title: Preparation of MS2-based nanoparticles as control and standard materials for the molecular detection of dengue virus serotypes.
abstract: To quantify dengue virus (DENV) and evaluate the performance of clinical laboratories using quantitative real-time polymerase chain reaction (qRT-PCR) assays, we constructed high-efficiency expression systems to produce DENV-1 to 4 nanoparticles and assessed their suitability as standard and control materials in 20 laboratories across China. Targeted gene sequences of DENV-1 to 4 were synthesized and inserted into pACYC-Duet 1-MS2 recombinant plasmids to generate corresponding nanoparticle expression systems. After collection, verification, and quantification by digital PCR (dPCR), DENV-1 to 4 nanoparticles were prepared as control and standard materials. Five positive and three negative samples of each DENV serotype in every panel were used for assessing the performance of the participating laboratories across China, as well as standard materials for the quantitative detection of DENV using qRT-PCR assays. The accuracy, sensitivity, and specificity of qRT-PCR used by the 20 evaluated laboratories were 89.6 (569/635), 85.1 (336/395), and 97.1% (233/240), respectively. Overall, sixteen (80.0%) laboratories were qualified in detecting DENV, among which five (25.0%) were designated as "competent", eleven (55.0%) were defined as "acceptable", and four (20%) were considered to be "improvable". The results generated from the DENV standard samples were significantly positively correlated with those generated by dPCR (r2=0.8698, P<0.001). In summary, DENV nanoparticles could potentially be used as controls for improving the performance of laboratories and as standards for the quantitative detection of DENV.
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; Department of Nuclear Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Clinical Laboratory, National Cancer Center/Cancer Hospital Chinese Academy of Medical Science & Peking Union Medical College, Beijing, People's Republic of China.
authors.forename: Guojing
authors.initials: G
authors.lastname: Wang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Wu
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Yang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China.
authors.forename: Kuo
authors.initials: K
authors.lastname: Zhang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China.
authors.forename: Guigao
authors.initials: G
authors.lastname: Lin
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China.
authors.forename: Yanxi
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Nuclear Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, People's Republic of China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Bao
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China.
authors.forename: Ziyang
authors.initials: Z
authors.lastname: Li
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China. Electronic address: jmli@nccl.org.cn.
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D015374:Biosensing Techniques / Q000379:methods / Q000592:standards*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D059021:Laboratory Proficiency Testing; D053758:Nanoparticles / Q000737:chemistry*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D065288:Serogroup; D012703:Serotyping / Q000379:methods*
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral
keywords: Control; Dengue fever; Digital polymerase chain reaction; Nanoparticle; Quantitative real-time polymerase chain reaction; Standard