Simple Low-Cost Production of DNA MS2 Virus-Like Particles As Molecular Diagnostic Controls.
Simple Low-Cost Production of DNA MS2 Virus-Like Particles As Molecular Diagnostic Controls.
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DOI：
10.1089/genbio.2022.0033
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发表时间：
2022-12-01
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--
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适当的控制对于诊断工作流程的验证和持续质量保证至关重要。质粒、DNA 或体外转录的 RNA 通常用于验证新型诊断工作流程，但它们对临床样本的代表性较差。与外源 RNA 包装的 RNA 噬菌体病毒样颗粒 (VLP) 已在临床诊断中用作工作流程控制，充当感染性病毒颗粒的替代品。 DNA 病毒的类似对照品的生产更具挑战性，因为类似的 DNA 噬菌体具有感染性，并且在 RNA 噬菌体中包装 DNA 需要复杂的纯化程序和昂贵的化学接头。我们提出了一种简单且廉价的方法来生产与 DNA 包装的 Emesvirus zinderi (MS2) VLP，该方法利用亲和层析来纯化和酶促生产适合包装的外源 DNA。生产的 VLP 与乙型肝炎病毒 DNA 一起包装，然后使用液滴数字 PCR 进行定量，并在认可的临床实验室中使用商业化验根据 WHO 国际标准进行校准。
Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA, or in vitro transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles (VLPs) packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce Emesvirus zinderi (MS2) VLPs, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced VLPs were packaged with hepatitis B virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory.
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影响因子：
9.4
作者：
Pasloske, BL;Walkerpeach, CR;DuBois, DB
通讯作者：
DuBois, DB
影响因子：
9.4
作者：
Dingle, KE;Crook, D;Jeffery, K
通讯作者：
Jeffery, K
影响因子：
5
作者：
Freystetter, Andrea;Paar, Christian;Berg, Joerg
通讯作者：
Berg, Joerg
DOI：
10.1128/genomea.01576-16
发表时间：
2017-02-01
期刊：
GENOME ANNOUNCEMENTS
影响因子：
--
作者：
Jenkins, Adrian;Minhas, Rehan;Berry, Neil
通讯作者：
Berry, Neil
影响因子：
9.4
作者：
Heermann, KH;Gerlich, WH;Thomssen, R
通讯作者：
Thomssen, R