title: Rapid visual identification of PCR amplified nucleic acids by centrifugal gel separation: Potential use for molecular point-of-care tests.
abstract: Recently, nucleic acid amplification and detection techniques have progressed based on advances in in microfluidics, microelectronics, and optical systems. Nucleic acids amplification based point-of-care test (POCT) in resource-limited settings requires simple visual detection methods. Several biosensing methods including lateral flow immunoassays (LFIA) were previously used to visually detect nucleic acids. However, prolonged assay time, several washing steps, and a need for specific antibodies limited their use. Here we developed a novel, rapid method to visualize amplified nucleic acids with naked eyes in clinical samples. First, we optimized conditions based on separation using very low centrifugal force and a density medium to detect human papillomavirus (HPV)-16 DNA in cervical specimens. After DNA extraction, HPV16 PCR was performed with biotin-labeled forward primer and Cy3-labeled reverse primer. PCR amplicon was mixed with streptavidin-magnetic beads, introduced into the density medium. After two-minute centrifugation, the result was visually identified. This system showed identical results with commercial HPV real-time PCR for 30 clinical samples and could detect up to 10(2)copies/mL of HPV DNA without any optical instruments. This robust and sensitive visual detection system is suitable for non-specialist personnel and point-of-care diagnosis in low-resource settings.
authors.affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea; Hematologic Malignancy Branch, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea. Electronic address: mindcatch@ncc.re.kr.
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Hwang
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea. Electronic address: kimde@konkuk.ac.kr.
authors.forename: Dong-Eun
authors.initials: DE
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea.
authors.forename: Ji-Hyun
authors.initials: JH
authors.lastname: Im
authors.affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea.
authors.forename: Su-Jin
authors.initials: SJ
authors.lastname: Kang
authors.affiliation: Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeonggi-do 410-769, Republic of Korea.
authors.forename: Do-Hoon
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Gachon University, Seongnam, Gyeonggi, and Gachon Medical Research Institute, Gil Medical Center, Inchon, Republic of Korea.
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Son
mesh_terms: D015374:Biosensing Techniques / Q000191:economics / Q000379:methods; D002232:Carbocyanines / Q000032:analysis; D002499:Centrifugation, Density Gradient / Q000191:economics / Q000379:methods*; D002584:Cervix Uteri / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D059346:Magnets / Q000737:chemistry; D061848:Optical Imaging; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D019095:Point-of-Care Systems / Q000191:economics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods
chemical_list: D002232:Carbocyanines; D004279:DNA, Viral; C085321:cyanine dye 5
keywords: Biosensing; Centrifugation; DNA separation; Gel-separation; Molecular POC; Visual detection