 650 | 4 | |a Infectious Disease | |
| 650 | 4 | |a 医药卫生科技 | |
| 650 | 4 | |a Medicine & Public Health | |
| 650 | 4 | |a 中东呼吸综合征冠状病毒 | |
| 650 | 4 | |a 假病毒 | |
| 650 | 4 | |a 病毒样颗粒 | |
| 650 | 4 | |a 阳性对照 | |
| 650 | 4 | |a MERS-CoV | |
| 650 | 4 | |a Pseudovirus | |
| 650 | 4 | |a Virus like particle | |
| 650 | 4 | |a Positive control | |
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| 700 | 0 | |a 罗洁 |4 oth | |
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