Exploring Viral Genomics at Lawrence Livermore National Laboratory

Kilpatrick, K ; Hiddessen, A

关键词: AMPLIFICATION;    DNA;    DYES;    ELECTROPHORESIS;    FOCUSING;    INFECTIOUS DISEASES;    LAWRENCE LIVERMORE NATIONAL LABORATORY;    MOLECULAR BIOLOGY;    NUCLEIC ACIDS;    POLYMERASE CHAIN RE;   

DOI  :  10.2172/924957
RP-ID  :  UCRL-TR-233957
PID  :  OSTI ID: 924957
Others  :  TRN: US200809%%712

美国|英语

来源: SciTech Connect

【 摘 要 】

This summer I had the privilege of working at Lawrence Livermore National Laboratory under the Nonproliferation, Homeland and International Security Directorate in the Chemical and Biological Countermeasures Division. I worked exclusively on the Viral Identification and Characterization Initiative (VICI) project focusing on the development of multiplexed polymerase chain reaction (PCR) assays. The goal of VICI is to combine several disciplines such as molecular biology, microfluidics, and bioinformatics in order to detect viruses and identify them in order to effectively and quickly counter infectious disease, natural or engineered. The difficulty in such a countermeasure is that little is known about viral diversity due to the ever changing nature of these organisms. In response, VICI is developing a new microfluidic bioanalytical platform to detect known and unknown viruses by analyzing every virus in a sample by isolating them into picoliter sized droplets on a microchip and individually analyzing them. The sample will be injected into a channel of oil to form droplets that will contain viral nucleic acids that will be amplified using PCR. The multiplexed PCR assay will produce a series of amplicons for a particular virus genome that provides an identifying signature. A device will then detect whether or not DNA is present in the droplet and will sort the empty droplets from the rest. From this point, the amplified DNA is released from the droplets and analyzed using capillary gel electrophoresis in order to read out the series of amplicons and thereby determine the identity of each virus. The following figure depicts the microfluidic process. For the abovementioned microfluidic process to work, a method for detecting amplification of target viral nucleic acids that does not interfere with the multiplexed biochemical reaction is required for downstream sorting and analysis. In this report, the successful development of a multiplexed PCR assay using SYBR Green I as a fluorescent dye to detect amplification of viral DNA that can later be integrated into microfluidic PCR system for sorting and analysis is shown.