Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays
Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays
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DOI：
10.1007/s00253-017-8436-9
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发表时间：
2017-10-01
影响因子：
5
通讯作者：
Berg, Joerg
中科院分区：
文献类型：
--
作者：
Freystetter, Andrea;Paar, Christian;Berg, Joerg
在分析和临床实验室中，基于分子聚合酶链式反应的分析方法的广泛使用带来了对检测特异性、稳定和可靠的外部控制(EC)以及标准和内部扩增控制(IC)的需求，以便获得一致的测试结果。此外，为质量保证项目生产和提供稳定的、具有良好特性的分子控制的需求也越来越大。在这项研究中，我们描述了一种新的方法来产生装甲双链DNA对照，这些对照被包裹在加州拟革兰氏菌多核多角体病毒的杆状病毒(BV)颗粒中。我们使用著名的BacPAK(TM)杆状病毒表达系统(Takara-Clontech)，去除了用于蛋白表达的多角体启动子，获得了重组BV装甲DNA。获得的BV-ARM DNA易于用标准的临床DNA提取方法提取，在我们的临床PCR分析中表现出良好的线性和良好的性能，抗DNase I消化，并在人血浆和血清中表现出显著的稳定性。BV-装甲DNA应用作分子分析中的ECs、量化标准和ICs，后者的应用允许对临床分子分析的样本充分性进行整体监测。BV-装甲DNA还可用于生产用于例如质量保证计划的双链DNA标准物质。生产BV-装甲DNA的简便性应该会使这种方法在广泛的分子应用中变得可行。最后，由于BV装甲DNA对哺乳动物不具传染性，它们的运输可能比临床标本更方便。
The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK (TM) Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.