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Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection

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The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4–5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.
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Keywords: Automated Nucleic Acid Extractor; Hepatitis B Virus (HBV); Hepatitis C Virus (HCV); Magnetic Nanoparticles (MNPs)

Document Type: Research Article

Affiliations: 1: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China 2: School of Applied Chemistry and Biotechnology, Shenzhen Polytechnic, Shenzhen 518055, China 3: Jiangsu Province Blood Center, Nanjing 210042, China 4: Department of Pathobiology, University of Arid Agriculture, Rawalpindi 46000, Pakistan

Publication date: February 1, 2017

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  • Journal for Nanoscience and Nanotechnology (JNN) is an international and multidisciplinary peer-reviewed journal with a wide-ranging coverage, consolidating research activities in all areas of nanoscience and nanotechnology into a single and unique reference source. JNN is the first cross-disciplinary journal to publish original full research articles, rapid communications of important new scientific and technological findings, timely state-of-the-art reviews with author's photo and short biography, and current research news encompassing the fundamental and applied research in all disciplines of science, engineering and medicine.
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