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A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids.

ABSTRACT: With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7079959 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids.

Zhang Lei L Sun Yu Y Chang Le L Jia Tingting T Wang Guojing G Zhang Rui R Zhang Kuo K Li Jinming J

Applied microbiology and biotechnology 20150519 17

With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successful ...[more]

PMID: 25981999

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Project description:Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

Dataset Information

A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids.

Publications

A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids.

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